# Patent

## Title
Rapid method for detection of pathogen

## Abstract

The present specification discloses methods of detecting a pathogen of interest and components useful in carrying out these methods, including a pre-enrichment media, and enrichment media and a detection solution.

## Background
This patent application claims priority pursuant to 35 U.S.C. §119(e) to U.S. Provisional Patent Application 61/927,401, filed Jan. 14, 2014, and claims priority pursuant to 35 U.S.C. §119(a) to Spanish Patent Application ES 201330064, filed Jan. 22, 2013, each of which is hereby incorporated by reference in its entirety.


Pathogens can contaminate food or food animals during production, processing and preparation. Similarly, pathogens can contaminate water sources or seafood harvested from such contaminated waters. Human exposure to pathogens can cause illness, most often gastroenteritis, but also potentially more serious diseases such as salmonellosis and hepatitis A. Exposure to pathogens can occur either by direct contact with, or ingestion of, contaminated foods or water or indirectly based on cross-contamination. Even though the United States has one of the safest food supplies in the world, there are still millions of cases of foodborne illness each year.
Early and rapid detection of pathogen contamination is vital to prevent widespread outbreaks of illness and ensure the health of the public at large. During the past 25 years, pathogen assays that increase sensitiviety and specificity as well as decrease the time involved to perform these assays have been developed by experts in applied microbiology and microbiological analysis. Although there is no universally accepted definition for these so-called rapid assays, these methods are simply performed faster than the traditional ones, or are easier to implement, or are more sensitive and specific. Despite the name, currently available rapid assays for pathogen detection still require several days to complete the assay and determine whether a food or water supply is contaminated with a pathogen.
The main obstacles associated with reducing the time needed to conduct pathogen detection assays is the balance between the sensitivity, specificity, and lower limit of detection of the assay on one hand and having enough pathogen present to detect its presence above contaminating microorganisms and background noise.
The present specification discloses a rapid method for the detection of pathogens which provides high sensitivity and specificity, a lower limit of detection, yet can be performed more quickly then currently available methods.
SUMMARY
Aspects of the present specification disclose a method of detecting a pathogen in a sample. In aspects, a method disclosed herein comprises the steps of a) incubation of the sample in a pre-enrichment comprising a low growth nutrient component, a growth inhibiting agent, and a surfactant with bacteriostatic or bactericidal action, wherein the incubation occurs at a specific temperature for a specific length of time; b) incubating an aliquot of pre-enrichment media from step (a) in an enrichment media, the enrichment media comprising a high growth nutrient component and a growth promoting agent, wherein the incubation occurs at a specific temperature for a specific length of time; and c) detecting the presence of absence of a pathogen by analyzing an aliquot of enrichment media from step (b).
Other aspects of the present specification disclose a pathogen analysis kit. In aspects, pathogen analysis kit comprising a pre-enrichment media and an enrichment media. In other aspects, the pathogen analysis kit may further comprise detection solution. In yet other aspects, the pathogen analysis kit may further comprise an immunopurification reagent system. In yet other aspects, the pathogen analysis kit may further comprise a label or an insert providing instructions on how to use the kit.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a graph of the proportionality between different densities of bacterial population and the signal obtained chronoamperometric.
FIG. 2 shows a graph of the proportionality between the registered electrochemical signal and progress in a particular bacterial load proliferation.
FIG. 3 shows a graph of the linear time-response curve of the presence of pathogen in fecal samples.
FIGS. 4A, 4B, and 4C show graphs of three independent experiments plotting the presence of target DNA amplified during each cycle of a PCR reaction.
FIG. 5 shows a graph of the proportionality between the registered electrochemical signal and progress in a particular bacterial load proliferation at pH 5.7. Bacterium were contacted with 1 mM pAPP, pH 5.7 for 30 minutes at 37° C. CA E=0.2 V t=60 s PalmSens PS06333.
FIG. 6 shows a graph of the proportionality between the registered electrochemical signal and progress in a particular bacterial load proliferation at pH 7.0. Bacterium were contacted with 1 mM pAPP, pH 5.7 for 30 minutes at 37° C. CA E=0.2 V t=60 s PalmSens PS06333.
FIG. 7 shows a graph of the proportionality between the registered electrochemical signal and progress in a particular bacterial load proliferation at pH 9.8. Bacterium were contacted with 1 mM pAPP, pH 5.7 for 30 minutes at 37° C. CA E=0.2 V t=60 s PalmSens PS06333.



DESCRIPTION
The present specification discloses a method of detecting a pathogen in a sample. The method comprises a pre-enrichment step and an enrichment step that use selective pre-growth and growth media. The combination of these steps, together with specific time and temperature conditions, progressively increases the population of the pathogen as well as an effective reduces the populations of unwanted organisms and/or other background noise which interferes with the detection of the pathogen. The selective increase of the pathogen population as well as the effectively removing unwanted organisms and/or other background noise allows for a more sensitive and precise detection of the pathogen then currently available methods. In addition, the method disclosed herein allows for a more rapid detection of a pathogen since it may be completed in about eight hours to about 30 hours; current pathogen detection methods require about 2 days to about 5 days to complete.
Aspect of the present specification disclose, in part, a pathogen. A pathogen refers to a microorganism that can cause disease in its host. Non-limiting examples of a pathogen include a prion, a virus, a bacterium, a fungus, a protozoan, a helimenth, and a parasite.
A prion is composed of a protein in a misfolded form. Prions reproduce by hijacking the functions of living cells and propagating a misfolded protein state by inducing existing, properly-folded proteins to convert into the disease-associated, prion form. As such, the prion acts as a template to guide the misfolding of more proteins into prion form. These newly formed prions can then go on to convert more proteins themselves, which triggers a chain reaction that produces large amounts of the prion form. Prions cause neurodegenerative disease by aggregating extracellularly within the central nervous system to form plaques known as amyloid, which disrupt the normal tissue structure. Prions are responsible for the transmissible spongiform encephalopathies in a variety of mammals, including bovine spongiform encephalopathy (BSE, also known as “mad cow disease”) in cattle, scrapies in sheep and goats, and chronic wasting disease in deer. In humans, prions cause Creutzfeldt-Jakob disorders, Gerstmann-Sträussler-Scheinker syndrome, Fatal Familial Insomnia, and kuru.
A virus is a small infectious agent typically range between 20-300 nanometers in length that replicates only inside the living cells of other organisms. Virus particles (known as virions) consist of two or three parts: i) the genetic material made from either DNA or RNA, long molecules that carry genetic information; ii) a protein coat that protects these genes; and in some cases iii) an envelope of lipids that surrounds the protein coat when they are outside a cell. The shapes of viruses range from simple helical and icosahedral forms to more complex structures. The average virus is about one one-hundredth the size of the average bacterium. Viruses can infect all types of life forms, from animals and plants to bacteria and archaea. Non-limiting examples of pathogenic viruses belong to the families Adenoviridae, Picornaviridae, Herpesviridae, Hepadnaviridae, Flaviviridae, Retroviridae, Orthomyxoviridae, Paramyxoviridae, Papovaviridae, Polyomavirus, Rhabdoviridae, and Togaviridae.
A bacteria is a single-celled prokaryotic microorganism characterized by the lack of a membrane-bound nucleus and membrane-bound organelles and can have a cell wall (Gram positive) or lack one (Gram negative). Morphologically, bacteria can be divided into rod-shaped (bacillus), round (coccus), spiral (spirillum), and incomplete spiral (vibrios). Non-limiting examples of pathogenic bacteria belong to the genera Bacillus, Bordetella, Borrelia, Brucella, Campylobacter, Chlamydia, Chlamydophila, Clostridium, Corynebacterium, Enterobacter, Enterococcus, Escherichia, Francisella, Haemophilus, Helicobacter, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Treponema, Vibrio, and Yersinia. Non-limiting examples of specific pathogenic bacterial species include a strain of Bacillus anthracis, a strain of a strain of Bordetella pertussis, a strain of a strain of Borrelia burgdorferi, a strain of a strain of Brucella abortus, a strain of a strain of Brucella canis, a strain of a strain of Brucella melitensis, a strain of a strain of Brucella suis, a strain of a strain of Campylobacter jejuni, a strain of Chlamydia pneumonia, a strain of Chlamydia trachomatis, a strain of Chlamydophila psittaci, a strain of Clostridium botulinum, a strain of Clostridium difficile, a strain of Clostridium perfringens, a strain of Clostridium tetani, a strain of Corynebacterium diphtheria, a strain of Enterobacter sakazakii, a strain of Enterococcus faecalis, a strain of Enterococcus faecium, a strain of Escherichia coli, a strain of Francisella tularensis, a strain of Haemophilus influenza, a strain of Helicobacter pylori, a strain of Legionella pneumophila, a strain of Leptospira interrogans, a strain of Listeria monocytogenes, a strain of Mycobacterium leprae, a strain of Mycobacterium tuberculosis, a strain of Mycobacterium ulcerans, a strain of Mycoplasma pneumonia, a strain of Neisseria gonorrhoeae, a strain of Neisseria meningitides, a strain of Pseudomonas aeruginosa, a strain of Rickettsia rickettsia, a strain of Salmonella typhi and Salmonella typhimurium, a strain of Shigella sonnei, a strain of Staphylococcus aureus, a strain of Staphylococcus epidermidis, a strain of Staphylococcus saprophyticus, a strain of Streptococcus agalactiae, a strain of Streptococcus pneumonia, a strain of Streptococcus pyogenes, a strain of Treponema pallidum, a strain of Vibrio cholera, a strain of Yersinia enterocolitica, and, a strain of Yersinia pestis. 
A fungus is a eukaryotic microorganism characterized by membrane-bound nucleus and organelles which lack chlorophyll have cell walls composed of chitin and reproduce by spores. Non-limiting examples of pathogenic fungi belong to the genera Asperfillus, Canidia, Cryptococcus, Histoplasma, Pneumocystis, and Stachybotrys. Non-limiting examples of specific pathogenic fungi species include a strain of Aspergillus clavatus, Aspergillus fumigatus, Aspergillus flavus, Canidia albicans, Cryptococcus albidus, Cryptococcus gattii, Cryptococcus laurentii, Cryptococcus neoformans, Histoplasma capsulatum, Pneumocystis jirovecii, Pneumocystis carinii, and Stachybotrys chartarum. 
A protozoa is a eukaryotic single-cell microorganism characterized by membrane-bound nucleus and organelles which lack chlorophyll and a cell wall and are motile. Protozoa commonly range from 10 to 52 micrometers, but can grow as large as 1 mm. Non-limiting examples of pathogenic protozoa belong to the genera Acanthamoeba, Balamuthia, Cryptosporidium, Dientamoeba, Endolimax, Entamoeba, Giardia, lodamoeba, Leishmania, Naegleria, Plasmodium, Sappinia, Toxoplasma, Trichomonas, and Trypanosoma. Non-limiting examples of specific pathogenic protozoa species include a strain of Acanthamoeba spp., Balamuthia mandrillaris, Cryptosporidium canis, Cryptosporidium felis, Cryptosporidium hominis, Cryptosporidium meleagridis, Cryptosporidium muris, Cryptosporidium parvum, Dientamoeba fragilis, Endolimax nana, Entamoeba dispar, Entamoeba hartmanni, Entamoeba histolytica, Entamoeba coli, Entamoeba moshkovskii, Giardia lamblia, lodamoeba butschlii, Leishmania aethiopica, Leishmania braziliensis, Leishmania chagasi, Leishmania donovani, Leishmania infantum, Leishmania major, Leishmania mexicana, Leishmania tropica, Naegleria fowleri, Plasmodium falciparum, Plasmodium knowlesi, Plasmodium malariae, Plasmodium ovale, Plasmodium vivax, Sappinia diploidea, Toxoplasma gondii, Trichomonas vaginalis, Trypanosoma brucei, and Trypanosoma cruzi. 
Aspect of the present specification disclose, in part, a sample. A variety of samples are useful in the methods disclosed herein. A sample refers to a biological matter that contains or potentially contains a pathogen. A sample encompasses but is not limited to a purified pathogen, a partially purified pathogen, a cell, a crude cell lysate, a partially purified cell lysate, a crude culture media, a partially purified culture media, a raw foodstuff, a partially-cooked foodstuff, a cooked foodstuff, a processed foodstuff; a dairy foodstuff, a beverage, an animal feed, a fecal sample, a vegetative sample, a soil sample, a water sample, a pond sediment, a human tissue sample, a raw livestock tissue sample, a processed livestock tissue sample, such as, e.g., leather.
Aspect of the present specification disclose, in part, a pre-enrichment step. A pre-enrichment step comprises incubating a sample disclosed herein in a pre-enrichment media for a defined time and at a defined temperature. A pre-enrichment media, also referred to as a pre-enrichment culture media is a buffered culture media that provides the nutrients necessary to sustain low-growth of the pathogen. In addition, a pre-enrichment media may also contain components which reduce or inhibit the growth of contaminating bacteria or other microorganisms. A pre-enrichment media comprises a low growth nutrient component, a surfactant, and optionally a growth inhibiting agent and/or a growth enhancing agent.
A pre-enrichment media typically comprises a low growth nutrient component used as a source of proteins, amino acids and nitrogen. Either a single low growth nutrient component may comprise a pre-enrichment media disclosed herein, or a plurality of low growth nutrient components may comprise a pre-enrichment media disclosed herein.
A non-limiting example of a low growth nutrient component is a peptone, such as, e.g., a peptide from an animal source and a peptone from a plant source. A peptone from an animal source includes, without limitation, an acid casein peptone, a bacteriological peptone, a beef extract powder, a casein peptone, a casein cc peptone, a gelatin peptone, a meat peptone, a polypeptone proteose peptone, and a proteose peptone No 3. A peptone from a plant source includes, without limitation, a malt extract, a soya peptone, and a yeast extract.
Any concentration of low growth nutrient component may be used, with the proviso that the concentration is useful to practice the methods disclosed herein. In aspects of this embodiment, a low growth nutrient component may be used at a concentration of, e.g., about 1 g/L, about 2 g/L, about 3 g/L, about 4 g/L, about 5 g/L, about 6 g/L, about 7 g/L, about 8 g/L, about 9 g/L, about 10 g/L, about 11 g/L, about 12 g/L, about 13 g/L, about 14 g/L, or about 15 g/L. In other aspects of this embodiment, a low growth nutrient component may be used at a concentration of, e.g., at least 1 g/L, at least 2 g/L, at least 3 g/L, at least 4 g/L, at least 5 g/L, at least 6 g/L, at least 7 g/L, at least 8 g/L, at least 9 g/L, at least 10 g/L, at least 11 g/L, at least 12 g/L, at least 13 g/L, at least 14 g/L, or at least 15 g/L. In yet other aspects of this embodiment, a low growth nutrient component may be used at a concentration of, e.g., at most 1 g/L, at most 2 g/L, at most 3 g/L, at most 4 g/L, at most 5 g/L, at most 6 g/L, at most 7 g/L, at most 8 g/L, at most 9 g/L, at most 10 g/L, at most 11 g/L, at most 12 g/L, at most 13 g/L, at most 14 g/L, or at most 15 g/L.
In yet other aspects of this embodiment, a low growth nutrient component may be used at a concentration of between, e.g., about 1 g/L to 2 g/L, about 1 g/L to 3 g/L, about 1 g/L to 4 g/L, about 1 g/L to 5 g/L, about 1 g/L to 6 g/L, about 1 g/L to 7 g/L, about 1 g/L to 8 g/L, about 1 g/L to 9 g/L, about 1 g/L to 10 g/L, about 1 g/L to 11 g/L, about 1 g/L to 12 g/L, about 1 g/L to 13 g/L, about 1 g/L to 14 g/L, about 1 g/L to 15 g/L, about 2 g/L to 3 g/L, about 2 g/L to 4 g/L, about 2 g/L to 5 g/L, about 2 g/L to 6 g/L, about 2 g/L to 7 g/L, about 2 g/L to 8 g/L, about 2 g/L to 9 g/L, about 2 g/L to 10 g/L, about 2 g/L to 11 g/L, about 2 g/L to 12 g/L, about 2 g/L to 13 g/L, about 2 g/L to 14 g/L, about 2 g/L to 15 g/L, about 3 g/L to 4 g/L, about 3 g/L to 5 g/L, about 3 g/L to 6 g/L, about 3 g/L to 7 g/L, about 3 g/L to 8 g/L, about 3 g/L to 9 g/L, about 3 g/L to 10 g/L, about 3 g/L to 11 g/L, about 3 g/L to 12 g/L, about 3 g/L to 13 g/L, about 3 g/L to 14 g/L, about 3 g/L to 15 g/L, about 4 g/L to 5 g/L, about 4 g/L to 6 g/L, about 4 g/L to 7 g/L, about 4 g/L to 8 g/L, about 4 g/L to 9 g/L, about 4 g/L to 10 g/L, about 4 g/L to 11 g/L, about 4 g/L to 12 g/L, about 4 g/L to 13 g/L, about 4 g/L to 14 g/L, about 4 g/L to 15 g/L, about 5 g/L to 6 g/L, about 5 g/L to 7 g/L, about 5 g/L to 8 g/L, about 5 g/L to 9 g/L, about 5 g/L to 10 g/L, about 5 g/L to 11 g/L, about 5 g/L to 12 g/L, about 5 g/L to 13 g/L, about 5 g/L to 14 g/L, about 5 g/L to 15 g/L, about 6 g/L to 7 g/L, about 6 g/L to 8 g/L, about 6 g/L to 9 g/L, about 6 g/L to 10 g/L, about 6 g/L to 11 g/L, about 6 g/L to 12 g/L, about 6 g/L to 13 g/L, about 6 g/L to 14 g/L, about 6 g/L to 15 g/L, about 7 g/L to 8 g/L, about 7 g/L to 9 g/L, about 7 g/L to 10 g/L, about 7 g/L to 11 g/L, about 7 g/L to 12 g/L, about 7 g/L to 13 g/L, about 7 g/L to 14 g/L, about 7 g/L to 15 g/L, about 8 g/L to 9 g/L, about 8 g/L to 10 g/L, about 8 g/L to 11 g/L, about 8 g/L to 12 g/L, about 8 g/L to 13 g/L, about 8 g/L to 14 g/L, about 8 g/L to 15 g/L, about 9 g/L to 10 g/L, about 9 g/L to 11 g/L, about 9 g/L to 12 g/L, about 9 g/L to 13 g/L, about 9 g/L to 14 g/L, about 9 g/L to 15 g/L, about 10 g/L to 11 g/L, about 10 g/L to 12 g/L, about 10 g/L to 13 g/L, about 10 g/L to 14 g/L, about 10 g/L to 15 g/L, about 11 g/L to 12 g/L, about 11 g/L to 13 g/L, about 11 g/L to 14 g/L, about 11 g/L to 15 g/L, about 12 g/L to 13 g/L, about 12 g/L to 14 g/L, about 12 g/L to 15 g/L, about 13 g/L to 14 g/L, about 13 g/L to 15 g/L, or about 14 g/L to 15 g/L.
A pre-enrichment media may also comprises a surfactant. Surfactants are compounds that lower the surface tension of a liquid, allowing easier spreading, and lowering of the interfacial tension between two liquids, or between a liquid and a solid. Either a single surfactant may comprise a pre-enrichment media disclosed herein, or a plurality of surfactants may comprise a pre-enrichment media disclosed herein. A surfactant disclosed herein provides bacteriostatic and bactericide action that retards or prevents growth of unwanted organisms contained in a sample disclosed herein. As non-limiting examples, a surfactant may retard or prevent growth of unwanted bacterial cells by disrupting the mechanisms of action of adsorption on the surface, interfering with the osmotic balance, preventing the intake of nutrients, denaturing proteins, inhibiting enzyme activity, and/or damaging the cell membrane.
Useful surfactants, include, without limitation, ionic surfactants, zwitterionic (amphoteric) surfactants, non-ionic surfactants, or any combination therein. The surfactant used in a method disclosed herein can be varied as appropriate by one skilled in the art and generally depends, in part, on the particular pre-enrichment media being used, the pathogen being detected, and/or the particular unwanted bacteria being removed. Ionic surfactants include anionic surfactants based on permanent (sulfate, sulfonate, phosphate) or pH dependent (carboxylate) anions. Anionic surfactants include, without limitation, alkyl sulfates like ammonium lauryl sulfate and sodium lauryl sulfate (SDS); alkyl ether sulfates like sodium laureth sulfate and sodium myreth sulfate; docusates like dioctyl sodium sulfosuccinate; tetradecyl sodium dodecyl sulfate; 7-ethyl-2-methyl-4-undecyl sodium sulphate; octadecyl sulphate; sulfonate fluorosurfactants like perfluorooctanesulfonate (PFOS) and perfluorobutanesulfonate; alkyl benzene sulfonates; alkyl aryl ether phosphates; alkyl ether phosphates; alkyl carboxylates like fatty acid salts and sodium stearate; sodium lauroyl sarcosinate; and carboxylate fluorosurfactants like perfluorononanoate and perfluorooctanoate.
Ionic surfactants also include cationic surfactants based on permanent or pH dependent cations. Cationic surfactants include, without limitation, alkyltrimethylammonium salts like cetyl trimethylammonium bromide (CTAB) and cetyl trimethylammonium chloride (CTAC); cetylpyridinium chloride (CPC); polyethoxylated tallow amine (POEA); benzalkonium chloride (BAC); benzethonium chloride (BZT); 5-Bromo-5-nitro-1,3-dioxane; dimethyldioctadecylammonium chloride; and dioctadecyldimethylammonium bromide (DODAB), as well as pH-dependent primary, secondary or tertiary amines like surfactants where the primary amines become positively charged at pH greater than 10, or the secondary amines become charged at pH less than 4, like octenidine dihydrochloride.
Zwitterionic surfactants are based on primary, secondary or tertiary amines or quaternary ammonium cation with a sulfonate, a carboxylate, or a phosphate. Zwitterionic surfactants include, without limitation, 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS); sultaines like cocamidopropyl hydroxysultaine; betaines like cocamidopropyl betaine; or lecithins.
Non-ionic surfactants are less denaturing and as such are useful to solubilize membrane proteins and lipids while retaining protein-protein interactions. Non-limiting examples of surfactants include polyoxyethylene glycol sorbitan alkyl esters like polysorbate 20 sorbitan monooleate (TWEEN® 20), polysorbate 40 sorbitan monooleate (TWEEN® 40), polysorbate 60 sorbitan monooleate (TWEEN® 60), polysorbate 61 sorbitan monooleate (TWEEN® 61), polysorbate 65 sorbitan monooleate (TWEEN® 65), polysorbate 80 sorbitan monooleate (TWEEN® 80), and polysorbate 81 sorbitan monooleate (TWEEN® 81); poloxamers (polyethylene-polypropylene copolymers), like Poloxamer 124 (PLURONIC® L44), Poloxamer 181 (PLURONIC® L61), Poloxamer 182 (PLURONIC® L62), Poloxamer 184 (PLURONIC® L64), Poloxamer 188 (PLURONIC® F68), Poloxamer 237 (PLURONIC® F87), Poloxamer 338 (PLURONIC® L108), and Poloxamer 407 (PLURONIC® F127); alkyl phenol polyglycol ethers; polyethylene glycol alkyl aryl ethers; polyoxyethylene glycol alkyl ethers, like octaethylene glycol monododecyl ether, pentaethylene glycol monododecyl ether, BRIJ® 30, and BRIJ® 35; 2-dodecoxyethanol (LUBROL®-PX); polyoxyethylene glycol octylphenol ethers like polyoxyethylene (4-5) p-t-octyl phenol (TRITON® X-45) and polyoxyethylene octyl phenyl ether (TRITON® X-100); nonylphenol ethoxylates like Nonoxynol-4 (TERGITOLTM NP-4), Nonoxynol-6 (TERGITOLTM NP-6), Nonoxynol-7 (TERGITOLTM NP-7), Nonoxynol-8 (TERGITOLTM NP-8), Nonoxynol-9 (TERGITOLTM NP-9), Nonoxynol-10 (TERGITOLTM NP-10), Nonoxynol-11 (TERGITOLTM NP-11), Nonoxynol-12 (TERGITOLTM NP-12), Nonoxynol-13 (TERGITOLTM NP-13), Nonoxynol-15 (TERGITOLTM NP-15), Nonoxynol-30 (TERGITOLTM NP-30), Nonoxynol-40 (TERGITOLTM NP-40), Nonoxynol-50 (TERGITOLTM NP-50), Nonoxynol-55 (TERGITOLTM NP-55), and Nonoxynol-70 (TERGITOLTM NP-70); phenoxypolyethoxylethanols like nonyl phenoxypolyethoxylethanol and octyl phenoxypolyethoxylethanol; glucoside alkyl ethers like octyl glucopyranoside; maltoside alkyl ethers like dodecyl maltopyranoside; thioglucoside alkyl ethers like heptyl thioglucopyranoside; digitonins; glycerol alkyl esters like glyceryl laurate; alkyl aryl polyether sulfates; alcohol sulfonates; sorbitan alkyl esters; cocamide ethanolamines like cocamide monoethanolamine and cocamide diethanolamine; sucrose monolaurate; dodecyl dimethylamine oxide, and sodium cholate. Other non-limiting examples of surfactants useful in the methods disclosed herein can be found in, e.g., Winslow, et al., Methods and Compositions for Simultaneously Isolating Hemoglobin from Red Blood Cells and Inactivating Viruses, U.S. 2008/0138790; Pharmaceutical Dosage Forms and Drug Delivery Systems (Howard C. Ansel et al., eds., Lippincott Williams & Wilkins Publishers, 7th ed. 1999); Remington: The Science and Practice of Pharmacy (Alfonso R. Gennaro ed., Lippincott, Williams & Wilkins, 20th ed. 2000); Goodman & Gilman's The Pharmacological Basis of Therapeutics (Joel G. Hardman et al., eds., McGraw-Hill Professional, 10th ed. 2001); and Handbook of Pharmaceutical Excipients (Raymond C. Rowe et al., APhA Publications, 4th edition 2003), each of which is hereby incorporated by reference in its entirety.
Any concentration of surfactant may be used, with the proviso that the concentration is useful to practice the methods disclosed herein. In aspects of this embodiment, a surfactant may be used at a concentration of, e.g., about 0.01% (v/v), about 0.05% (v/v), about 0.075% (v/v), about 0.1% (v/v), about 0.2% (v/v), about 0.3% (v/v), about 0.4% (v/v), about 0.5% (v/v), about 0.6% (v/v), about 0.7% (v/v), about 0.8% (v/v), about 0.9% (v/v), about 1.0% (v/v), about 2.0% (v/v), about 3.0% (v/v), about 4.0% (v/v), about 5.0% (v/v), about 6.0% (v/v), about 7.0% (v/v), about 8.0% (v/v), about 9.0% (v/v), or about 10.0% (v/v). In other aspects of this embodiment, a surfactant may be used at a concentration of, e.g., at least 0.01% (v/v), at least 0.05% (v/v), at least 0.075% (v/v), at least 0.1% (v/v), at least 0.25% (v/v), at least 0.5% (v/v), at least 0.75% (v/v), at least 1.0% (v/v), at least 2.5% (v/v), at least 5.0% (v/v), at least 7.5% (v/v), or at least 10.0% (v/v). In yet other aspects of this embodiment, a surfactant may be used at a concentration of, e.g., at most 0.01% (v/v), at most 0.05% (v/v), at most 0.075% (v/v), at most 0.1% (v/v), at most 0.25% (v/v), at most 0.5% (v/v), at most 0.75% (v/v), at most 1.0% (v/v), at most 2.5% (v/v), at most 5.0% (v/v), at most 7.5% (v/v), or at most 10.0% (v/v).
In still other aspects of this embodiment, a surfactant may be used at a concentration of between, e.g., about 0.01% (v/v) to about 0.05% (v/v), about 0.01% (v/v) to about 0.1% (v/v), about 0.01% (v/v) to about 0.5% (v/v), about 0.01% (v/v) to about 1.0% (v/v), about 0.01% (v/v) to about 2.0% (v/v), about 0.01% (v/v) to about 3.0% (v/v), about 0.01% (v/v) to about 4.0% (v/v), about 0.01% (v/v) to about 5.0% (v/v), about 0.05% (v/v) to about 0.1% (v/v), about 0.05% (v/v) to about 0.5% (v/v), about 0.05% (v/v) to about 1.0% (v/v), about 0.05% (v/v) to about 2.0% (v/v), about 0.05% (v/v) to about 3.0% (v/v), about 0.05% (v/v) to about 4.0% (v/v), about 0.05% (v/v) to about 5.0% (v/v), about 0.1% (v/v) to about 0.5% (v/v), about 0.1% (v/v) to about 1.0% (v/v), about 0.1% (v/v) to about 2.0% (v/v), about 0.2% (v/v) to about 0.5% (v/v), about 0.2% (v/v) to about 1.0% (v/v), about 0.2% (v/v) to about 2.0% (v/v), about 0.2% (v/v) to about 3.0% (v/v), about 0.2% (v/v) to about 4.0% (v/v), about 0.2% (v/v) to about 5.0% (v/v), about 0.5% (v/v) to about 1.0% (v/v), about 0.5% (v/v) to about 2.0% (v/v), about 0.5% (v/v) to about 3.0% (v/v), about 0.5% (v/v) to about 4.0% (v/v), about 0.5% (v/v) to about 5.0% (v/v), about 0.5% (v/v) to about 6.0% (v/v), about 0.5% (v/v) to about 7.0% (v/v), about 0.5% (v/v) to about 8.0% (v/v), about 0.5% (v/v) to about 9.0% (v/v), about 0.5% (v/v) to about 10.0% (v/v), about 1.0% (v/v) to about 2.0% (v/v), about 1.0% (v/v) to about 3.0% (v/v), about 1.0% (v/v) to about 4.0% (v/v), about 1.0% (v/v) to about 5.0% (v/v), about 1.0% (v/v) to about 6.0% (v/v), about 1.0% (v/v) to about 7.0% (v/v), about 1.0% (v/v) to about 8.0% (v/v), about 1.0% (v/v) to about 9.0% (v/v), or about 1.0% (v/v) to about 10.0% (v/v).
In aspects of this embodiment, the surfactant may be used at a concentration of, e.g., about 0.001 g/L, about 0.002 g/L, about 0.003 g/L, about 0.004 g/L, about 0.005 g/L, about 0.006 g/L, about 0.007 g/L, about 0.008 g/L, about 0.009 g/L, about 0.01 g/L, about 0.02 g/L, about 0.03 g/L, about 0.04 g/L, about 0.05 g/L, about 0.06 g/L, about 0.07 g/L, about 0.08 g/L, about 0.09 g/L, or about 0.1 g/L. In other aspects of this embodiment, the surfactant may be used at a concentration of, e.g., at least 0.001 g/L, at least 0.002 g/L, at least 0.003 g/L, at least 0.004 g/L, at least 0.005 g/L, at least 0.006 g/L, at least 0.007 g/L, at least 0.008 g/L, at least 0.009 g/L, at least 0.01 g/L, at least 0.02 g/L, at least 0.03 g/L, at least 0.04 g/L, at least 0.05 g/L, at least 0.06 g/L, at least 0.07 g/L, at least 0.08 g/L, at least 0.09 g/L, or at least 0.1 g/L. In yet other aspects of this embodiment, the surfactant may be used at a concentration of, e.g., at most 0.001 g/L, at most 0.002 g/L, at most 0.003 g/L, at most 0.004 g/L, at most 0.005 g/L, at most 0.006 g/L, at most 0.007 g/L, at most 0.008 g/L, at most 0.009 g/L, at most 0.01 g/L, at most 0.02 g/L, at most 0.03 g/L, at most 0.04 g/L, at most 0.05 g/L, at most 0.06 g/L, at most 0.07 g/L, at most 0.08 g/L, at most 0.09 g/L, or at most 0.1 g/L.
In still other aspects of this embodiment, the surfactant may be used at a concentration of, e.g., about 0.001 g/L to about 0.005 g/L, about 0.001 g/L to about 0.006 g/L, about 0.001 g/L to about 0.007 g/L, about 0.001 g/L to about 0.008 g/L, about 0.001 g/L to about 0.009 g/L, about 0.001 g/L to about 0.01 g/L, about 0.001 g/L to about 0.02 g/L, about 0.001 g/L to about 0.03 g/L, about 0.001 g/L to about 0.04 g/L, about 0.001 g/L to about 0.05 g/L, about 0.001 g/L to about 0.06 g/L, about 0.001 g/L to about 0.07 g/L, about 0.001 g/L to about 0.08 g/L, about 0.001 g/L to about 0.09 g/L, about 0.001 g/L to about 0.1 g/L, about 0.005 g/L to about 0.01 g/L, about 0.005 g/L to about 0.02 g/L, about 0.005 g/L to about 0.03 g/L, about 0.005 g/L to about 0.04 g/L, about 0.005 g/L to about 0.05 g/L, about 0.005 g/L to about 0.06 g/L, about 0.005 g/L to about 0.07 g/L, about 0.005 g/L to about 0.08 g/L, about 0.005 g/L to about 0.09 g/L, about 0.005 g/L to about 0.1 g/L, about 0.01 g/L to about 0.05 g/L, about 0.01 g/L to about 0.06 g/L, about 0.01 g/L to about 0.07 g/L, about 0.01 g/L to about 0.08 g/L, about 0.01 g/L to about 0.09 g/L, about 0.01 g/L to about 0.1 g/L, or about 0.05 g/L to about 0.1 g/L.
A pre-enrichment media may optionally comprises a growth inhibiting agent. A growth inhibiting agent typically comprises a component that reduces or inhibits the growth of contaminating bacteria or other contaminating microorganisms. In addition, a growth inhibiting agent may not affect the growth of a pathogen of interest or affects it to a lesser extent that the contaminating bacteria or other contaminating microorganisms. Either a single growth inhibiting agent may comprise a pre-enrichment media disclosed herein, or a plurality of growth inhibiting agents may comprise a pre-enrichment media disclosed herein. Non-limiting examples of a growth inhibiting agent include an anti-microbial compound, iodine, magneusium, and a triarylmethane dye.
An anti-microbial compound is one that is antagonistic to the growth of a microorganism. These compounds can be divided into two broad categories of microbicidal agents which kill a microorganism and microbiostatic agents which slow down or stall the growth of a microorganism. An anti-microbial compound is commonly classified based on its mechanism of action, chemical structure, or spectrum of activity. Most target bacterial functions or growth processes. Those that target the bacterial cell wall (penicillins and cephalosporins) or the cell membrane (polymyxins), or interfere with essential bacterial enzymes (rifamycins, lipiarmycins, quinolones, and sulfonamides) have bactericidal activities. Those that target protein synthesis (macrolides, lincosamides and tetracyclines) are usually bacteriostatic (with the exception of bactericidal aminoglycosides). Further categorization is based on their target specificity. “Narrow-spectrum” antibacterial antibiotics target specific types of bacteria, such as Gram-negative or Gram-positive bacteria, whereas broad-spectrum antibiotics affect a wide range of bacteria. An anti-microbial compound includes, without limitation, an aminoglycoside, an ansamycin, a carbacephem, a carbapenen, a cephalosporin, a cyclic lipopeptide, a glycopeptide, a glycylcycline, a lincosamide, a lipiamycin, a lipopeptide, a macrolide, a monobactam, a nitrofuran, an oxazolidonome, a penicillin, a qunolones, a sulfonamide, and a tetracycline.
Iodine combines with amino acids like tyrosine or histidine by a simple chemical reaction that denatures protiens having these amino acids exposed to the extra-cellular environment.
Magnesium known to have anti-bacterial activity. In aspects of this embodiment, magnesium is magnesium chloride and magnesium sulfate.
A triarylmethane dye is a group of synthetic organic compounds containing triphenylmethane backbones that produce an intense, pH-dependent color. Triarylmethane dyes can be grouped into families according to the nature of the substituents on the aryl groups. Methyl violet dyes have dimethylamino groups at the p-positions of two aryl groups and include, without limitation, methyl violet 2B, methyl violet 6B, and methyl violet 10B. Fuchsine dyes have amine (NH2 or NHMe) functional groups at the p-positions of each aryl group and include, without limitation, pararosaniline, fuchsine, new fuchsine, fuchsin basic violet, and fuchine acid. Phenol dyes have hydroxyl groups at the p positions of each aryl group and include, without limitation, phenol red, chlorophenol red, and cresol red. Malachite green dyes are related to the methyl violet dyes, except that they contain one phenyl (C6H5) group and include, without limitation, malachite green and brilliant green. A triarylmethane dye includes, without limitation, aluminon, aniline Blue WS, aurin, aurintricarboxylic acid, brilliant blue FCF, brilliant green, bromocresol green, bromocresol purple, bromophenol blue, bromopyrogallol red, bromothymol blue, bromsulphthalein, chlorophenol red, coomassie brilliant blue, cresol red, crystal violet, crystal violet lactone, ethyl green, fast green FCF, fluoran, fuchsine, fuchsine acid, gentian, green S, light green SF yellowish, malachite green, methyl blue, methyl violet, new fuchsine, pararosaniline, patent blue V, phenol red, phenolphthalein, rose bengal, thymolphthalein, victoria blue BO, water blue, xylene cyanol, and xylenol orange.
Any concentration of growth inhibiting agent may be used, with the proviso that the concentration is useful to practice the methods disclosed herein. In aspects of this embodiment, a growth inhibiting agent may be used at a concentration of, e.g., about 0.01% (v/v), about 0.05% (v/v), about 0.075% (v/v), about 0.1% (v/v), about 0.2% (v/v), about 0.3% (v/v), about 0.4% (v/v), about 0.5% (v/v), about 0.6% (v/v), about 0.7% (v/v), about 0.8% (v/v), about 0.9% (v/v), about 1.0% (v/v), about 2.0% (v/v), about 3.0% (v/v), about 4.0% (v/v), about 5.0% (v/v), about 6.0% (v/v), about 7.0% (v/v), about 8.0% (v/v), about 9.0% (v/v), or about 10.0% (v/v). In other aspects of this embodiment, a growth inhibiting agent may be used at a concentration of, e.g., at least 0.01% (v/v), at least 0.05% (v/v), at least 0.075% (v/v), at least 0.1% (v/v), at least 0.25% (v/v), at least 0.5% (v/v), at least 0.75% (v/v), at least 1.0% (v/v), at least 2.5% (v/v), at least 5.0% (v/v), at least 7.5% (v/v), or at least 10.0% (v/v). In yet other aspects of this embodiment, a growth inhibiting agent may be used at a concentration of, e.g., at most 0.01% (v/v), at most 0.05% (v/v), at most 0.075% (v/v), at most 0.1% (v/v), at most 0.25% (v/v), at most 0.5% (v/v), at most 0.75% (v/v), at most 1.0% (v/v), at most 2.5% (v/v), at most 5.0% (v/v), at most 7.5% (v/v), or at most 10.0% (v/v).
In still other aspects of this embodiment, a growth inhibiting agent may be used at a concentration of between, e.g., about 0.01% (v/v) to about 0.05% (v/v), about 0.01% (v/v) to about 0.1% (v/v), about 0.01% (v/v) to about 0.5% (v/v), about 0.01% (v/v) to about 1.0% (v/v), about 0.01% (v/v) to about 2.0% (v/v), about 0.01% (v/v) to about 3.0% (v/v), about 0.01% (v/v) to about 4.0% (v/v), about 0.01% (v/v) to about 5.0% (v/v), about 0.05% (v/v) to about 0.1% (v/v), about 0.05% (v/v) to about 0.5% (v/v), about 0.05% (v/v) to about 1.0% (v/v), about 0.05% (v/v) to about 2.0% (v/v), about 0.05% (v/v) to about 3.0% (v/v), about 0.05% (v/v) to about 4.0% (v/v), about 0.05% (v/v) to about 5.0% (v/v), about 0.1% (v/v) to about 0.5% (v/v), about 0.1% (v/v) to about 1.0% (v/v), about 0.1% (v/v) to about 2.0% (v/v), about 0.2% (v/v) to about 0.5% (v/v), about 0.2% (v/v) to about 1.0% (v/v), about 0.2% (v/v) to about 2.0% (v/v), about 0.2% (v/v) to about 3.0% (v/v), about 0.2% (v/v) to about 4.0% (v/v), about 0.2% (v/v) to about 5.0% (v/v), about 0.5% (v/v) to about 1.0% (v/v), about 0.5% (v/v) to about 2.0% (v/v), about 0.5% (v/v) to about 3.0% (v/v), about 0.5% (v/v) to about 4.0% (v/v), about 0.5% (v/v) to about 5.0% (v/v), about 0.5% (v/v) to about 6.0% (v/v), about 0.5% (v/v) to about 7.0% (v/v), about 0.5% (v/v) to about 8.0% (v/v), about 0.5% (v/v) to about 9.0% (v/v), about 0.5% (v/v) to about 10.0% (v/v), about 1.0% (v/v) to about 2.0% (v/v), about 1.0% (v/v) to about 3.0% (v/v), about 1.0% (v/v) to about 4.0% (v/v), about 1.0% (v/v) to about 5.0% (v/v), about 1.0% (v/v) to about 6.0% (v/v), about 1.0% (v/v) to about 7.0% (v/v), about 1.0% (v/v) to about 8.0% (v/v), about 1.0% (v/v) to about 9.0% (v/v), or about 1.0% (v/v) to about 10.0% (v/v).
A pre-enrichment media may optionally comprises a growth enhancing agent. A growth enhancing agent promotes rapid growth of a pathogen by reducing the lag phase in culture media, thereby reactivating dormant pathogen.
Non-limiting examples of a growth enhancing agent include a siderophore. A siderophore is a high-affinity iron chelating compound that acts to sequester and solubilize the iron. These compounds are important to a pathogen for its acquisition of iron in order to maintain cellular respiration and DNA synthesis. This is because under most culture environment, the amount of free iron (approximately 1×10−9 M) is below the concentration required by most bacterial pathogens for growth. Non-limiting examples of a siderophore include Aerobactin, Alcaligin, Azotobactin, Bacillibactin, Desferrioxamine B, Desferrioxamine E, Enterobactin, Ferrichrome, Ferrioxiamina-B, Ferrioxiamina-E, Fusarinine C, Mycobactin, Ornibactin, Petrobactin, Pyoverdine, Pyochelin, Salmochelin, Staphyloferring A, Vibriobactin, and Yersiniabactin.
Any concentration of growth enhancing agent may be used, with the proviso that the concentration is useful to practice the methods disclosed herein. In aspects of this embodiment, a growth enhancing agent may be used at a concentration of, e.g., about 0.01 μM, about 0.05 μM, about 0.075 μM, about 0.1 μM, about 0.2 μM, about 0.3 μM, about 0.4 μM, about 0.5 μM, about 0.6 μM, about 0.7 μM, about 0.8 μM, about 0.9 μM, about 1.0 μM, about 2.0 μM, about 3.0 μM, about 4.0 μM, about 5.0 μM, about 6.0 μM, about 7.0 μM, about 8.0 μM, about 9.0 μM, or about 10.0% (v/v). In other aspects of this embodiment, a growth enhancing agent may be used at a concentration of, e.g., at least 0.01 μM, at least 0.05 μM, at least 0.075 μM, at least 0.1 μM, at least 0.25 μM, at least 0.5 μM, at least 0.75 μM, at least 1.0 μM, at least 2.5 μM, at least 5.0 μM, at least 7.5 μM, or at least 10.0% (v/v). In yet other aspects of this embodiment, a growth enhancing agent may be used at a concentration of, e.g., at most 0.01 μM, at most 0.05 μM, at most 0.075 μM, at most 0.1 μM, at most 0.25 μM, at most 0.5 μM, at most 0.75 μM, at most 1.0 μM, at most 2.5 μM, at most 5.0 μM, at most 7.5 μM, or at most 10.0 μM
In still other aspects of this embodiment, a growth enhancing agent may be used at a concentration of between, e.g., about 0.01% (v/v) to about 0.05 μM, about 0.01% (v/v) to about 0.1 μM, about 0.01% (v/v) to about 0.5 μM, about 0.01% (v/v) to about 1.0 μM, about 0.01% (v/v) to about 2.0 μM, about 0.01% (v/v) to about 3.0 μM, about 0.01% (v/v) to about 4.0 μM, about 0.01% (v/v) to about 5.0 μM, about 0.05% (v/v) to about 0.1 μM, about 0.05% (v/v) to about 0.5 μM, about 0.05% (v/v) to about 1.0 μM, about 0.05% (v/v) to about 2.0 μM, about 0.05% (v/v) to about 3.0 μM, about 0.05% (v/v) to about 4.0 μM, about 0.05% (v/v) to about 5.0 μM, about 0.1% (v/v) to about 0.5 μM, about 0.1% (v/v) to about 1.0 μM, about 0.1% (v/v) to about 2.0 μM, about 0.2% (v/v) to about 0.5 μM, about 0.2% (v/v) to about 1.0 μM, about 0.2% (v/v) to about 2.0 μM, about 0.2% (v/v) to about 3.0 μM, about 0.2% (v/v) to about 4.0 μM, about 0.2% (v/v) to about 5.0 μM, about 0.5% (v/v) to about 1.0 μM, about 0.5% (v/v) to about 2.0 μM, about 0.5% (v/v) to about 3.0 μM, about 0.5% (v/v) to about 4.0 μM, about 0.5% (v/v) to about 5.0 μM, about 0.5% (v/v) to about 6.0 μM, about 0.5% (v/v) to about 7.0 μM, about 0.5% (v/v) to about 8.0 μM, about 0.5% (v/v) to about 9.0 μM, about 0.5% (v/v) to about 10.0 μM, about 1.0% (v/v) to about 2.0 μM, about 1.0% (v/v) to about 3.0 μM, about 1.0% (v/v) to about 4.0 μM, about 1.0% (v/v) to about 5.0 μM, about 1.0% (v/v) to about 6.0 μM, about 1.0% (v/v) to about 7.0 μM, about 1.0% (v/v) to about 8.0 μM, about 1.0% (v/v) to about 9.0 μM, or about 1.0% (v/v) to about 10.0 μM).
In an embodiment, a pre-enrichment media comprises 2 g/L to 12 g/L of a peptone, 0.05 to 3% (v/v) of a growth inhibiting agent and 0.05 to 5% (v/v) of a surfactant. In aspects of this embodiment, a pre-enrichment media comprises 2 g/L to 12 g/L of a soy peptone, 0.05 to 3% (v/v) of an anti-microbial compound and 0.05 to 5% (v/v) of a surfactant. In other aspects of this embodiment, a pre-enrichment media comprises 2 g/L to 12 g/L of a soy peptone, 0.05 to 3% (v/v) of an anti-microbial compound and 0.05 to 5% (v/v) of a non-ionic surfactant. In yet other aspects of this embodiment, a pre-enrichment media comprises 2 g/L to 12 g/L of a soy peptone, 0.05 to 3% (v/v) of an anti-microbial compound and 0.05 to 5% (v/v) of an ionic surfactant.
In aspects of this embodiment, a pre-enrichment media comprises 2 g/L to 12 g/L of a soy peptone, 0.05 to 3% (v/v) of a triarylmethane dye and 0.05 to 5% (v/v) of a non-ionic surfactant. In other aspects of this embodiment, a pre-enrichment media comprises 2 g/L to 12 g/L of a soy peptone, 0.05 to 3% (v/v) of triarylmethane dye and 0.05 to 5% (v/v) of an ionic surfactant. In yet other aspects of this embodiment, a pre-enrichment media comprises 2 g/L to 12 g/L of a soy peptone, 0.05 to 3% (v/v) of triarylmethane dye and 0.05 to 5% (v/v) of an nonylphenol ethoxylate.
In aspects of this embodiment, a pre-enrichment media comprises 4 g/L to 8 g/L of a soy peptone, 0.05 to 3% (v/v) of a Malachite green dye and 0.05 to 5% (v/v) of an nonylphenol ethoxylate. In other aspects of this embodiment, a pre-enrichment media comprises 4 g/L to 8 g/L of a soy peptone, 0.05 to 3% (v/v) of brilliant green and 0.05 to 5% (v/v) of an nonylphenol ethoxylate. In aspects of this embodiment, a pre-enrichment media comprises 4 g/L to 8 g/L of a soy peptone, 0.05 to 3% (v/v) of a Malachite green dye and 0.05 to 5% (v/v) of an octadecyl sulfate. In other aspects of this embodiment, a pre-enrichment media comprises 4 g/L to 8 g/L of a soy peptone, 0.05 to 3% (v/v) of brilliant green and 0.05 to 5% (v/v) of an octadecyl sulfate.
In aspects of this embodiment, a pre-enrichment media comprises 4 g/L to 8 g/L of a soy peptone, 0.05 to 3% (v/v) of a Malachite green dye and 0.05 to 5% (v/v) of a Nonoxynol-4 (Tergitol-4). In other aspects of this embodiment, a pre-enrichment media comprises 4 g/L to 8 g/L of a soy peptone, 0.05 to 3% (v/v) of Brilliant Green and 0.05 to 5% (v/v) of a Nonoxynol-4 (Tergitol-4). In aspects of this embodiment, a pre-enrichment media comprises 4 g/L to 8 g/L of a soy peptone, 0.05 to 3% (v/v) of a Malachite green dye and 0.05 to 5% (v/v) of a tetradecyl sodium dodecyl sulfate. In other aspects of this embodiment, a pre-enrichment media comprises 4 g/L to 8 g/L of a soy peptone, 0.05 to 3% (v/v) of Brilliant Green and 0.05 to 5% (v/v) of a tetradecyl sodium dodecyl sulfate. In aspects of this embodiment, a pre-enrichment media comprises 4 g/L to 8 g/L of a soy peptone, 0.05 to 3% (v/v) of a Malachite green dye and 0.05 to 5% (v/v) of a 7-ethyl-2-methyl-4-undecyll sodium sulphate. In other aspects of this embodiment, a pre-enrichment media comprises 4 g/L to 8 g/L of a soy peptone, 0.05 to 3% (v/v) of Brilliant Green and 0.05 to 5% (v/v) of a 7-ethyl-2-methyl-4-undecyll sodium sulphate.
In an embodiment, a pre-enrichment media comprises 5 g/L to 15 g/L of a peptone, 2 g/L to 6 g/L Bile Salts, 0.001 g/L to 0.0012 g/L of a growth inhibiting agent and 0.05 mL/L to 6 mL/L of a surfactant. In aspects of this embodiment, a pre-enrichment media comprises 5 g/L to 15 g/L of a Caseine Peptone, 2 g/L to 6 g/L Bile Salts, 0.001 g/L to 0.0012 g/L of an anti-microbial compound and 0.05 mL/L to 6 mL/L of a surfactant. In other aspects of this embodiment, a pre-enrichment media comprises 5 g/L to 15 g/L of a Caseine Peptone, 2 g/L to 6 g/L Bile Salts, 0.001 g/L to 0.0012 g/L of an anti-microbial compound and 0.05 mL/L to 6 mL/L of a non-ionic surfactant. In yet other aspects of this embodiment, a pre-enrichment media comprises 5 g/L to 15 g/L of a Caseine Peptone, 2 g/L to 6 g/L Bile Salts, 0.001 g/L to 0.0012 g/L of an anti-microbial compound and 0.05 mL/L to 6 mL/L of an ionic surfactant.
In aspects of this embodiment, a pre-enrichment media comprises 5 g/L to 15 g/L of a Caseine Peptone, 2 g/L to 6 g/L Bile Salts, 0.001 g/L to 0.0012 g/L of a triarylmethane dye and 0.05 mL/L to 6 mL/L of a surfactant. In other aspects of this embodiment, a pre-enrichment media comprises 5 g/L to 15 g/L of a Caseine Peptone, 2 g/L to 6 g/L Bile Salts, 0.001 g/L to 0.0012 g/L of a triarylmethane dye and 0.05 mL/L to 6 mL/L of a non-ionic surfactant. In yet other aspects of this embodiment, a pre-enrichment media comprises 5 g/L to 15 g/L of a Caseine Peptone, 2 g/L to 6 g/L Bile Salts, 0.001 g/L to 0.0012 g/L of a triarylmethane dye and 0.05 mL/L to 6 mL/L of an ionic surfactant. In aspects of this embodiment, a pre-enrichment media comprises 5 g/L to 15 g/L of a Caseine Peptone, 2 g/L to 6 g/L Bile Salts, 0.001 g/L to 0.0012 g/L of a triarylmethane dye and 0.05 mL/L to 6 mL/L of a nonylphenol ethoxylate.
In aspects of this embodiment, a pre-enrichment media comprises 8 g/L to 12 g/L of a Caseine Peptone, 3 g/L to 5 g/L Bile Salts, 0.004 g/L to 0.0008 g/L of a Malachite green dye and 1.0 mL/L to 3 mL/L of a nonylphenol ethoxylate. In aspects of this embodiment, a pre-enrichment media comprises 8 g/L to 12 g/L of a Caseine Peptone, 3 g/L to 5 g/L Bile Salts, 0.004 g/L to 0.0008 g/L of Brilliant Green and 1.0 mL/L to 3 mL/L of a nonylphenol ethoxylate.
In aspects of this embodiment, a pre-enrichment media comprises 8 g/L to 12 g/L of a Caseine Peptone, 3 g/L to 5 g/L Bile Salts, 0.004 g/L to 0.0008 g/L of a Malachite green dye and 1.0 mL/L to 3 mL/L of Nonoxynol-4 (Tergitol-4). In other aspects of this embodiment, a pre-enrichment media comprises 8 g/L to 12 g/L of a Caseine Peptone, 3 g/L to 5 g/L Bile Salts, 0.004 g/L to 0.0008 g/L of Brilliant Green and 1.0 mL/L to 3 mL/L of Nonoxynol-4 (Tergitol-4).
In another aspect of this embodiment, a pre-enrichment media comprises 10 g/L Caseine Peptone, 4 g/L Bile Salts, 3.5 g/L Disodium Phosphate, 1.5 g/L Potassium Phosphate, 0.006 g/L Brilliant Green, and 2 mL/L Nonoxynol-4 (Tergitol-4). In another aspect of this embodiment, a pre-enrichment media comprises 10 g/L Caseine Peptone, 4 g/L Bile Salts, 3.5 g/L Disodium Phosphate, 1.5 g/L Potassium Phosphate, 0.002 g/L Brilliant Green, and 2 mL/L Nonoxynol-4 (Tergitol-4).
In an embodiment, a pre-enrichment media comprises 5 g/L to 15 g/L of a peptone, 2 g/L to 6 g/L of Bile Salts, 2 g/L to 6 g/L of Meat Extract, 2 g/L to 6 g/L of a first growth inhibiting agent, and 0.001 g/L to 0.0012 g/L of a second growth inhibiting agent. In aspects of this embodiment, a pre-enrichment media comprises 5 g/L to 15 g/L of a Casien peptone, 2 g/L to 6 g/L of Bile Salts, 2 g/L to 6 g/L of Meat Extract, 2 g/L to 6 g/L of an iodine compound, and 0.001 g/L to 0.0012 g/L of an anti-microbial compound. In other aspects of this embodiment, a pre-enrichment media comprises 5 g/L to 15 g/L of a Casien peptone, 2 g/L to 6 g/L of Bile Salts, 2 g/L to 6 g/L of Meat Extract, 2 g/L to 6 g/L of an iodine compound, and 0.001 g/L to 0.0012 g/L of a triarylmethane dye.
In an embodiment, a pre-enrichment media comprises 5 g/L to 15 g/L of a peptone, 2 g/L to 6 g/L of Bile Salts, 2 g/L to 6 g/L of Meat Extract, 2 g/L to 6 g/L of a first growth inhibiting agent, 2 g/L to 6 g/L of a second growth inhibiting agent, and 0.001 g/L to 0.0012 g/L of a third growth inhibiting agent. In aspects of this embodiment, a pre-enrichment media comprises 5 g/L to 15 g/L of a Caseine peptone, 2 g/L to 6 g/L of Bile Salts, 2 g/L to 6 g/L of Meat Extract, 2 g/L to 6 g/L of a first iodine compound, 2 g/L to 6 g/L of a second iodine compound, and 0.001 g/L to 0.0012 g/L of an anti-microbial compound. In other aspects of this embodiment, a pre-enrichment media comprises 5 g/L to 15 g/L of a Caseine peptone, 2 g/L to 6 g/L of Bile Salts, 2 g/L to 6 g/L of Meat Extract, 2 g/L to 6 g/L of a first iodine compound, 2 g/L to 6 g/L of a second iodine compound, and 0.001 g/L to 0.0012 g/L of a triarylmethane dye.
In an embodiment, a pre-enrichment media comprises 5 g/L to 15 g/L of a peptone, 2 g/L to 6 g/L of Bile Salts, 2 g/L to 6 g/L of Meat Extract, 2 g/L to 6 g/L of a first growth inhibiting agent, 2 g/L to 6 g/L of a second growth inhibiting agent, 0.001 g/L to 0.0012 g/L of a third growth inhibiting agent, and 0.001 g/L to 0.0012 g/L of a fourth growth inhibiting agent. In aspects of this embodiment, a pre-enrichment media comprises 5 g/L to 15 g/L of a Caseine peptone, 2 g/L to 6 g/L of Bile Salts, 2 g/L to 6 g/L of Meat Extract, 2 g/L to 6 g/L of a first iodine compound, 2 g/L to 6 g/L of a second iodine compound, 0.001 g/L to 0.0012 g/L of an anti-microbial compound, and 0.001 g/L to 0.0012 g/L of a triarylmethane dye.
In aspects of this embodiment, a pre-enrichment media comprises 7 g/L to 11 g/L of a Caseine peptone, 3.5 g/L to 5.5 g/L Bile Salts, 3 g/L to 5 g/L Meat Extract, 3 g/L to 5 g/L of a first iodine compound, 3 g/L to 5 g/L of a second iodine compound, 0.006 g/L to 0.0010 g/L of an anti-microbial compound, 0.006 g/L to 0.0010 g/L of a triarylmethane dye. In other aspects of this embodiment, a pre-enrichment media comprises 7 g/L to 11 g/L of a Caseine peptone, 3.5 g/L to 5.5 g/L Bile Salts, 3 g/L to 5 g/L Meat Extract, 3 g/L to 5 g/L of Iodine, 3 g/L to 5 g/L of Potassium Iodide, 0.006 g/L to 0.0010 g/L of an aminocoumarin antibiotic, 0.006 g/L to 0.0010 g/L of a Malachite green dye. In yet other aspects of this embodiment, a pre-enrichment media comprises 7 g/L to 11 g/L of a Caseine peptone, 3.5 g/L to 5.5 g/L Bile Salts, 3 g/L to 5 g/L Meat Extract, 3 g/L to 5 g/L of Iodine, 3 g/L to 5 g/L of Potassium Iodide, 0.006 g/L to 0.0010 g/L of Novobiocin, 0.006 g/L to 0.0010 g/L of Brilliant Green.
In another aspect of this embodiment, a pre-enrichment media comprises 4.7 g/L Bile Salts, 4.3 g/L Meat Extract, 8.6 g/L Caseine Peptone, 2.6 g/L NaCl, 38.7 g/L, CaCO3, 30.5 g/L, Na2S2O3, 4 g/L Iodine, 4 g/L Potassium Iodide, 0.008 g/L Novobiocin, and 0.008 g/L Brilliant Green.
In aspects of this embodiment, a pre-enrichment media comprises 7 g/L to 11 g/L of a Caseine peptone, 3.5 g/L to 5.5 g/L Bile Salts, 3 g/L to 5 g/L Meat Extract, 3 g/L to 5 g/L of a first iodine compound, 3 g/L to 5 g/L of a second iodine compound, 0.002 g/L to 0.006 g/L of an anti-microbial compound, 0.002 g/L to 0.006 g/L of a triarylmethane dye. In other aspects of this embodiment, a pre-enrichment media comprises 7 g/L to 11 g/L of a Caseine peptone, 3.5 g/L to 5.5 g/L Bile Salts, 3 g/L to 5 g/L Meat Extract, 3 g/L to 5 g/L of Iodine, 3 g/L to 5 g/L of Potassium Iodide, 0.002 g/L to 0.006 g/L of an aminocoumarin antibiotic, 0.002 g/L to 0.006 g/L of a Malachite green dye. In yet other aspects of this embodiment, a pre-enrichment media comprises 7 g/L to 11 g/L of a Caseine peptone, 3.5 g/L to 5.5 g/L Bile Salts, 3 g/L to 5 g/L Meat Extract, 3 g/L to 5 g/L of Iodine, 3 g/L to 5 g/L of Potassium Iodide, 0.002 g/L to 0.006 g/L of Novobiocin, 0.002 g/L to 0.006 g/L of Brilliant Green.
In another aspect of this embodiment, a pre-enrichment media comprises 4.7 g/L Bile Salts, 4.3 g/L Meat Extract, 8.6 g/L Caseine Peptone, 2.6 g/L NaCl, 38.7 g/L, CaCO3, 30.5 g/L, Na2S2O3, 4 g/L Iodine, 4 g/L Potassium Iodide, 0.008 g/L Novobiocin, and 0.008 g/L Brilliant Green. In another aspect of this embodiment, a pre-enrichment media comprises 4.7 g/L Bile Salts, 4.3 g/L Meat Extract, 8.6 g/L Caseine Peptone, 2.6 g/L NaCl, 38.7 g/L, CaCO3, 30.5 g/L, Na2S2O3, 4 g/L Iodine, 4 g/L Potassium Iodide, 0.004 g/L Novobiocin, and 0.004 g/L Brilliant Green. In another aspect of this embodiment, a pre-enrichment media comprises 2.4 g/L Bile Salts, 2.1 g/L Meat Extract, 4.3 g/L Caseine Peptone, 1.3 g/L NaCl, 19.3 g/L, CaCO3, 15.2 g/L, Na2S2O3, 2 g/L Iodine, 2 g/L Potassium Iodide, 0.004 g/L Novobiocin, and 0.004 g/L Brilliant Green. In another aspect of this embodiment, a pre-enrichment media comprises 2.4 g/L Bile Salts, 4.3 g/L Soy Peptone, 1.3 g/L NaCl, 19.3 g/L, CaCO3, 15.2 g/L, Na2S2O3, 2 g/L Iodine, 2 g/L Potassium Iodide, 0.004 g/L Novobiocin, and 0.004 g/L Brilliant Green.
In an embodiment, a pre-enrichment media comprises 5 g/L to 15 g/L of a peptone, 2 g/L to 6 g/L of Bile Salts, 2 g/L to 6 g/L of a first growth inhibiting agent, 2 g/L to 6 g/L of a second growth inhibiting agent, and 0.001 g/L to 0.0012 g/L of a third growth inhibiting agent. In aspects of this embodiment, a pre-enrichment media comprises 5 g/L to 15 g/L of a Caseine peptone, 2 g/L to 6 g/L of Bile Salts, 2 g/L to 6 g/L of a first iodine compound, 2 g/L to 6 g/L of a second iodine compound, and 0.001 g/L to 0.0012 g/L of an anti-microbial compound. In other aspects of this embodiment, a pre-enrichment media comprises 5 g/L to 15 g/L of a Caseine peptone, 2 g/L to 6 g/L of Bile Salts, 2 g/L to 6 g/L of a first iodine compound, 2 g/L to 6 g/L of a second iodine compound, and 0.001 g/L to 0.0012 g/L of a triarylmethane dye.
In aspects of this embodiment, a pre-enrichment media comprises 8 g/L to 12 g/L of a Caseine peptone, 3 g/L to 5 g/L Bile Salts, 3 g/L to 5 g/L of a first iodine compound, 3 g/L to 5 g/L of a second iodine compound, and 0.002 g/L to 0.006 g/L of an anti-microbial compound. In other aspects of this embodiment, a pre-enrichment media comprises 8 g/L to 12 of a Caseine peptone, 3 g/L to 5 g/L Bile Salts, 3 g/L to 5 g/L of Iodine, 3 g/L to 5 g/L of Potassium Iodide, and 0.002 g/L to 0.006 g/L of an aminocoumarin antibiotic. In yet other aspects of this embodiment, a pre-enrichment media comprises 8 g/L to 12 of a Caseine peptone, 3 g/L to 5 g/L Bile Salts, 3 g/L to 5 g/L of Iodine, 3 g/L to 5 g/L of Potassium Iodide, and 0.002 g/L to 0.006 g/L of Novobiocin.
In another aspect of this embodiment, a pre-enrichment media comprises 10 g/L Caseine Peptone, 4 g/L Bile Salts, 3.5 g/L Disodium Phosphate, 1.5 g/L Potassium Phosphate, 7.6 g/L NaCl, 30.5 g/L, Na2S2O3, 4 g/L Iodide, 4 g/L Potassium Iodide, and 0.004 g/L Novobiocin. In another aspect of this embodiment, a pre-enrichment media comprises 10 g/L Caseine Peptone, 2.4 g/L Bile Salts, 3.5 g/L Disodium Phosphate, 1.5 g/L Potassium Phosphate, 6.3 g/L NaCl, 15.2 g/L, Na2S2O3, 4 g/L Iodide, 4 g/L Potassium Iodide, 0.004 g/L Novobiocin, In another aspect of this embodiment, a pre-enrichment media comprises 10 g/L Caseine Peptone, 2.4 g/L Bile Salts, 3.5 g/L Disodium Phosphate, 1.5 g/L Potassium Phosphate, 6.3 g/L NaCl, 19.3 g/L, CaCO3, 15.2 g/L, Na2S2O3, 4 g/L Iodide, 4 g/L Potassium Iodide, and 0.004 g/L Novobiocin.
In an embodiment, a pre-enrichment media comprises 3 g/L to 7 g/L of a Tryptone and 0.5 g/L to 3 g/L of a growth inhibiting agent. In aspects of this embodiment, a pre-enrichment media comprises 3 g/L to 7 g/L of a Tryptone and 0.5 g/L to 3 g/L of an anti-microbial compound. In other aspects of this embodiment, a pre-enrichment media comprises 3 g/L to 7 g/L of a Tryptone and 0.5 g/L to 3 g/L of a selenium compound. In yet other aspects of this embodiment, a pre-enrichment media comprises 3 g/L to 7 g/L of a Tryptone and 0.5 g/L to 3 g/L of Sodium Hydrogen Selenite. In still other aspects of this embodiment, a pre-enrichment media comprises 4 g/L to 6 g/L of a Tryptone and 1 g/L to 2 g/L of Sodium Hydrogen Selenite. In another aspect of this embodiment, a pre-enrichment media comprises 5 g/L Tryptone, 4 g/L Lactose, 10 g/L Disodium Phosphate, 0.01 g/L L-Cystine, and 1.51 g/L Sodium Hydrogen Selenite.
In an embodiment, a pre-enrichment media comprises 3 g/L to 7 g/L of a Tryptone, 1 g/L to 3 g/L of Bile Salts, 2 g/L to 6 g/L of a first growth inhibiting agent, 2 g/L to 6 g/L of a second growth inhibiting agent, and 0.001 g/L to 0.0012 g/L of a third growth inhibiting agent. In aspects of this embodiment, a pre-enrichment media comprises 3 g/L to 7 g/L of a Tryptone, 1 g/L to 3 g/L of Bile Salts, 2 g/L to 6 g/L of a first iodine compound, 2 g/L to 6 g/L of a second iodine compound, and 0.001 g/L to 0.0012 g/L of an anti-microbial compound. In other aspects of this embodiment, a pre-enrichment media comprises 3 g/L to 7 g/L of a Tryptone, 1 g/L to 3 g/L of Bile Salts, 2 g/L to 6 g/L of a first iodine compound, 2 g/L to 6 g/L of a second iodine compound, and 0.001 g/L to 0.0012 g/L of a triarylmethane dye.
In aspects of this embodiment, a pre-enrichment media comprises 4 g/L to 6 g/L of a Tryptone, 1.5 g/L to 3.5 g/L Bile Salts, 3 g/L to 5 g/L of a first iodine compound, 3 g/L to 5 g/L of a second iodine compound, and 0.002 g/L to 0.006 g/L of an anti-microbial compound. In other aspects of this embodiment, a pre-enrichment media comprises 4 g/L to 6 g/L of a Tryptone, 1.5 g/L to 3.5 g/L Bile Salts, 3 g/L to 5 g/L of Iodine, 3 g/L to 5 g/L of Potassium Iodide, and 0.002 g/L to 0.006 g/L of an aminocoumarin antibiotic. In yet other aspects of this embodiment, a pre-enrichment media comprises 4 g/L to 6 g/L of a Tryptone, 1.5 g/L to 3.5 g/L Bile Salts, 3 g/L to 5 g/L of Iodine, 3 g/L to 5 g/L of Potassium Iodide, and 0.002 g/L to 0.006 g/L of Novobiocin.
In another aspect of this embodiment, a pre-enrichment media comprises 5 g/L Tryptone, 4 g/L Lactose, 2.4 g/L Bile Salts, 10 g/L Disodium Phosphate, 0.01 g/L L-Cystine, 19.3 g/L, CaCO3, 30.5 g/L, Na2S2O3, 4 g/L Iodide, 4 g/L Potassium Iodide, and 0.004 g/L Novobiocin. In another aspect of this embodiment, a pre-enrichment media comprises 5 g/L Tryptone, 4 g/L Lactose, 2.4 g/L Bile Salts, 10 g/L Disodium Phosphate, 0.01 g/L L-Cystine, 30.5 g/L, Na2S2O3, 4 g/L Iodide, 4 g/L Potassium Iodide, and 0.004 g/L Novobiocin.
Aspect of the present specification disclose, in part, incubation of a sample in a pre-enrichment media. Incubation of a sample is performed under temperature and time parameters that facilitate the growth of the pathogen in the sample, retard the growth of an unwanted organism in the sample, and/or establish conditions that otherwise increase the population of the pathogen in the sample and/or retard the growth of an unwanted organism in the sample. Incubation of pre-enrichment media may be performed under constant rotation, reversal or agitation.
Any temperature may be used during incubation of a sample in a pre-enrichment media, with the proviso that the temperature is useful to practice the methods disclosed herein. In aspects of this embodiment, a temperature used to incubate of a sample in a pre-enrichment media may be, e.g., about 25° C., about 26° C., about 27° C., about 28° C., about 29° C., about 30° C., about 31° C., about 32° C., about 33° C., about 34° C., about 35° C., about 36° C., about 37° C., about 38° C., about 39° C., about 40° C., about 41° C., or about 42° C. In other aspects of this embodiment, a temperature used to incubate of a sample in a pre-enrichment media may be, e.g., at least 25° C., at least 26° C., at least 27° C., at least 28° C., at least 29° C., at least 30° C., at least 31° C., at least 32° C., at least 33° C., at least 34° C., at least 35° C., at least 36° C., at least 37° C., at least 38° C., at least 39° C., at least 40° C., at least 41° C., or at least 42° C. In yet other aspects of this embodiment, a temperature used to incubate of a sample in a pre-enrichment media may be, e.g., at most 25° C., at most 26° C., at most 27° C., at most 28° C., at most 29° C., at most 30° C., at most 31° C., at most 32° C., at most 33° C., at most 34° C., at most 35° C., at most 36° C., at most 37° C., at most 38° C., at most 39° C., at most 40° C., at most 41° C., or at most 42° C. In still other aspects of this embodiment, a temperature used to incubate of a sample in a pre-enrichment media may be, e.g., about 25° C. to about 29° C., about 26° C. to about 30° C., about 27° C. to about 31° C., about 28° C. to about 32° C., about 29° C. to about 33° C., about 30° C. to about 34° C., about 31° C. to about 35° C., about 32° C. to about 36° C., about 33° C. to about 37° C., about 34° C. to about 38° C., about 35° C. to about 39° C., about 36° C. to about 40° C., about 37° C. to about 41° C., about 38° C. to about 42° C., about 39° C. to about 43° C., or about 40° C. to about 44° C.
Any time may be used during incubation of a sample in a pre-enrichment media, with the proviso that the time is useful to practice the methods disclosed herein. In aspects of this embodiment, a time used to incubate of a sample in a pre-enrichment media may be, e.g., about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours. In other aspects of this embodiment, a time used to incubate of a sample in a pre-enrichment media may be, e.g., at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, at least 23 hours, or at least 24 hours. In yet other aspects of this embodiment, a time used to incubate of a sample in a pre-enrichment media may be, e.g., at most 4 hours, at most 5 hours, at most 6 hours, at most 7 hours, at most 8 hours, at most 9 hours, at most 10 hours, at most 11 hours, at most 12 hours, at most 13 hours, at most 14 hours, at most 15 hours, at most 16 hours, at most 17 hours, at most 18 hours, at most 19 hours, at most 20 hours, at most 21 hours, at most 22 hours, at most 23 hours, or at most 24 hours. In yet other aspects of this embodiment, a time used to incubate of a sample in a pre-enrichment media may be, e.g., about 4 hours to about 6 hours, about 5 hours to about 7 hours, about 6 hours to about 8 hours, about 7 hours to about 9 hours, about 8 hours to about 10 hours, about 9 hours to about 11 hours, about 10 hours to about 12 hours, about 11 hours to about 13 hours, about 12 hours to about 14 hours, about 13 hours to about 15 hours, about 14 hours to about 16 hours, about 15 hours to about 17 hours, about 16 hours to about 18 hours, about 17 hours to about 19 hours, about 18 hours to about 20 hours, about 19 hours to about 21 hours, about 20 hours to about 22 hours, about 21 hours to about 23 hours, about 22 hours to about 24 hours, about 23 hours to about 25 hours, or about 24 hours to about 26 hours.
In aspects of this embodiment, a sample in a pre-enrichment media may be incubated at a temperature of, e.g., about 25° C., about 26° C., about 27° C., about 28° C., about 29° C., about 30° C., about 31° C., about 32° C., about 33° C., about 34° C., about 35° C., about 36° C., about 37° C., about 38° C., about 39° C., about 40° C., about 41° C., or about 42° C. for a time of, e.g., about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours. Incubation of pre-enrichment media may be performed under constant rotation, reversal or agitation.
In other aspects of this embodiment, a sample in a pre-enrichment media may be incubated at a temperature of, e.g., at least 25° C., at least 26° C., at least 27° C., at least 28° C., at least 29° C., at least 30° C., at least 31° C., at least 32° C., at least 33° C., at least 34° C., at least 35° C., at least 36° C., at least 37° C., at least 38° C., at least 39° C., at least 40° C., at least 41° C., or at least 42° C. for a time of, e.g., at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, at least 23 hours, or at least 24 hours. Incubation of pre-enrichment media may be performed under constant rotation, reversal or agitation.
In yet other aspects of this embodiment, a sample in a pre-enrichment media may be incubated at a temperature of, e.g., at most 25° C., at most 26° C., at most 27° C., at most 28° C., at most 29° C., at most 30° C., at most 31° C., at most 32° C., at most 33° C., at most 34° C., at most 35° C., at most 36° C., at most 37° C., at most 38° C., at most 39° C., at most 40° C., at most 41° C., or at most 42° C. for a time of, e.g., at most 4 hours, at most 5 hours, at most 6 hours, at most 7 hours, at most 8 hours, at most 9 hours, at most 10 hours, at most 11 hours, at most 12 hours, at most 13 hours, at most 14 hours, at most 15 hours, at most 16 hours, at most 17 hours, at most 18 hours, at most 19 hours, at most 20 hours, at most 21 hours, at most 22 hours, at most 23 hours, or at most 24 hours. Incubation of pre-enrichment media may be performed under constant rotation, reversal or agitation.
In still other aspects of this embodiment, a sample in a pre-enrichment media may be incubated at a temperature of, e.g., about 25° C. to about 29° C., about 26° C. to about 30° C., about 27° C. to about 31° C., about 28° C. to about 32° C., about 29° C. to about 33° C., about 30° C. to about 34° C., about 31° C. to about 35° C., about 32° C. to about 36° C., about 33° C. to about 37° C., about 34° C. to about 38° C., about 35° C. to about 39° C., about 36° C. to about 40° C., about 37° C. to about 41° C., about 38° C. to about 42° C., about 39° C. to about 43° C., or about 40° C. to about 44° C. for a time of, e.g., about 4 hours to about 6 hours, about 5 hours to about 7 hours, about 6 hours to about 8 hours, about 7 hours to about 9 hours, about 8 hours to about 10 hours, about 9 hours to about 11 hours, about 10 hours to about 12 hours, about 11 hours to about 13 hours, about 12 hours to about 14 hours, about 13 hours to about 15 hours, about 14 hours to about 16 hours, about 15 hours to about 17 hours, about 16 hours to about 18 hours, about 17 hours to about 19 hours, about 18 hours to about 20 hours, about 19 hours to about 21 hours, about 20 hours to about 22 hours, about 21 hours to about 23 hours, about 22 hours to about 24 hours, about 23 hours to about 25 hours, or about 24 hours to about 26 hours. Incubation of pre-enrichment media may be performed under constant rotation, reversal or agitation.
A method for the detection of a pathogen in a sample comprises the step of incubation of the sample in an enrichment media. After a pre-enrichment media step disclosed herein is completed, an aliquot of the pre-enrichment media is transferred to an enrichment media for subsequent growth of the pathogen. An enrichment step comprises incubating an aliquot of the pre-enrichment media in an enrichment media for a defined time and at a defined temperature.
Any volume of an aliquot of pre-enrichment media may be used during incubation of a pre-enrichment media in a enrichment media, with the proviso that the volume is useful to practice the methods disclosed herein. In aspects of this embodiment, an aliquot volume of pre-enrichment media transferred to an enrichment media may be, e.g., about 1/50, about 1/75, about 1/100, about 1/125, about 1/150, about 1/175, about 1/200, about 1/225, about 1/250, about 1/275, about 1/300, about 1/325, about 1/350, about 1/375, about 1/400, about 1/425, about 1/450, about 1/475, about 1/500, about 1/525, about 1/550, about 1/575, about 1/600, about 1/625, about 1/650, about 1/675, about 1/700, about 1/725, about 1/750, about 1/775, about 1/800, about 1/825, about 1/850, about 1/875, about 1/900, about 1/925, about 1/950, about 1/975, or about 1/1,000 the volume of an enrichment media used in an enrichment step. In other aspects of this embodiment, an aliquot volume of pre-enrichment media transferred to an enrichment media may be, e.g., at least 1/50, at least 1/75, at least 1/100, at least 1/125, at least 1/150, at least 1/175, at least 1/200, at least 1/225, at least 1/250, at least 1/275, at least 1/300, at least 1/325, at least 1/350, at least 1/375, at least 1/400, at least 1/425, at least 1/450, at least 1/475, at least 1/500, at least 1/525, at least 1/550, at least 1/575, at least 1/600, at least 1/625, at least 1/650, at least 1/675, at least 1/700, at least 1/725, at least 1/750, at least 1/775, at least 1/800, at least 1/825, at least 1/850, at least 1/875, at least 1/900, at least 1/925, at least 1/950, at least 1/975, or at least 1/1,000 the volume of an enrichment media used in an enrichment step. In yet other aspects of this embodiment, an aliquot volume of pre-enrichment media transferred to an enrichment media may be, e.g., at most 1/50, at most 1/75, at most 1/100, at most 1/125, at most 1/150, at most 1/175, at most 1/200, at most 1/225, at most 1/250, at most 1/275, at most 1/300, at most 1/325, at most 1/350, at most 1/375, at most 1/400, at most 1/425, at most 1/450, at most 1/475, at most 1/500, at most 1/525, at most 1/550, at most 1/575, at most 1/600, at most 1/625, at most 1/650, at most 1/675, at most 1/700, at most 1/725, at most 1/750, at most 1/775, at most 1/800, at most 1/825, at most 1/850, at most 1/875, at most 1/900, at most 1/925, at most 1/950, at most 1/975, or at most 1/1,000 the volume of an enrichment media used in an enrichment step.
In yet other aspects of this embodiment, an aliquot volume of pre-enrichment media transferred to an enrichment media may be, e.g., about 1/50 to about 1/100, about 1/50 to about 1/150, about 1/50 to about 1/200, about 1/50 to about 1/250, about 1/50 to about 1/300, about 1/50 to about 1/350, about 1/50 to about 1/400, about 1/50 to about 1/450, about 1/50 to about 1/500, about 1/550 to about 1/600, about 1/50 to about 1/650, about 1/50 to about 1/700, about 1/50 to about 1/750, about 1/50 to about 1/800, about 1/50 to about 1/850, about 1/50 to about 1/900, about 1/50 to about 1/950, about 1/50 to about 1/1,000, about 1/100 to about 1/150, about 1/100 to about 1/200, about 1/100 to about 1/250, about 1/100 to about 1/300, about 1/100 to about 1/350, about 1/100 to about 1/400, about 1/100 to about 1/450, about 1/100 to about 1/500, about 1/100 to about 1/550, about 1/100 to about 1/600, about 1/100 to about 1/650, about 1/100 to about 1/700, about 1/100 to about 1/750, about 1/100 to about 1/800, about 1/100 to about 1/850, about 1/100 to about 1/900, about 1/100 to about 1/950, about 1/100 to about 1/1,000, about 1/200 to about 1/250, about 1/200 to about 1/300, about 1/200 to about 1/350, about 1/200 to about 1/400, about 1/200 to about 1/450, about 1/200 to about 1/500, about 1/200 to about 1/550, about 1/200 to about 1/600, about 1/200 to about 1/650, about 1/200 to about 1/700, about 1/200 to about 1/750, about 1/200 to about 1/800, about 1/200 to about 1/850, about 1/200 to about 1/900, about 1/200 to about 1/950, about 1/200 to about 1/1,000, about 1/300 to about 1/350, about 1/300 to about 1/400, about 1/300 to about 1/450, about 1/300 to about 1/500, about 1/300 to about 1/550, about 1/300 to about 1/600, about 1/300 to about 1/650, about 1/300 to about 1/700, about 1/300 to about 1/750, about 1/300 to about 1/800, about 1/300 to about 1/850, about 1/300 to about 1/900, about 1/300 to about 1/950, about 1/300 to about 1/1,000, about 1/400 to about 1/450, about 1/400 to about 1/500, about 1/400 to about 1/550, about 1/400 to about 1/600, about 1/400 to about 1/650, about 1/400 to about 1/700, about 1/400 to about 1/750, about 1/400 to about 1/800, about 1/400 to about 1/850, about 1/400 to about 1/900, about 1/400 to about 1/950, about 1/400 to about 1/1,000, about 1/500 to about 1/550, about 1/500 to about 1/600, about 1/500 to about 1/650, about 1/500 to about 1/700, about 1/500 to about 1/750, about 1/500 to about 1/800, about 1/500 to about 1/850, about 1/500 to about 1/900, about 1/500 to about 1/950, or about 1/500 to about 1/1,000.
In aspects of this embodiment, a ratio of pre-enrichment media to enrichment media may used in an enrichment step may be, e.g., about 1:50, about 1:75, about 1:100, about 1:125, about 1:150, about 1:175, about 1:200, about 1:225, about 1:250, about 1:275, about 1:300, about 1:325, about 1:350, about 1:375, about 1:400, about 1:425, about 1:450, about 1:475, about 1:500, about 1:525, about 1:550, about 1:575, about 1:600, about 1:625, about 1:650, about 1:675, about 1:700, about 1:725, about 1:750, about 1:775, about 1:800, about 1:825, about 1:850, about 1:875, about 1:900, about 1:925, about 1:950, about 1:975, or about 1:1,000. In other aspects of this embodiment, a ratio of pre-enrichment media to enrichment media may used in an enrichment step may be, e.g., at least 1:50, at least 1:75, at least 1:100, at least 1:125, at least 1:150, at least 1:175, at least 1:200, at least 1:225, at least 1:250, at least 1:275, at least 1:300, at least 1:325, at least 1:350, at least 1:375, at least 1:400, at least 1:425, at least 1:450, at least 1:475, at least 1:500, at least 1:525, at least 1:550, at least 1:575, at least 1:600, at least 1:625, at least 1:650, at least 1:675, at least 1:700, at least 1:725, at least 1:750, at least 1:775, at least 1:800, at least 1:825, at least 1:850, at least 1:875, at least 1:900, at least 1:925, at least 1:950, at least 1:975, or at least 1:1,000. In yet other aspects of this embodiment, a ratio of pre-enrichment media to enrichment media may used in an enrichment step may be, e.g., at most 1:50, at most 1:75, at most 1:100, at most 1:125, at most 1:150, at most 1:175, at most 1:200, at most 1:225, at most 1:250, at most 1:275, at most 1:300, at most 1:325, at most 1:350, at most 1:375, at most 1:400, at most 1:425, at most 1:450, at most 1:475, at most 1:500, at most 1:525, at most 1:550, at most 1:575, at most 1:600, at most 1:625, at most 1:650, at most 1:675, at most 1:700, at most 1:725, at most 1:750, at most 1:775, at most 1:800, at most 1:825, at most 1:850, at most 1:875, at most 1:900, at most 1:925, at most 1:950, at most 1:975, or at most 1:1,000.
In still other aspects of this embodiment, a ratio of pre-enrichment media to enrichment media may used in an enrichment step may be, e.g., about 1:50 to about 1:100, about 1:50 to about 1:150, about 1:50 to about 1:200, about 1:50 to about 1:250, about 1:50 to about 1:300, about 1:50 to about 1:350, about 1:50 to about 1:400, about 1:50 to about 1:450, about 1:50 to about 1:500, about 1:50 to about 1:550, about 1:50 to about 1:600, about 1:50 to about 1:650, about 1:50 to about 1:700, about 1:50 to about 1:750, about 1:50 to about 1:800, about 1:50 to about 1:850, about 1:50 to about 1:900, about 1:50 to about 1:950, about 1:50 to about 1:1,000, about 1:100 to about 1:200, about 1:100 to about 1:250, about 1:100 to about 1:300, about 1:100 to about 1:350, about 1:100 to about 1:400, about 1:100 to about 1:450, about 1:100 to about 1:500, about 1:100 to about 1:550, about 1:100 to about 1:600, about 1:100 to about 1:650, about 1:100 to about 1:700, about 1:100 to about 1:750, about 1:100 to about 1:800, about 1:100 to about 1:850, about 1:100 to about 1:900, about 1:100 to about 1:950, about 1:100 to about 1:1,000, about 1:200 to about 1:250, about 1:200 to about 1:300, about 1:200 to about 1:350, about 1:200 to about 1:400, about 1:200 to about 1:450, about 1:200 to about 1:500, about 1:200 to about 1:550, about 1:200 to about 1:600, about 1:200 to about 1:650, about 1:200 to about 1:700, about 1:200 to about 1:750, about 1:200 to about 1:800, about 1:200 to about 1:850, about 1:200 to about 1:900, about 1:200 to about 1:950, about 1:200 to about 1:1,000, about 1:300 to about 1:350, about 1:300 to about 1:400, about 1:300 to about 1:450, about 1:300 to about 1:500, about 1:300 to about 1:550, about 1:300 to about 1:600, about 1:300 to about 1:650, about 1:300 to about 1:700, about 1:300 to about 1:750, about 1:300 to about 1:800, about 1:300 to about 1:850, about 1:300 to about 1:900, about 1:300 to about 1:950, about 1:300 to about 1:1,000, about 1:400 to about 1:450, about 1:400 to about 1:500, about 1:400 to about 1:550, about 1:400 to about 1:600, about 1:400 to about 1:650, about 1:400 to about 1:700, about 1:400 to about 1:750, about 1:400 to about 1:800, about 1:400 to about 1:850, about 1:400 to about 1:900, about 1:400 to about 1:950, about 1:400 to about 1:1,000, about 1:500 to about 1:550, about 1:500 to about 1:600, about 1:500 to about 1:650, about 1:500 to about 1:700, about 1:500 to about 1:750, about 1:500 to about 1:800, about 1:500 to about 1:850, about 1:500 to about 1:900, about 1:500 to about 1:950, about 1:500 to about 1:1,000, about 1:600 to about 1:650, about 1:600 to about 1:700, about 1:600 to about 1:750, about 1:600 to about 1:800, about 1:600 to about 1:850, about 1:600 to about 1:900, about 1:600 to about 1:950, about 1:600 to about 1:1,000, about 1:700 to about 1:750, about 1:700 to about 1:800, about 1:700 to about 1:850, about 1:700 to about 1:900, about 1:700 to about 1:950, about 1:700 to about 1:1,000, about 1:800 to about 1:850, about 1:800 to about 1:900, about 1:800 to about 1:950, about 1:800 to about 1:1,000, about 1:900 to about 1:950, about 1:900 to about 1:1,000, or about 1:950 to about 1:1,000.
Aspect of the present specification disclose, in part, an enrichment media. An enrichment media, also referred to as an enrichment culture media is a buffered culture media that provides the nutrients necessary to sustain high-growth of the pathogen. This is typically done by tailoring the media particularly conducive to the growth of the pathogen such as, e.g., considering survivable osmotic pressure ranges, survivable pH ranges, resistance to selective compounds, minimal nutritional requirements. Non-limiting examples of an enrichment media include a Rappaport Vassiliadis Soya Medium (RVS), a McConkey Broth, a Fraser Broth, a Soy-Triptone Broth (TSB), a Reinforce Clostridium Broth, a Campylobacter Thioglycolate Medium, a Nitrate Broth, a Triple Sugar Iron Broth (TSI), a Sodium Hippurate Broth, a Selenite Cystine Broth, a GN Broth, a Todd Hewitt Broth, a Malt Extract Broth, an Azide Dextrose Broth, and a Hektoen Broth.
In an aspect of this embodiment, when the pathogen is a Salmonella sp., the enrichment media may be a Rappaport Vassiliadis Soya Medium, a Selenite Cystine Broth, or a GN Broth. In another aspect of this embodiment, when the pathogen is an Escherichia coli, the enrichment media may be a McConkey Broth. In yet another aspect of this embodiment, when the pathogen is a Listeria monocytogenes, the enrichment media may be a Fraser Broth. In still another aspect of this embodiment, when the pathogen is a Staphylococcus aureus, the enrichment media may be a Soy-Triptone Broth (TSB). In another aspect of this embodiment, when the pathogen is a Clostridium sp., the enrichment media may be a Reinforce Clostridium Broth. In yet another aspect of this embodiment, when the pathogen is a Campylobacter sp., the enrichment media may be a Campylobacter Thioglycolate Medium.
In another aspect of this embodiment, when the pathogen is a Pseudomonas sp., the enrichment media may be a Nitrate Broth or a Triple Sugar Iron Broth (TSI). In yet another aspect of this embodiment, when the pathogen is an Acinetobacter sp., the enrichment media may be a Nitrate Broth or a Triple Sugar Iron Broth (TSI). In still another aspect of this embodiment, when the pathogen is a Legionella sp., the enrichment media may be a Sodium Hippurate Broth. In another aspect of this embodiment, when the pathogen is a Shigella sp., the enrichment media may be a Selenite Cystine Broth or a GN Broth. In yet another aspect of this embodiment, when the pathogen is a Streptococcus sp., the enrichment media may be a Todd Hewitt Broth. In another aspect of this embodiment, when the pathogen is an Enterococcus sp., the enrichment media may be an Azide Dextrose Broth.
In another aspect of this embodiment, when the pathogen is a fungus, the enrichment media may be a Malt Extract Broth. In yet another aspect of this embodiment, when the pathogen is a yeast, the enrichment media may be a Malt Extract Broth. In still another aspect of this embodiment, when the pathogen is a mold, the enrichment media may be a Malt Extract Broth. In another aspect of this embodiment, when the pathogen is a Proteus, the enrichment media may be a Hektoen Broth.
An enrichment media typically comprises a high growth nutrient component used as a source of proteins, amino acids and nitrogen. Either a single high growth nutrient component may comprise an enrichment media disclosed herein, or a plurality of high growth nutrient components may comprise an enrichment media disclosed herein. A non-limiting example of a high growth nutrient component is a peptone as disclosed herein.
Any concentration of high growth nutrient component may be used, with the proviso that the concentration is useful to practice the methods disclosed herein. In aspects of this embodiment, a high growth nutrient component may be used at a concentration of, e.g., about 1 g/L, about 2 g/L, about 3 g/L, about 4 g/L, about 5 g/L, about 6 g/L, about 7 g/L, about 8 g/L, about 9 g/L, about 10 g/L, about 11 g/L, about 12 g/L, about 13 g/L, about 14 g/L, or about 15 g/L. In other aspects of this embodiment, a high growth nutrient component may be used at a concentration of, e.g., at least 1 g/L, at least 2 g/L, at least 3 g/L, at least 4 g/L, at least 5 g/L, at least 6 g/L, at least 7 g/L, at least 8 g/L, at least 9 g/L, at least 10 g/L, at least 11 g/L, at least 12 g/L, at least 13 g/L, at least 14 g/L, or at least 15 g/L. In yet other aspects of this embodiment, a high growth nutrient component may be used at a concentration of, e.g., at most 1 g/L, at most 2 g/L, at most 3 g/L, at most 4 g/L, at most 5 g/L, at most 6 g/L, at most 7 g/L, at most 8 g/L, at most 9 g/L, at most 10 g/L, at most 11 g/L, at most 12 g/L, at most 13 g/L, at most 14 g/L, or at most 15 g/L.
In yet other aspects of this embodiment, a high growth nutrient component may be used at a concentration of between, e.g., about 1 g/L to 2 g/L, about 1 g/L to 3 g/L, about 1 g/L to 4 g/L, about 1 g/L to 5 g/L, about 1 g/L to 6 g/L, about 1 g/L to 7 g/L, about 1 g/L to 8 g/L, about 1 g/L to 9 g/L, about 1 g/L to 10 g/L, about 1 g/L to 11 g/L, about 1 g/L to 12 g/L, about 1 g/L to 13 g/L, about 1 g/L to 14 g/L, about 1 g/L to 15 g/L, about 2 g/L to 3 g/L, about 2 g/L to 4 g/L, about 2 g/L to 5 g/L, about 2 g/L to 6 g/L, about 2 g/L to 7 g/L, about 2 g/L to 8 g/L, about 2 g/L to 9 g/L, about 2 g/L to 10 g/L, about 2 g/L to 11 g/L, about 2 g/L to 12 g/L, about 2 g/L to 13 g/L, about 2 g/L to 14 g/L, about 2 g/L to 15 g/L, about 3 g/L to 4 g/L, about 3 g/L to 5 g/L, about 3 g/L to 6 g/L, about 3 g/L to 7 g/L, about 3 g/L to 8 g/L, about 3 g/L to 9 g/L, about 3 g/L to 10 g/L, about 3 g/L to 11 g/L, about 3 g/L to 12 g/L, about 3 g/L to 13 g/L, about 3 g/L to 14 g/L, about 3 g/L to 15 g/L, about 4 g/L to 5 g/L, about 4 g/L to 6 g/L, about 4 g/L to 7 g/L, about 4 g/L to 8 g/L, about 4 g/L to 9 g/L, about 4 g/L to 10 g/L, about 4 g/L to 11 g/L, about 4 g/L to 12 g/L, about 4 g/L to 13 g/L, about 4 g/L to 14 g/L, about 4 g/L to 15 g/L, about 5 g/L to 6 g/L, about 5 g/L to 7 g/L, about 5 g/L to 8 g/L, about 5 g/L to 9 g/L, about 5 g/L to 10 g/L, about 5 g/L to 11 g/L, about 5 g/L to 12 g/L, about 5 g/L to 13 g/L, about 5 g/L to 14 g/L, about 5 g/L to 15 g/L, about 6 g/L to 7 g/L, about 6 g/L to 8 g/L, about 6 g/L to 9 g/L, about 6 g/L to 10 g/L, about 6 g/L to 11 g/L, about 6 g/L to 12 g/L, about 6 g/L to 13 g/L, about 6 g/L to 14 g/L, about 6 g/L to 15 g/L, about 7 g/L to 8 g/L, about 7 g/L to 9 g/L, about 7 g/L to 10 g/L, about 7 g/L to 11 g/L, about 7 g/L to 12 g/L, about 7 g/L to 13 g/L, about 7 g/L to 14 g/L, about 7 g/L to 15 g/L, about 8 g/L to 9 g/L, about 8 g/L to 10 g/L, about 8 g/L to 11 g/L, about 8 g/L to 12 g/L, about 8 g/L to 13 g/L, about 8 g/L to 14 g/L, about 8 g/L to 15 g/L, about 9 g/L to 10 g/L, about 9 g/L to 11 g/L, about 9 g/L to 12 g/L, about 9 g/L to 13 g/L, about 9 g/L to 14 g/L, about 9 g/L to 15 g/L, about 10 g/L to 11 g/L, about 10 g/L to 12 g/L, about 10 g/L to 13 g/L, about 10 g/L to 14 g/L, about 10 g/L to 15 g/L, about 11 g/L to 12 g/L, about 11 g/L to 13 g/L, about 11 g/L to 14 g/L, about 11 g/L to 15 g/L, about 12 g/L to 13 g/L, about 12 g/L to 14 g/L, about 12 g/L to 15 g/L, about 13 g/L to 14 g/L, about 13 g/L to 15 g/L, or about 14 g/L to 15 g/L.
An enrichment media typically comprises a growth promoting agent. Non-limiting examples of a growth promoting agent is an iron containing compound that can be used by a pathogen of interest as an iron source. In an aspect of this embodiment, a growth promoting agent is ammonium ferric citrate.
Any concentration of ammonium ferric citrate may be used, with the proviso that the concentration is useful to practice the methods disclosed herein. In aspects of this embodiment, ammonium ferric citrate may be used at a concentration of, e.g., about 0.01 mg/mL, about 0.02 mg/mL, about 0.03 mg/mL, about 0.04 mg/mL, about 0.05 mg/mL, about 0.06 mg/mL, about 0.07 mg/mL, about 0.08 mg/mL, about 0.09 mg/mL, about 0.1 mg/mL, about 0.2 mg/mL, about 0.3 mg/mL, about 0.4 mg/mL, about 0.5 mg/mL, about 0.6 mg/mL, about 0.7 mg/mL, about 0.8 mg/mL, about 0.9 mg/mL, about 1.0 mg/mL, about 2.0 mg/mL, about 3.0 mg/mL, about 4.0 mg/mL, about 5.0 mg/mL, about 6.0 mg/mL, about 7.0 mg/mL, about 8.0 mg/mL, about 9.0 mg/mL, about 10 mg/mL, about 11 mg/mL, about 12 mg/mL, about 13 mg/mL, about 14 mg/mL, or about 15 mg/mL. In other aspects of this embodiment, ammonium ferric citrate may be used at a concentration of, e.g., at least 0.01 mg/mL, at least 0.02 mg/mL, at least 0.03 mg/mL, at least 0.04 mg/mL, at least 0.05 mg/mL, at least 0.06 mg/mL, at least 0.07 mg/mL, at least 0.08 mg/mL, at least 0.09 mg/mL, at least 0.1 mg/mL, at least 0.2 mg/mL, at least 0.3 mg/mL, at least 0.4 mg/mL, at least 0.5 mg/mL, at least 0.6 mg/mL, at least 0.7 mg/mL, at least 0.8 mg/mL, at least 0.9 mg/mL, at least 1.0 mg/mL, at least 2.0 mg/mL, at least 3.0 mg/mL, at least 4.0 mg/mL, at least 5.0 mg/mL, at least 6.0 mg/mL, at least 7.0 mg/mL, at least 8.0 mg/mL, at least 9.0 mg/mL, at least 10 mg/mL, at least 11 mg/mL, at least 12 mg/mL, at least 13 mg/mL, at least 14 mg/mL, or at least 15 mg/mL. In yet other aspects of this embodiment, ammonium ferric citrate may be used at a concentration of, e.g., at most 0.01 mg/mL, at most 0.02 mg/mL, at most 0.03 mg/mL, at most 0.04 mg/mL, at most 0.05 mg/mL, at most 0.06 mg/mL, at most 0.07 mg/mL, at most 0.08 mg/mL, at most 0.09 mg/mL, at most 0.1 mg/mL, at most 0.2 mg/mL, at most 0.3 mg/mL, at most 0.4 mg/mL, at most 0.5 mg/mL, at most 0.6 mg/mL, at most 0.7 mg/mL, at most 0.8 mg/mL, at most 0.9 mg/mL, at most 1.0 mg/mL, at most 2.0 mg/mL, at most 3.0 mg/mL, at most 4.0 mg/mL, at most 5.0 mg/mL, at most 6.0 mg/mL, at most 7.0 mg/mL, at most 8.0 mg/mL, at most 9.0 mg/mL, at most 10 mg/mL, at most 11 mg/mL, at most 12 mg/mL, at most 13 mg/mL, at most 14 mg/mL, or at most 15 mg/mL.
In still other aspects of this embodiment, ammonium ferric citrate may be used at a concentration of, e.g., about 0.01 mg/mL to about 0.05 mg/mL about 0.01 mg/mL to about 0.1 mg/mL, about 0.01 mg/mL to about 0.5 mg/mL, about 0.05 mg/mL to about 0.1 mg/mL, about 0.05 mg/mL to about 0.5 mg/mL, about 0.05 mg/mL to about 1.0 mg/mL, about 0.05 mg/mL to about 5.0 mg/mL, about 0.1 mg/mL to about 0.5 mg/mL, about 0.1 mg/mL to about 1.0 mg/mL, about 0.1 mg/mL to about 5.0 mg/mL, about 0.1 mg/mL to about 10 mg/mL, about 0.1 mg/mL to about 15 mg/mL, about 0.5 mg/mL to about 1.0 mg/mL, about 0.5 mg/mL to about 5.0 mg/mL, about 0.5 mg/mL to about 10 mg/mL, about 0.5 mg/mL to about 15 mg/mL, about 1.0 mg/mL to about 5.0 mg/mL, about 1.0 mg/mL to about 10 mg/mL, about 1.0 mg/mL to about 15 mg/mL, about 5.0 mg/mL to about 10 mg/mL, about 5.0 mg/mL to about 15 mg/mL, or about 10 mg/mL to about 15 mg/mL.
An enrichment media typically comprises a growth enhancing agent as disclosed herein in the concentration ranges disclosed herein.
In aspects of this embodiment, an enrichment media comprises 4.5 g/L Soy Peptone, 7.2 g/L Sodium Chloride, 1.26 g/L Potassium Phosphate (monobasic), 0.18 g/L Potassium Phosphate (dibasic), 13.58 g/L Magnesium Chloride (anhydrous), 0.036 g/L Malachite Green, and 0.62 g/L Ammonium Ferric Citrate. In aspects of this embodiment, an enrichment media comprises 4.5 g/L Soy Peptone, 7.2 g/L Sodium Chloride, 1.26 g/L Potassium Phosphate (monobasic), 0.18 g/L Potassium Phosphate (dibasic), 29.0 g/L Magnesium Chloride (hexahydrate), 0.036 g/L Malachite Green, and 0.62 g/L Ammonium Ferric Citrate.
Aspect of the present specification disclose, in part, incubation of an aliquiot of pre-enrichment media in an enrichment media. Incubation of an aliquiot of pre-enrichment media is performed under temperature and time parameters that facilitate the growth of the pathogen in the sample, retard the growth of an unwanted organism in the sample, and/or establish conditions that otherwise increase the population of the pathogen in the sample and/or retard the growth of an unwanted organism in the sample. Incubation of enrichment media may be performed under constant rotation, reversal or agitation.
Any temperature may be used during incubation of an aliquiot of pre-enrichment media in an enrichment media, with the proviso that the temperature is useful to practice the methods disclosed herein. In aspects of this embodiment, a temperature used to incubate an aliquiot of pre-enrichment media in an enrichment media may be, e.g., about 15° C., about 16° C., about 17° C., about 18° C., about 19° C., about 20° C., about 21° C., about 22° C., about 23° C., about 24° C., about 25° C., about 26° C., about 27° C., about 28° C., about 29° C., about 30° C., about 31° C., about 32° C., about 33° C., about 34° C., about 35° C., about 36° C., about 37° C., about 38° C., about 39° C., about 40° C., about 41° C., about 42° C., about 43° C., about 44° C., about 45° C., about 46° C., about 47° C., about 48° C., about 49° C., or about 50° C. In other aspects of this embodiment, a temperature used to incubate an aliquiot of pre-enrichment media in an enrichment media may be, e.g., at least 15° C., at least 16° C., at least 17° C., at least 18° C., at least 19° C., at least 20° C., at least 21° C., at least 22° C., at least 23° C., at least 24° C., at least 25° C., at least 26° C., at least 27° C., at least 28° C., at least 29° C., at least 30° C., at least 31° C., at least 32° C., at least 33° C., at least 34° C., at least 35° C., at least 36° C., at least 37° C., at least 38° C., at least 39° C., at least 40° C., at least 41° C., at least 42° C., at least 43° C., at least 44° C., at least 45° C., at least 46° C., at least 47° C., at least 48° C., at least 49° C., or at least 50° C. In yet other aspects of this embodiment, a temperature used to incubate an aliquiot of pre-enrichment media in an enrichment media may be, e.g., at most 15° C., at most 16° C., at most 17° C., at most 18° C., at most 19° C., at most 20° C., at most 21° C., at most 22° C., at most 23° C., at most 24° C., at most 25° C., at most 26° C., at most 27° C., at most 28° C., at most 29° C., at most 30° C., at most 31° C., at most 32° C., at most 33° C., at most 34° C., at most 35° C., at most 36° C., at most 37° C., at most 38° C., at most 39° C., at most 40° C., at most 41° C., at most 42° C., at most 43° C., at most 44° C., at most 45° C., at most 46° C., at most 47° C., at most 48° C., at most 49° C., or at most 50° C. In still other aspects of this embodiment, a temperature used to incubate an aliquiot of pre-enrichment media in an enrichment media may be, e.g., about 15° C. to about 19° C., about 16° C. to about 20° C., about 17° C. to about 21° C., about 18° C. to about 22° C., about 19° C. to about 23° C., about 20° C. to about 24° C., about 21° C. to about 25° C., about 22° C. to about 26° C., about 23° C. to about 27° C., about 24° C. to about 28° C., about 25° C. to about 29° C., about 26° C. to about 30° C., about 27° C. to about 31° C., about 28° C. to about 32° C., about 29° C. to about 33° C., about 30° C. to about 34° C., about 31° C. to about 35° C., about 32° C. to about 36° C., about 33° C. to about 37° C., about 34° C. to about 38° C., about 35° C. to about 39° C., about 36° C. to about 40° C., about 37° C. to about 41° C., about 38° C. to about 42° C., about 39° C. to about 43° C., about 40° C. to about 44° C., about 41° C. to about 45° C., about 42° C. to about 46° C., about 43° C. to about 47° C., about 44° C. to about 48° C., about 45° C. to about 49° C., about 46° C. to about 50° C., about 47° C. to about 51° C., about 48° C. to about 52° C., about 49° C. to about 53° C., or about 50° C. to about 54° C.
Any time may be used during incubation of an aliquiot of pre-enrichment media in an enrichment media, with the proviso that the time is useful to practice the methods disclosed herein. In aspects of this embodiment, a time used to incubate an aliquiot of pre-enrichment media in an enrichment media may be, e.g., about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours. In other aspects of this embodiment, a time used to incubate an aliquiot of pre-enrichment media in an enrichment media may be, e.g., at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, at least 23 hours, or at least 24 hours. In yet other aspects of this embodiment, a time used to incubate an aliquiot of pre-enrichment media in an enrichment media may be, e.g., at most 2 hours, at most 3 hours, at most 4 hours, at most 5 hours, at most 6 hours, at most 7 hours, at most 8 hours, at most 9 hours, at most 10 hours, at most 11 hours, at most 12 hours, at most 13 hours, at most 14 hours, at most 15 hours, at most 16 hours, at most 17 hours, at most 18 hours, at most 19 hours, at most 20 hours, at most 21 hours, at most 22 hours, at most 23 hours, or at most 24 hours. In yet other aspects of this embodiment, a time used to incubate an aliquiot of pre-enrichment media in an enrichment media may be, e.g., about 2 hours to about 4 hours, about 3 hours to about 5 hours, about 4 hours to about 6 hours, about 5 hours to about 7 hours, about 6 hours to about 8 hours, about 7 hours to about 9 hours, about 8 hours to about 10 hours, about 9 hours to about 11 hours, about 10 hours to about 12 hours, about 11 hours to about 13 hours, about 12 hours to about 14 hours, about 13 hours to about 15 hours, about 14 hours to about 16 hours, about 15 hours to about 17 hours, about 16 hours to about 18 hours, about 17 hours to about 19 hours, about 18 hours to about 20 hours, about 19 hours to about 21 hours, about 20 hours to about 22 hours, about 21 hours to about 23 hours, about 22 hours to about 24 hours, about 23 hours to about 25 hours, or about 24 hours to about 26 hours.
In aspects of this embodiment, an aliquiot of pre-enrichment media in an enrichment media may be incubated at a temperature of, e.g., about 15° C., about 16° C., about 17° C., about 18° C., about 19° C., about 20° C., about 21° C., about 22° C., about 23° C., about 24° C., about 25° C., about 26° C., about 27° C., about 28° C., about 29° C., about 30° C., about 31° C., about 32° C., about 33° C., about 34° C., about 35° C., about 36° C., about 37° C., about 38° C., about 39° C., about 40° C., about 41° C., about 42° C., about 43° C., about 44° C., about 45° C., about 46° C., about 47° C., about 48° C., about 49° C., or about 50° C. for a time of, e.g., about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours. Incubation of the enrichment media may be under constant rotation, reversal or agitation.
In other aspects of this embodiment, an aliquiot of pre-enrichment media in an enrichment media may be incubated at a temperature of, e.g., at least 15° C., at least 16° C., at least 17° C., at least 18° C., at least 19° C., at least 20° C., at least 21° C., at least 22° C., at least 23° C., at least 24° C., at least 25° C., at least 26° C., at least 27° C., at least 28° C., at least 29° C., at least 30° C., at least 31° C., at least 32° C., at least 33° C., at least 34° C., at least 35° C., at least 36° C., at least 37° C., at least 38° C., at least 39° C., at least 40° C., at least 41° C., at least 42° C., at least 43° C., at least 44° C., at least 45° C., at least 46° C., at least 47° C., at least 48° C., at least 49° C., or at least 50° C. for a time of, e.g., at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, at least 23 hours, or at least 24 hours. Incubation of the enrichment media may be under constant rotation, reversal or agitation.
In yet other aspects of this embodiment, an aliquiot of pre-enrichment media in an enrichment media may be incubated at a temperature of, e.g., at most 15° C., at most 16° C., at most 17° C., at most 18° C., at most 19° C., at most 20° C., at most 21° C., at most 22° C., at most 23° C., at most 24° C., at most 25° C., at most 26° C., at most 27° C., at most 28° C., at most 29° C., at most 30° C., at most 31° C., at most 32° C., at most 33° C., at most 34° C., at most 35° C., at most 36° C., at most 37° C., at most 38° C., at most 39° C., at most 40° C., at most 41° C., at most 42° C., at most 43° C., at most 44° C., at most 45° C., at most 46° C., at most 47° C., at most 48° C., at most 49° C., or at most 50° C. for a time of, e.g., at most 2 hours, at most 3 hours, at most 4 hours, at most 5 hours, at most 6 hours, at most 7 hours, at most 8 hours, at most 9 hours, at most 10 hours, at most 11 hours, at most 12 hours, at most 13 hours, at most 14 hours, at most 15 hours, at most 16 hours, at most 17 hours, at most 18 hours, at most 19 hours, at most 20 hours, at most 21 hours, at most 22 hours, at most 23 hours, or at most 24 hours. Incubation of the enrichment media may be under constant rotation, reversal or agitation.
In still other aspects of this embodiment, an aliquiot of pre-enrichment media in an enrichment media may be incubated at a temperature of, e.g., about 15° C. to about 19° C., about 16° C. to about 20° C., about 17° C. to about 21° C., about 18° C. to about 22° C., about 19° C. to about 23° C., about 20° C. to about 24° C., about 21° C. to about 25° C., about 22° C. to about 26° C., about 23° C. to about 27° C., about 24° C. to about 28° C., about 25° C. to about 29° C., about 26° C. to about 30° C., about 27° C. to about 31° C., about 28° C. to about 32° C., about 29° C. to about 33° C., about 30° C. to about 34° C., about 31° C. to about 35° C., about 32° C. to about 36° C., about 33° C. to about 37° C., about 34° C. to about 38° C., about 35° C. to about 39° C., about 36° C. to about 40° C., about 37° C. to about 41° C., about 38° C. to about 42° C., about 39° C. to about 43° C., about 40° C. to about 44° C., about 41° C. to about 45° C., about 42° C. to about 46° C., about 43° C. to about 47° C., about 44° C. to about 48° C., about 45° C. to about 49° C., about 46° C. to about 50° C., about 47° C. to about 51° C., about 48° C. to about 52° C., about 49° C. to about 53° C., or about 50° C. to about 54° C. for a time of, e.g., about 2 hours to about 4 hours, about 3 hours to about 5 hours, about 4 hours to about 6 hours, about 5 hours to about 7 hours, about 6 hours to about 8 hours, about 7 hours to about 9 hours, about 8 hours to about 10 hours, about 9 hours to about 11 hours, about 10 hours to about 12 hours, about 11 hours to about 13 hours, about 12 hours to about 14 hours, about 13 hours to about 15 hours, about 14 hours to about 16 hours, about 15 hours to about 17 hours, about 16 hours to about 18 hours, about 17 hours to about 19 hours, about 18 hours to about 20 hours, about 19 hours to about 21 hours, about 20 hours to about 22 hours, about 21 hours to about 23 hours, about 22 hours to about 24 hours, about 23 hours to about 25 hours, or about 24 hours to about 26 hours. Incubation of the enrichment media may be under constant rotation, reversal or agitation.
Aspect of the present specification disclose, in part, a purification step. After being enriched, a pathogen may be subsequently purified from an enrichment media. Purification of a pathogen includes capture of the pathogen to a more concentrated form and/or removal contaminating microorganism, impurities, and debris. As such a purification step disclosed herein increases detection of a pathogen by increasing the concentration of pathogen and/or decreasing contaminants, thereby increasing the level of detectable signal measured in a subsequent detection step. Common purification procedures used to capture a pathogen and/or remove contaminating microorganism, impurities, and debris include affinity chromatography, ion-exchange chromatography, size exclusion chromatography, hydrophobic-interaction chromatography, ceramic hydroxyapatite chromatography, reverse-phase HPLC, gel filtration, precipitation, immuno-precipitation, diafiltration, chromatofocusing.
In one embodiment, a pathogen is purified from an enrichment media using an immuno-precipitation using antibodies or aptamers for a pathogen of interest. Immunoprecipitation is the technique of precipitating an antigen out of solution using an antibody or aptamer that specifically binds to that antigen. Immunoprecipitation requires that the antibody be coupled to a solid substrate at some point in the procedure. This is typically done using standard coupling procedures known in the art. Examples of solid substrates include agarose particles and magnetic particles. In aspects of this embodiment, the immuno-precipitation method employs antibodies or aptamers for a pathogen linked to magnetic particles.
Any concentration of magnetic particles linked with an antibody or apatmer may be used during its incubation during a purification step, with the proviso that the concentration is useful to practice the methods disclosed herein. In aspects of this embodiment, a concentration of magnetic particles linked with an antibody or apatmer used during a purification step may be, e.g., about 1×104 immunmagnetic particles/mL of enrichment media comprising the pathogen of interest, about 1×105 immunmagnetic particles/mL of enrichment media comprising the pathogen of interest, about 1×106 immunmagnetic particles/mL of enrichment media comprising the pathogen of interest, about 1×107 immunmagnetic particles/mL of enrichment media comprising the pathogen of interest, or about 1×108 immunmagnetic particles/mL of enrichment media comprising the pathogen of interest. In other aspects of this embodiment, a concentration of magnetic particles linked with an antibody or apatmer used during a purification step may be, e.g., at least 1×104 immunmagnetic particles/mL of enrichment media comprising the pathogen of interest, at least 1×105 immunmagnetic particles/mL of enrichment media comprising the pathogen of interest, at least 1×106 immunmagnetic particles/mL of enrichment media comprising the pathogen of interest, at least 1×107 immunmagnetic particles/mL of enrichment media comprising the pathogen of interest, or at least 1×108 immunmagnetic particles/mL of enrichment media comprising the pathogen of interest. In yet other aspects of this embodiment, a concentration of magnetic particles linked with an antibody or apatmer used during a purification step may be, e.g., at most 1×104 immunmagnetic particles/mL of enrichment media comprising the pathogen of interest, at most 1×105 immunmagnetic particles/mL of enrichment media comprising the pathogen of interest, at most 1×106 immunmagnetic particles/mL of enrichment media comprising the pathogen of interest, at most 1×107 immunmagnetic particles/mL of enrichment media comprising the pathogen of interest, or at most 1×108 immunmagnetic particles/mL of enrichment media comprising the pathogen of interest.
In still other aspects of this embodiment, a concentration of magnetic particles linked with an antibody or apatmer used during a purification step may be, e.g., about 1×104 to about 1×105 immunmagnetic particles/mL of enrichment media comprising the pathogen of interest, about 1×104 to about 1×106 immunmagnetic particles/mL of enrichment media comprising the pathogen of interest, about 1×104 to about 1×107 immunmagnetic particles/mL of enrichment media comprising the pathogen of interest, about 1×104 to about 1×108 immunmagnetic particles/mL of enrichment media comprising the pathogen of interest, about 1×105 to about 1×106 immunmagnetic particles/mL of enrichment media comprising the pathogen of interest, about 1×105 to about 1×107 immunmagnetic particles/mL of enrichment media comprising the pathogen of interest, about 1×105 to about 1×108 immunmagnetic particles/mL of enrichment media comprising the pathogen of interest, about 1×106 to about 1×107 immunmagnetic particles/mL of enrichment media comprising the pathogen of interest, about 1×106 to about 1×108 immunmagnetic particles/mL of enrichment media comprising the pathogen of interest, or about 1×107 to about 1×108 immunmagnetic particles/mL of enrichment media comprising the pathogen of interest.
Aspect of the present specification disclose, in part, incubation of immunmagnetic particles with enrichment media comprising the pathogen of interest. Incubation of immunmagnetic particles with enrichment media comprising the pathogen of interest is performed under temperature and time parameters that facilitate binding of the pathogen to the magnetic particles linked with an antibody or apatmer for the pathogen of interest. Incubation of immunmagnetic particles with enrichment media comprising the pathogen of interest may be performed under agitation/rotation.
Any temperature may be used during incubation of immunmagnetic particles with enrichment media comprising the pathogen of interest, with the proviso that the temperature is useful to practice the methods disclosed herein. In aspects of this embodiment, a temperature used to incubate immunmagnetic particles with enrichment media comprising the pathogen of interest may be, e.g., about 15° C., about 16° C., about 17° C., about 18° C., about 19° C., about 20° C., about 21° C., about 22° C., about 23° C., about 24° C., about 25° C., about 26° C., about 27° C., about 28° C., about 29° C., about 30° C., about 31° C., about 32° C., about 33° C., about 34° C., about 35° C., about 36° C., about 37° C., about 38° C., about 39° C., about 40° C., about 41° C., about 42° C., about 43° C., about 44° C., about 45° C., about 46° C., about 47° C., about 48° C., about 49° C., or about 50° C. In other aspects of this embodiment, a temperature used to incubate immunmagnetic particles with enrichment media comprising the pathogen of interest may be, e.g., at least 15° C., at least 16° C., at least 17° C., at least 18° C., at least 19° C., at least 20° C., at least 21° C., at least 22° C., at least 23° C., at least 24° C., at least 25° C., at least 26° C., at least 27° C., at least 28° C., at least 29° C., at least 30° C., at least 31° C., at least 32° C., at least 33° C., at least 34° C., at least 35° C., at least 36° C., at least 37° C., at least 38° C., at least 39° C., at least 40° C., at least 41° C., at least 42° C., at least 43° C., at least 44° C., at least 45° C., at least 46° C., at least 47° C., at least 48° C., at least 49° C., or at least 50° C. In yet other aspects of this embodiment, a temperature used to incubate immunmagnetic particles with enrichment media comprising the pathogen of interest may be, e.g., at most 15° C., at most 16° C., at most 17° C., at most 18° C., at most 19° C., at most 20° C., at most 21° C., at most 22° C., at most 23° C., at most 24° C., at most 25° C., at most 26° C., at most 27° C., at most 28° C., at most 29° C., at most 30° C., at most 31° C., at most 32° C., at most 33° C., at most 34° C., at most 35° C., at most 36° C., at most 37° C., at most 38° C., at most 39° C., at most 40° C., at most 41° C., at most 42° C., at most 43° C., at most 44° C., at most 45° C., at most 46° C., at most 47° C., at most 48° C., at most 49° C., or at most 50° C. In still other aspects of this embodiment, a temperature used to incubate immunmagnetic particles with enrichment media comprising the pathogen of interest may be, e.g., about 15° C. to about 19° C., about 16° C. to about 20° C., about 17° C. to about 21° C., about 18° C. to about 22° C., about 19° C. to about 23° C., about 20° C. to about 24° C., about 21° C. to about 25° C., about 22° C. to about 26° C., about 23° C. to about 27° C., about 24° C. to about 28° C., about 25° C. to about 29° C., about 26° C. to about 30° C., about 27° C. to about 31° C., about 28° C. to about 32° C., about 29° C. to about 33° C., about 30° C. to about 34° C., about 31° C. to about 35° C., about 32° C. to about 36° C., about 33° C. to about 37° C., about 34° C. to about 38° C., about 35° C. to about 39° C., about 36° C. to about 40° C., about 37° C. to about 41° C., about 38° C. to about 42° C., about 39° C. to about 43° C., about 40° C. to about 44° C., about 41° C. to about 45° C., about 42° C. to about 46° C., about 43° C. to about 47° C., about 44° C. to about 48° C., about 45° C. to about 49° C., about 46° C. to about 50° C., about 47° C. to about 51° C., about 48° C. to about 52° C., about 49° C. to about 53° C., or about 50° C. to about 54° C.
Any time may be used during incubation of immunmagnetic particles with enrichment media comprising the pathogen of interest, with the proviso that the time is useful to practice the methods disclosed herein. In aspects of this embodiment, a time used to incubate immunmagnetic particles with enrichment media comprising the pathogen of interest may be, e.g., about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 70 minutes, about 80 minutes, about 90 minutes, about 100 minutes, about 110 minutes, about 120 minutes, about 130 minutes, about 140 minutes, or about 150 minutes. In other aspects of this embodiment, a time used to incubate immunmagnetic particles with enrichment media comprising the pathogen of interest may be, e.g., at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, at least 60 minutes, at least 70 minutes, at least 80 minutes, at least 90 minutes, at least 100 minutes, at least 110 minutes, at least 120 minutes, at least 130 minutes, at least 140 minutes, or at least 150 minutes. In yet other aspects of this embodiment, a time used to incubate immunmagnetic particles with enrichment media comprising the pathogen of interest may be, e.g., at most 5 minutes, at most 10 minutes, at most 15 minutes, at most 20 minutes, at most 30 minutes, at most 40 minutes, at most 50 minutes, at most 60 minutes, at most 70 minutes, at most 80 minutes, at most 90 minutes, at most 100 minutes, at most 110 minutes, at most 120 minutes, at most 130 minutes, at most 140 minutes, or at most 150 minutes.
In yet other aspects of this embodiment, a time used to incubate immunmagnetic particles with enrichment media comprising the pathogen of interest may be, e.g., about 5 minutes to about 20 minutes, about 5 minutes to about 30 minutes, about 5 minutes to about 40 minutes, about 5 minutes to about 50 minutes, about 5 minutes to about 60 minutes, about 5 minutes to about 70 minutes, about 5 minutes to about 80 minutes, about 5 minutes to about 90 minutes, about 5 minutes to about 100 minutes, about 5 minutes to about 110 minutes, about 5 minutes to about 120 minutes, about 5 minutes to about 130 minutes, about 5 minutes to about 140 minutes, about 5 minutes to about 150 minutes, about 10 minutes to about 20 minutes, about 10 minutes to about 30 minutes, about 10 minutes to about 40 minutes, about 10 minutes to about 50 minutes, about 10 minutes to about 60 minutes, about 10 minutes to about 70 minutes, about 10 minutes to about 80 minutes, about 10 minutes to about 90 minutes, about 10 minutes to about 100 minutes, about 10 minutes to about 110 minutes, about 10 minutes to about 120 minutes, about 10 minutes to about 130 minutes, about 10 minutes to about 140 minutes, about 10 minutes to about 150 minutes, about 20 minutes to about 30 minutes, about 20 minutes to about 40 minutes, about 20 minutes to about 50 minutes, about 20 minutes to about 60 minutes, about 20 minutes to about 70 minutes, about 20 minutes to about 80 minutes, about 20 minutes to about 90 minutes, about 20 minutes to about 100 minutes, about 20 minutes to about 110 minutes, about 20 minutes to about 120 minutes, about 20 minutes to about 130 minutes, about 20 minutes to about 140 minutes, about 20 minutes to about 150 minutes, about 30 minutes to about 40 minutes, about 30 minutes to about 50 minutes, about 30 minutes to about 60 minutes, about 30 minutes to about 70 minutes, about 30 minutes to about 80 minutes, about 30 minutes to about 90 minutes, about 30 minutes to about 100 minutes, about 30 minutes to about 110 minutes, about 30 minutes to about 120 minutes, about 30 minutes to about 130 minutes, about 30 minutes to about 140 minutes, about 30 minutes to about 150 minutes, about 60 minutes to about 70 minutes, about 60 minutes to about 80 minutes, about 60 minutes to about 90 minutes, about 60 minutes to about 100 minutes, about 60 minutes to about 110 minutes, about 60 minutes to about 120 minutes, about 60 minutes to about 130 minutes, about 60 minutes to about 140 minutes, about 60 minutes to about 150 minutes, about 90 minutes to about 100 minutes, about 90 minutes to about 110 minutes, about 90 minutes to about 120 minutes, about 90 minutes to about 130 minutes, about 90 minutes to about 140 minutes, about 90 minutes to about 150 minutes, about 120 minutes to about 130 minutes, about 120 minutes to about 140 minutes, or about 120 minutes to about 150 minutes.
In aspects of this embodiment, immunmagnetic particles may be incubated with enrichment media comprising the pathogen of interest at a temperature of, e.g., about 15° C., about 16° C., about 17° C., about 18° C., about 19° C., about 20° C., about 21° C., about 22° C., about 23° C., about 24° C., about 25° C., about 26° C., about 27° C., about 28° C., about 29° C., about 30° C., about 31° C., about 32° C., about 33° C., about 34° C., about 35° C., about 36° C., about 37° C., about 38° C., about 39° C., about 40° C., about 41° C., about 42° C., about 43° C., about 44° C., about 45° C., about 46° C., about 47° C., about 48° C., about 49° C., or about 50° C. for a time of, e.g., about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 70 minutes, about 80 minutes, about 90 minutes, about 100 minutes, about 110 minutes, about 120 minutes, about 130 minutes, about 140 minutes, or about 150 minutes.
In other aspects of this embodiment, immunmagnetic particles may be incubated with enrichment media comprising the pathogen of interest at a temperature of, e.g., at least 15° C., at least 16° C., at least 17° C., at least 18° C., at least 19° C., at least 20° C., at least 21° C., at least 22° C., at least 23° C., at least 24° C., at least 25° C., at least 26° C., at least 27° C., at least 28° C., at least 29° C., at least 30° C., at least 31° C., at least 32° C., at least 33° C., at least 34° C., at least 35° C., at least 36° C., at least 37° C., at least 38° C., at least 39° C., at least 40° C., at least 41° C., at least 42° C., at least 43° C., at least 44° C., at least 45° C., at least 46° C., at least 47° C., at least 48° C., at least 49° C., or at least 50° C. for a time of, e.g., at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, at least 60 minutes, at least 70 minutes, at least 80 minutes, at least 90 minutes, at least 100 minutes, at least 110 minutes, at least 120 minutes, at least 130 minutes, at least 140 minutes, or at least 150 minutes.
In yet other aspects of this embodiment, immunmagnetic particles may be incubated with enrichment media comprising the pathogen of interest at a temperature of, e.g., at most 15° C., at most 16° C., at most 17° C., at most 18° C., at most 19° C., at most 20° C., at most 21° C., at most 22° C., at most 23° C., at most 24° C., at most 25° C., at most 26° C., at most 27° C., at most 28° C., at most 29° C., at most 30° C., at most 31° C., at most 32° C., at most 33° C., at most 34° C., at most 35° C., at most 36° C., at most 37° C., at most 38° C., at most 39° C., at most 40° C., at most 41° C., at most 42° C., at most 43° C., at most 44° C., at most 45° C., at most 46° C., at most 47° C., at most 48° C., at most 49° C., or at most 50° C. for a time of, e.g., at most 5 minutes, at most 10 minutes, at most 15 minutes, at most 20 minutes, at most 30 minutes, at most 40 minutes, at most 50 minutes, at most 60 minutes, at most 70 minutes, at most 80 minutes, at most 90 minutes, at most 100 minutes, at most 110 minutes, at most 120 minutes, at most 130 minutes, at most 140 minutes, or at most 150 minutes.
In still other aspects of this embodiment, immunmagnetic particles may be incubated with enrichment media comprising the pathogen of interest at a temperature of, e.g., about 15° C. to about 19° C., about 16° C. to about 20° C., about 17° C. to about 21° C., about 18° C. to about 22° C., about 19° C. to about 23° C., about 20° C. to about 24° C., about 21° C. to about 25° C., about 22° C. to about 26° C., about 23° C. to about 27° C., about 24° C. to about 28° C., about 25° C. to about 29° C., about 26° C. to about 30° C., about 27° C. to about 31° C., about 28° C. to about 32° C., about 29° C. to about 33° C., about 30° C. to about 34° C., about 31° C. to about 35° C., about 32° C. to about 36° C., about 33° C. to about 37° C., about 34° C. to about 38° C., about 35° C. to about 39° C., about 36° C. to about 40° C., about 37° C. to about 41° C., about 38° C. to about 42° C., about 39° C. to about 43° C., about 40° C. to about 44° C., about 41° C. to about 45° C., about 42° C. to about 46° C., about 43° C. to about 47° C., about 44° C. to about 48° C., about 45° C. to about 49° C., about 46° C. to about 50° C., about 47° C. to about 51° C., about 48° C. to about 52° C., about 49° C. to about 53° C., or about 50° C. to about 54° C. for a time of, e.g., about 5 minutes to about 20 minutes, about 5 minutes to about 30 minutes, about 5 minutes to about 40 minutes, about 5 minutes to about 50 minutes, about 5 minutes to about 60 minutes, about 5 minutes to about 70 minutes, about 5 minutes to about 80 minutes, about 5 minutes to about 90 minutes, about 5 minutes to about 100 minutes, about 5 minutes to about 110 minutes, about 5 minutes to about 120 minutes, about 5 minutes to about 130 minutes, about 5 minutes to about 140 minutes, about 5 minutes to about 150 minutes, about 10 minutes to about 20 minutes, about 10 minutes to about 30 minutes, about 10 minutes to about 40 minutes, about 10 minutes to about 50 minutes, about 10 minutes to about 60 minutes, about 10 minutes to about 70 minutes, about 10 minutes to about 80 minutes, about 10 minutes to about 90 minutes, about 10 minutes to about 100 minutes, about 10 minutes to about 110 minutes, about 10 minutes to about 120 minutes, about 10 minutes to about 130 minutes, about 10 minutes to about 140 minutes, about 10 minutes to about 150 minutes, about 20 minutes to about 30 minutes, about 20 minutes to about 40 minutes, about 20 minutes to about 50 minutes, about 20 minutes to about 60 minutes, about 20 minutes to about 70 minutes, about 20 minutes to about 80 minutes, about 20 minutes to about 90 minutes, about 20 minutes to about 100 minutes, about 20 minutes to about 110 minutes, about 20 minutes to about 120 minutes, about 20 minutes to about 130 minutes, about 20 minutes to about 140 minutes, about 20 minutes to about 150 minutes, about 30 minutes to about 40 minutes, about 30 minutes to about 50 minutes, about 30 minutes to about 60 minutes, about 30 minutes to about 70 minutes, about 30 minutes to about 80 minutes, about 30 minutes to about 90 minutes, about 30 minutes to about 100 minutes, about 30 minutes to about 110 minutes, about 30 minutes to about 120 minutes, about 30 minutes to about 130 minutes, about 30 minutes to about 140 minutes, about 30 minutes to about 150 minutes, about 60 minutes to about 70 minutes, about 60 minutes to about 80 minutes, about 60 minutes to about 90 minutes, about 60 minutes to about 100 minutes, about 60 minutes to about 110 minutes, about 60 minutes to about 120 minutes, about 60 minutes to about 130 minutes, about 60 minutes to about 140 minutes, about 60 minutes to about 150 minutes, about 90 minutes to about 100 minutes, about 90 minutes to about 110 minutes, about 90 minutes to about 120 minutes, about 90 minutes to about 130 minutes, about 90 minutes to about 140 minutes, about 90 minutes to about 150 minutes, about 120 minutes to about 130 minutes, about 120 minutes to about 140 minutes, or about 120 minutes to about 150 minutes.
After completion of incubation, the pathogen-bound immunoparticles may be isolated from the enrichment media. In aspects of this embodiment, immunoparticles may be isolated from the enrichment media using a magnetic separator which concentrates the immunoparticles in a specific location allowing for the enrichment media to be removed. Alternatively, immunoparticles may be isolated from the enrichment media by centrifuged to concentrate the immunoparticles, thereby allowing for the enrichment media to be removed. After removal of the enrichment media, the isolated pathogen-bound immunoparticles may be washed one or more times using a buffered solution. Subsequent isolation of the immunoparticles may be accomplished using a magnetic separator or centrifugation and removal of the buffered solution.
However, a purification step disclosed herein is optional. Thus, in one embodiment, a method of detecting a pathogen disclosed herein does not comprise a purification of a pathogen from a pre-enrichment media, an enrichment media, or both a pre-enrichment media and an enrichment media.
Aspect of the present specification disclose, in part, a detection step. After being enriched, presence or absence of a pathogen of interest may be determined by qualitatively or quantitatively measuring the amount of pathogen contained within the enrichment media. In one embodiment, detection presence or absence of a pathogen of interest occurs after completion of an enrichment step disclosed herein and without the need of a purification step disclosed herein. In one embodiment, detection presence or absence of a pathogen of interest occurs after completion of a purification step disclosed herein. However, an enrichment media comprising a pathogen of interest may be processed to remove debris and other contaminants without any pathogen purification. In one embodiment, an enrichment media comprising a pathogen of interest is centrifuged to remove debris. In another embodiment no such centrifugation step is required. In addition, use of a secondary antibody to boast a detection signal is optional. Thus, in one embodiment, a method of detecting a pathogen does not comprise use of a secondary antibody to boast a detection signal.
In aspects of this embodiment, a method disclosed herein may qualitatively or quantitatively detect pathogen having a concentration in a sample disclosed herein of, e.g., about 1×10−−5 cfu/mL, about 1×10−4 cfu/mL, about 1×10−3 cfu/mL, about 1×10−2 cfu/mL, about 1×10−1 cfu/mL, about 1×100 cfu/mL, about 1×101 cfu/mL, about 1×102 cfu/mL, about 1×103 cfu/mL, about 1×104 cfu/mL, about 1×105 cfu/mL, about 1×106 cfu/mL, about 1×107 cfu/mL, about 1×108 cfu/mL, about 1×109 cfu/mL, or about 1×1010 cfu/mL. In other aspects of this embodiment, a method disclosed herein may qualitatively or quantitatively detect pathogen having a concentration in a sample disclosed herein of, e.g., at least 1×10−5 cfu/mL, at least 1×10−4 cfu/mL, at least 1×10−3 cfu/mL, at least 1×10−2 cfu/mL, at least 1×10−1 cfu/mL, at least 1×100 cfu/mL, at least 1×101 cfu/mL, at least 1×102 cfu/mL, at least 1×103 cfu/mL, at least 1×104 cfu/mL, at least 1×105 cfu/mL, at least 1×106 cfu/mL, at least 1×107 cfu/mL, at least 1×106 cfu/mL, at least 1×109 cfu/mL, or at least 1×1010 cfu/mL. In yet other aspects of this embodiment, a method disclosed herein may qualitatively or quantitatively detect pathogen having a concentration in a sample disclosed herein of, e.g., at most 1×10−5 cfu/mL, at most 1×10−4 cfu/mL, at most 1×10−3 cfu/mL, at most 1×10−2 cfu/mL, at most 1×10−1 cfu/mL, at most 1×100 cfu/mL, at most 1×101 cfu/mL, at most 1×102 cfu/mL, at most 1×103 cfu/mL, at most 1×104 cfu/mL, at most 1×105 cfu/mL, at most 1×106 cfu/mL, at most 1×107 cfu/mL, at most 1×108 cfu/mL, at most 1×109 cfu/mL, or at most 1×1010 cfu/mL.
In still other aspects of this embodiment, a method disclosed herein may qualitatively or quantitatively detect pathogen having a concentration in a sample disclosed herein of, e.g., about 1×10−5 cfu/mL to about 1×10−4 cfu/mL, about 1×10−5 cfu/mL to about 1×10−3 cfu/mL, about 1×10−5 cfu/mL to about 1×10−2 cfu/mL, about 1×10−5 cfu/mL to about 1×10−1 cfu/mL, about 1×10−5 cfu/mL to about 1×100 cfu/mL, about 1×10−5 cfu/mL to about 1×101 cfu/mL, about 1×10−5 cfu/mL to about 1×102 cfu/mL, about 1×10−5 cfu/mL to about 1×103 cfu/mL, about 1×10−5 cfu/mL to about 1×104 cfu/mL, about 1×10−5 cfu/mL to about 1×105 cfu/mL, about 1×10−5 cfu/mL to about 1×106 cfu/mL, about 1×10−5 cfu/mL to about 1×107 cfu/mL, about 1×10−5 cfu/mL to about 1×106 cfu/mL, about 1×10−5 cfu/mL to about 1×109 cfu/mL, about 1×10−5 cfu/mL to about 1×1010 cfu/mL, about 1×10−4 cfu/mL to about 1×10−3 cfu/mL, about 1×10−4 cfu/mL to about 1×10−2 cfu/mL, about 1×10−4 cfu/mL to about 1×10−1 cfu/mL, about 1×10−4 cfu/mL to about 1×100 cfu/mL, about 1×10−4 cfu/mL to about 1×101 cfu/mL, about 1×10−4 cfu/mL to about 1×102 cfu/mL, about 1×10−4 cfu/mL to about 1×103 cfu/mL, about 1×10−4 cfu/mL to about 1×104 cfu/mL, about 1×10−4 cfu/mL to about 1×105 cfu/mL, about 1×10−4 cfu/mL to about 1×106 cfu/mL, about 1×10−4 cfu/mL to about 1×107 cfu/mL, about 1×10−4 cfu/mL to about 1×106 cfu/mL, about 1×10−4 cfu/mL to about 1×109 cfu/mL, about 1×10−4 cfu/mL to about 1×1010 cfu/mL, about 1×10−3 cfu/mL to about 1×10−2 cfu/mL, about 1×10−3 cfu/mL to about 1×10−1 cfu/mL, about 1×10−3 cfu/mL to about 1×100 cfu/mL, about 1×10−3 cfu/mL to about 1×101 cfu/mL, about 1×10−3 cfu/mL to about 1×102 cfu/mL, about 1×10−3 cfu/mL to about 1×103 cfu/mL, about 1×10−3 cfu/mL to about 1×104 cfu/mL, about 1×10−3 cfu/mL to about 1×105 cfu/mL, about 1×10−3 cfu/mL to about 1×106 cfu/mL, about 1×10−3 cfu/mL to about 1×107 cfu/mL, about 1×10−3 cfu/mL to about 1×106 cfu/mL, about 1×10−3 cfu/mL to about 1×109 cfu/mL, about 1×10−3 cfu/mL to about 1×1010 cfu/mL, about 1×10−2 cfu/mL to about 1×10−1 cfu/mL, about 1×10−2 cfu/mL to about 1×102 cfu/mL, about 1×10−2 cfu/mL to about 1×101 cfu/mL, about 1×10−2 cfu/mL to about 1×102 cfu/mL, about 1×10−2 cfu/mL to about 1×103 cfu/mL, about 1×10−2 cfu/mL to about 1×104 cfu/mL, about 1×10−2 cfu/mL to about 1×105 cfu/mL, about 1×10−2 cfu/mL to about 1×106 cfu/mL, about 1×10−2 cfu/mL to about 1×107 cfu/mL, about 1×10−2 cfu/mL to about 1×108 cfu/mL, about 1×10−2 cfu/mL to about 1×109 cfu/mL, about 1×10−2 cfu/mL to about 1×1010 cfu/mL, about 1×10−1 cfu/mL to about 1×100 cfu/mL, about 1×10−1 cfu/mL to about 1×101 cfu/mL, about 1×10−1 cfu/mL to about 1×102 cfu/mL, about 1×10−1 cfu/mL to about 1×103 cfu/mL, about 1×10−1 cfu/mL to about 1×104 cfu/mL, about 1×10−1 cfu/mL to about 1×105 cfu/mL, about 1×10−1 cfu/mL to about 1×106 cfu/mL, about 1×10−1 cfu/mL to about 1×107 cfu/mL, about 1×10−1 cfu/mL to about 1×108 cfu/mL, about 1×10−1 cfu/mL to about 1×109 cfu/mL, about 1×10−1 cfu/mL to about 1×1010 cfu/mL, about 1×100 cfu/mL to about 1×101 cfu/mL, about 1×100 cfu/mL to about 1×102 cfu/mL, about 1×100 cfu/mL to about 1×103 cfu/mL, about 1×100 cfu/mL to about 1×104 cfu/mL, about 1×100 cfu/mL to about 1×105 cfu/mL, about 1×100 cfu/mL to about 1×106 cfu/mL, about 1×100 cfu/mL to about 1×107 cfu/mL, about 1×100 cfu/mL to about 1×108 cfu/mL, about 1×100 cfu/mL to about 1×109 cfu/mL, about 1×100 cfu/mL to about 1×1010 cfu/mL, about 1×101 cfu/mL to about 1×102 cfu/mL, about 1×101 cfu/mL to about 1×103 cfu/mL, about 1×101 cfu/mL to about 1×104 cfu/mL, about 1×101 cfu/mL to about 1×105 cfu/mL, about 1×101 cfu/mL to about 1×106 cfu/mL, about 1×101 cfu/mL to about 1×107 cfu/mL, about 1×101 cfu/mL to about 1×108 cfu/mL, about 1×101 cfu/mL to about 1×109 cfu/mL, about 1×101 cfu/mL to about 1×1010 cfu/mL, about 1×102 cfu/mL to about 1×103 cfu/mL, about 1×102 cfu/mL to about 1×104 cfu/mL, about 1×102 cfu/mL to about 1×105 cfu/mL, about 1×102 cfu/mL to about 1×106 cfu/mL, about 1×102 cfu/mL to about 1×107 cfu/mL, about 1×102 cfu/mL to about 1×108 cfu/mL, about 1×102 cfu/mL to about 1×109 cfu/mL, about 1×102 cfu/mL to about 1×1010 cfu/mL, about 1×103 cfu/mL to about 1×104 cfu/mL, about 1×103 cfu/mL to about 1×105 cfu/mL, about 1×103 cfu/mL to about 1×106 cfu/mL, about 1×103 cfu/mL to about 1×107 cfu/mL, about 1×103 cfu/mL to about 1×108 cfu/mL, about 1×103 cfu/mL to about 1×109 cfu/mL, about 1×103 cfu/mL to about 1×1010 cfu/mL, about 1×104 cfu/mL to about 1×105 cfu/mL, about 1×104 cfu/mL to about 1×106 cfu/mL, about 1×104 cfu/mL to about 1×107 cfu/mL, about 1×104 cfu/mL to about 1×108 cfu/mL, about 1×104 cfu/mL to about 1×109 cfu/mL, or about 1×104 cfu/mL to about 1×1010 cfu/mL.
In aspects of this embodiment, a method disclosed herein may qualitatively or quantitatively detect pathogen having a concentration after the pre-enrichment step of, e.g., about 1×10−5 cfu/mL, about 1×10−4 cfu/mL, about 1×10−3 cfu/mL, about 1×10−2 cfu/mL, about 1×10−1 cfu/mL, about 1×100 cfu/mL, about 1×101 cfu/mL, about 1×102 cfu/mL, about 1×103 cfu/mL, about 1×104 cfu/mL, about 1×105 cfu/mL, about 1×106 cfu/mL, about 1×107 cfu/mL, about 1×108 cfu/mL, about 1×109 cfu/mL, or about 1×1010 cfu/mL. In other aspects of this embodiment, a method disclosed herein may qualitatively or quantitatively detect pathogen having a concentration after the pre-enrichment step of, e.g., at least 1×10−5 cfu/mL, at least 1×10−4 cfu/mL, at least 1×10−3 cfu/mL, at least 1×10−2 cfu/mL, at least 1×10−1 cfu/mL, at least 1×100 cfu/mL, at least 1×101 cfu/mL, at least 1×102 cfu/mL, at least 1×103 cfu/mL, at least 1×104 cfu/mL, at least 1×105 cfu/mL, at least 1×106 cfu/mL, at least 1×107 cfu/mL, at least 1×108 cfu/mL, at least 1×109 cfu/mL, or at least 1×1010 cfu/mL. In yet other aspects of this embodiment, a method disclosed herein may qualitatively or quantitatively detect pathogen having a concentration after the pre-enrichment step of, e.g., at most 1×10−5 cfu/mL, at most 1×10−4 cfu/mL, at most 1×10−3 cfu/mL, at most 1×10−2 cfu/mL, at most 1×10−1 cfu/mL, at most 1×100 cfu/mL, at most 1×101 cfu/mL, at most 1×102 cfu/mL, at most 1×103 cfu/mL, at most 1×104 cfu/mL, at most 1×105 cfu/mL, at most 1×106 cfu/mL, at most 1×107 cfu/mL, at most 1×108 cfu/mL, at most 1×109 cfu/mL, or at most 1×1010 cfu/mL.
In still other aspects of this embodiment, a method disclosed herein may qualitatively or quantitatively detect pathogen having a concentration after the pre-enrichment step of f, e.g., about 1×10−5 cfu/mL to about 1×10−4 cfu/mL, about 1×10−5 cfu/mL to about 1×10−3 cfu/mL, about 1×10−5 cfu/mL to about 1×10−2 cfu/mL, about 1×10−5 cfu/mL to about 1×10−1 cfu/mL, about 1×10−5 cfu/mL to about 1×100 cfu/mL, about 1×10−5 cfu/mL to about 1×101 cfu/mL, about 1×10−5 cfu/mL to about 1×102 cfu/mL, about 1×10−5 cfu/mL to about 1×103 cfu/mL, about 1×10−5 cfu/mL to about 1×104 cfu/mL, about 1×10−5 cfu/mL to about 1×105 cfu/mL, about 1×10−5 cfu/mL to about 1×106 cfu/mL, about 1×10−5 cfu/mL to about 1×107 cfu/mL, about 1×10−5 cfu/mL to about 1×108 cfu/mL, about 1×10−5 cfu/mL to about 1×109 cfu/mL, about 1×10−5 cfu/mL to about 1×1010 cfu/mL, about 1×10−4 cfu/mL to about 1×10−3 cfu/mL, about 1×10−4 cfu/mL to about 1×10−2 cfu/mL, about 1×10−4 cfu/mL to about 1×10−1 cfu/mL, about 1×10−4 cfu/mL to about 1×100 cfu/mL, about 1×10−4 cfu/mL to about 1×101 cfu/mL, about 1×10−4 cfu/mL to about 1×102 cfu/mL, about 1×10−4 cfu/mL to about 1×103 cfu/mL, about 1×10−4 cfu/mL to about 1×104 cfu/mL, about 1×10−4 cfu/mL to about 1×105 cfu/mL, about 1×10−4 cfu/mL to about 1×106 cfu/mL, about 1×10−4 cfu/mL to about 1×107 cfu/mL, about 1×10−4 cfu/mL to about 1×108 cfu/mL, about 1×10−4 cfu/mL to about 1×109 cfu/mL, about 1×10−4 cfu/mL to about 1×1010 cfu/mL, about 1×10−3 cfu/mL to about 1×10−2 cfu/mL, about 1×10−3 cfu/mL to about 1×10−1 cfu/mL, about 1×10−3 cfu/mL to about 1×100 cfu/mL, about 1×10−3 cfu/mL to about 1×101 cfu/mL, about 1×10−3 cfu/mL to about 1×102 cfu/mL, about 1×10−3 cfu/mL to about 1×103 cfu/mL, about 1×10−3 cfu/mL to about 1×104 cfu/mL, about 1×10−3 cfu/mL to about 1×105 cfu/mL, about 1×10−3 cfu/mL to about 1×106 cfu/mL, about 1×10−3 cfu/mL to about 1×107 cfu/mL, about 1×10−3 cfu/mL to about 1×108 cfu/mL, about 1×10−3 cfu/mL to about 1×109 cfu/mL, about 1×10−3 cfu/mL to about 1×1010 cfu/mL, about 1×10−2 cfu/mL to about 1×10−1 cfu/mL, about 1×10−2 cfu/mL to about 1×100 cfu/mL, about 1×10−2 cfu/mL to about 1×101 cfu/mL, about 1×10−2 cfu/mL to about 1×102 cfu/mL, about 1×10−2 cfu/mL to about 1×103 cfu/mL, about 1×10−2 cfu/mL to about 1×104 cfu/mL, about 1×10−2 cfu/mL to about 1×105 cfu/mL, about 1×10−2 cfu/mL to about 1×106 cfu/mL, about 1×10−2 cfu/mL to about 1×107 cfu/mL, about 1×10−2 cfu/mL to about 1×108 cfu/mL, about 1×10−2 cfu/mL to about 1×109 cfu/mL, about 1×10−2 cfu/mL to about 1×1010 cfu/mL, about 1×10−1 cfu/mL to about 1×100 cfu/mL, about 1×10−1 cfu/mL to about 1×101 cfu/mL, about 1×10−1 cfu/mL to about 1×102 cfu/mL, about 1×10−1 cfu/mL to about 1×103 cfu/mL, about 1×10−1 cfu/mL to about 1×104 cfu/mL, about 1×10−1 cfu/mL to about 1×105 cfu/mL, about 1×10−1 cfu/mL to about 1×106 cfu/mL, about 1×10−1 cfu/mL to about 1×107 cfu/mL, about 1×10−1 cfu/mL to about 1×108 cfu/mL, about 1×10−1 cfu/mL to about 1×109 cfu/mL, about 1×10−1 cfu/mL to about 1×1010 cfu/mL, about 1×100 cfu/mL to about 1×101 cfu/mL, about 1×100 cfu/mL to about 1×102 cfu/mL, about 1×100 cfu/mL to about 1×103 cfu/mL, about 1×100 cfu/mL to about 1×104 cfu/mL, about 1×100 cfu/mL to about 1×105 cfu/mL, about 1×100 cfu/mL to about 1×106 cfu/mL, about 1×100 cfu/mL to about 1×107 cfu/mL, about 1×100 cfu/mL to about 1×108 cfu/mL, about 1×100 cfu/mL to about 1×109 cfu/mL, about 1×100 cfu/mL to about 1×1010 cfu/mL, about 1×101 cfu/mL to about 1×102 cfu/mL, about 1×101 cfu/mL to about 1×103 cfu/mL, about 1×101 cfu/mL to about 1×104 cfu/mL, about 1×101 cfu/mL to about 1×105 cfu/mL, about 1×101 cfu/mL to about 1×106 cfu/mL, about 1×101 cfu/mL to about 1×107 cfu/mL, about 1×101 cfu/mL to about 1×108 cfu/mL, about 1×101 cfu/mL to about 1×109 cfu/mL, about 1×101 cfu/mL to about 1×1010 cfu/mL, about 1×102 cfu/mL to about 1×103 cfu/mL, about 1×102 cfu/mL to about 1×104 cfu/mL, about 1×102 cfu/mL to about 1×105 cfu/mL, about 1×102 cfu/mL to about 1×106 cfu/mL, about 1×102 cfu/mL to about 1×107 cfu/mL, about 1×102 cfu/mL to about 1×108 cfu/mL, about 1×102 cfu/mL to about 1×109 cfu/mL, about 1×102 cfu/mL to about 1×1010 cfu/mL, about 1×103 cfu/mL to about 1×104 cfu/mL, about 1×103 cfu/mL to about 1×105 cfu/mL, about 1×103 cfu/mL to about 1×106 cfu/mL, about 1×103 cfu/mL to about 1×107 cfu/mL, about 1×103 cfu/mL to about 1×108 cfu/mL, about 1×103 cfu/mL to about 1×109 cfu/mL, about 1×103 cfu/mL to about 1×1010 cfu/mL, about 1×104 cfu/mL to about 1×105 cfu/mL, about 1×104 cfu/mL to about 1×106 cfu/mL, about 1×104 cfu/mL to about 1×107 cfu/mL, about 1×104 cfu/mL to about 1×108 cfu/mL, about 1×104 cfu/mL to about 1×109 cfu/mL, or about 1×104 cfu/mL to about 1×1010 cfu/mL.
In aspects of this embodiment, a method disclosed herein may qualitatively or quantitatively detect pathogen having a concentration after the enrichment step of, e.g., about 1×10−5 cfu/mL, about 1×10−4 cfu/mL, about 1×10−3 cfu/mL, about 1×10−2 cfu/mL, about 1×10−1 cfu/mL, about 1×100 cfu/mL, about 1×101 cfu/mL, about 1×102 cfu/mL, about 1×103 cfu/mL, about 1×104 cfu/mL, about 1×105 cfu/mL, about 1×106 cfu/mL, about 1×107 cfu/mL, about 1×108 cfu/mL, about 1×109 cfu/mL, or about 1×1010 cfu/mL. In other aspects of this embodiment, a method disclosed herein may qualitatively or quantitatively detect pathogen having a concentration after the enrichment step of, e.g., at least 1×10−5 cfu/mL, at least 1×10−4 cfu/mL, at least 1×10−3 cfu/mL, at least 1×10−2 cfu/mL, at least 1×10−1 cfu/mL, at least 1×100 cfu/mL, at least 1×101 cfu/mL, at least 1×102 cfu/mL, at least 1×103 cfu/mL, at least 1×104 cfu/mL, at least 1×105 cfu/mL, at least 1×106 cfu/mL, at least 1×107 cfu/mL, at least 1×108 cfu/mL, at least 1×109 cfu/mL, or at least 1×1010 cfu/mL. In yet other aspects of this embodiment, a method disclosed herein may qualitatively or quantitatively detect pathogen having a concentration after the enrichment step of, e.g., at most 1×10−5 cfu/mL, at most 1×10−4 cfu/mL, at most 1×10−3 cfu/mL, at most 1×10−2 cfu/mL, at most 1×10−1 cfu/mL, at most 1×100 cfu/mL, at most 1×101 cfu/mL, at most 1×102 cfu/mL, at most 1×103 cfu/mL, at most 1×104 cfu/mL, at most 1×105 cfu/mL, at most 1×106 cfu/mL, at most 1×107 cfu/mL, at most 1×108 cfu/mL, at most 1×109 cfu/mL, or at most 1×1010 cfu/mL.
In still other aspects of this embodiment, a method disclosed herein may qualitatively or quantitatively detect pathogen having a concentration after the enrichment step of f, e.g., about 1×10−5 cfu/mL to about 1×10−4 cfu/mL, about 1×10−5 cfu/mL to about 1×10−3 cfu/mL, about 1×10−5 cfu/mL to about 1×10−2 cfu/mL, about 1×10−5 cfu/mL to about 1×10−1 cfu/mL, about 1×10−5 cfu/mL to about 1×100 cfu/mL, about 1×10−5 cfu/mL to about 1×101 cfu/mL, about 1×10−5 cfu/mL to about 1×102 cfu/mL, about 1×10−5 cfu/mL to about 1×103 cfu/mL, about 1×10−5 cfu/mL to about 1×104 cfu/mL, about 1×10−5 cfu/mL to about 1×105 cfu/mL, about 1×10−5 cfu/mL to about 1×106 cfu/mL, about 1×10−5 cfu/mL to about 1×107 cfu/mL, about 1×10−5 cfu/mL to about 1×108 cfu/mL, about 1×10−5 cfu/mL to about 1×109 cfu/mL, about 1×10−5 cfu/mL to about 1×1010 cfu/mL, about 1×10−4 cfu/mL to about 1×10−3 cfu/mL, about 1×10−4 cfu/mL to about 1×10−2 cfu/mL, about 1×10−4 cfu/mL to about 1×10−1 cfu/mL, about 1×10−4 cfu/mL to about 1×100 cfu/mL, about 1×10−4 cfu/mL to about 1×101 cfu/mL, about 1×10−4 cfu/mL to about 1×102 cfu/mL, about 1×10−4 cfu/mL to about 1×103 cfu/mL, about 1×10−4 cfu/mL to about 1×104 cfu/mL, about 1×10−4 cfu/mL to about 1×105 cfu/mL, about 1×10−4 cfu/mL to about 1×106 cfu/mL, about 1×10−4 cfu/mL to about 1×107 cfu/mL, about 1×10−4 cfu/mL to about 1×108 cfu/mL, about 1×10−4 cfu/mL to about 1×109 cfu/mL, about 1×10−4 cfu/mL to about 1×1010 cfu/mL, about 1×10−3 cfu/mL to about 1×10−2 cfu/mL, about 1×10−3 cfu/mL to about 1×10−1 cfu/mL, about 1×10−3 cfu/mL to about 1×100 cfu/mL, about 1×10−3 cfu/mL to about 1×101 cfu/mL, about 1×10−3 cfu/mL to about 1×102 cfu/mL, about 1×10−3 cfu/mL to about 1×103 cfu/mL, about 1×10−3 cfu/mL to about 1×104 cfu/mL, about 1×10−3 cfu/mL to about 1×105 cfu/mL, about 1×10−3 cfu/mL to about 1×106 cfu/mL, about 1×10−3 cfu/mL to about 1×107 cfu/mL, about 1×10−3 cfu/mL to about 1×108 cfu/mL, about 1×10−3 cfu/mL to about 1×109 cfu/mL, about 1×10−3 cfu/mL to about 1×1010 cfu/mL, about 1×10−2 cfu/mL to about 1×10−1 cfu/mL, about 1×10−2 cfu/mL to about 1×100 cfu/mL, about 1×10−2 cfu/mL to about 1×101 cfu/mL, about 1×10−2 cfu/mL to about 1×102 cfu/mL, about 1×10−2 cfu/mL to about 1×103 cfu/mL, about 1×10−2 cfu/mL to about 1×104 cfu/mL, about 1×10−2 cfu/mL to about 1×105 cfu/mL, about 1×10−2 cfu/mL to about 1×106 cfu/mL, about 1×10−2 cfu/mL to about 1×107 cfu/mL, about 1×10−2 cfu/mL to about 1×108 cfu/mL, about 1×10−2 cfu/mL to about 1×109 cfu/mL, about 1×10−2 cfu/mL to about 1×1010 cfu/mL, about 1×10−1 cfu/mL to about 1×100 cfu/mL, about 1×10−1 cfu/mL to about 1×101 cfu/mL, about 1×10−1 cfu/mL to about 1×102 cfu/mL, about 1×10−1 cfu/mL to about 1×103 cfu/mL, about 1×10−1 cfu/mL to about 1×104 cfu/mL, about 1×10−1 cfu/mL to about 1×108 cfu/mL, about 1×10−1 cfu/mL to about 1×106 cfu/mL, about 1×10−1 cfu/mL to about 1×107 cfu/mL, about 1×10−1 cfu/mL to about 1×108 cfu/mL, about 1×10−1 cfu/mL to about 1×109 cfu/mL, about 1×10−1 cfu/mL to about 1×1010 cfu/mL, about 1×100 cfu/mL to about 1×101 cfu/mL, about 1×100 cfu/mL to about 1×102 cfu/mL, about 1×100 cfu/mL to about 1×103 cfu/mL, about 1×100 cfu/mL to about 1×104 cfu/mL, about 1×100 cfu/mL to about 1×108 cfu/mL, about 1×100 cfu/mL to about 1×106 cfu/mL, about 1×100 cfu/mL to about 1×107 cfu/mL, about 1×100 cfu/mL to about 1×108 cfu/mL, about 1×100 cfu/mL to about 1×109 cfu/mL, about 1×100 cfu/mL to about 1×1010 cfu/mL, about 1×101 cfu/mL to about 1×102 cfu/mL, about 1×101 cfu/mL to about 1×103 cfu/mL, about 1×101 cfu/mL to about 1×104 cfu/mL, about 1×101 cfu/mL to about 1×108 cfu/mL, about 1×101 cfu/mL to about 1×106 cfu/mL, about 1×101 cfu/mL to about 1×107 cfu/mL, about 1×101 cfu/mL to about 1×108 cfu/mL, about 1×101 cfu/mL to about 1×109 cfu/mL, about 1×101 cfu/mL to about 1×1010 cfu/mL, about 1×102 cfu/mL to about 1×103 cfu/mL, about 1×102 cfu/mL to about 1×104 cfu/mL, about 1×102 cfu/mL to about 1×108 cfu/mL, about 1×102 cfu/mL to about 1×106 cfu/mL, about 1×102 cfu/mL to about 1×107 cfu/mL, about 1×102 cfu/mL to about 1×108 cfu/mL, about 1×102 cfu/mL to about 1×109 cfu/mL, about 1×102 cfu/mL to about 1×1010 cfu/mL, about 1×103 cfu/mL to about 1×104 cfu/mL, about 1×103 cfu/mL to about 1×108 cfu/mL, about 1×103 cfu/mL to about 1×106 cfu/mL, about 1×103 cfu/mL to about 1×107 cfu/mL, about 1×103 cfu/mL to about 1×108 cfu/mL, about 1×103 cfu/mL to about 1×109 cfu/mL, about 1×103 cfu/mL to about 1×1010 cfu/mL, about 1×104 cfu/mL to about 1×108 cfu/mL, about 1×104 cfu/mL to about 1×106 cfu/mL, about 1×104 cfu/mL to about 1×107 cfu/mL, about 1×104 cfu/mL to about 1×108 cfu/mL, about 1×104 cfu/mL to about 1×109 cfu/mL, or about 1×104 cfu/mL to about 1×1010 cfu/mL.
Common detection procedures used to determine the presence or absence of a pathogen include nucleic acid-based detection methods, protein-based detection methods, activity-based detection methods, growth-based detection methods, and sensor-based detection methods.
In one embodiment, detection presence or absence of a pathogen of interest occurs using a nucleic acid-based detection method. Non-limiting examples of nucleic acid-based detection method include DNA-based detection methods and RNA-based detection methods. DNA-based detection methods include, without limitation, like Southern blot analysis, PCR-based assays, sequence analysis, immuno-based detection assays, and hybridization assays using FRET, polarization or other fluorescent, chemiluminescent or bioluminescent detection methods. RNA-based detection methods include, without limitation, like Northern blot analysis, RT-PCR-based assays, RNA sequencing, immuno-based detection assays, and hybridization assays using FRET, polarization or other fluorescent, chemiluminescent or bioluminescent detection methods.
In one embodiment, detection presence or absence of a pathogen of interest occurs using a protein-based detection method. Non-limiting examples of protein-based detection methods include gel-based detection methods, immuno-based detection methods and protein-interaction-based methods. Gel-based detection methods include, without limitation polyacrylamide gel electrophoresis and SDS-PAGE. Immuno-based detection methods include, without limitation, Western blot analysis, ELISA, and immunoprecipitation. Protein-interaction-based methods include, without limitation, protein-protein interaction-based assays, protein-DNA interaction-based assays, and protein-RNA interaction-based assays.
In one embodiment, detection presence or absence of a pathogen of interest occurs using an activity-based detection method. Non-limiting examples of activity-based detection methods include enzymatic activity assays and assays based on protein function. An enzymatic activity assays typically involves incubating an aliquot from an enrichment media or from the supernatant taken after an immunopurification step in a buffered solution containing a suitable substrate. If the desired enzyme is present in the aliquot, then is will catalyze the conversion of the substrate into a product. Measuring either the loss of substrate or the formation of product can then be either qualitatively or quantitatively correlated to the amount of enzyme present and hence the amount of pathogen in the enrichment media. Similarly, assays based on protein function measure the amount of function present in the sample and extrapolate the amount of pathogen in the enrichment media based on this measurement.
In one embodiment, detection presence or absence of a pathogen of interest occurs using a growth-based detection method. Non-limiting examples of growth-based detection method include plating assays measuring colony formation with or without growth selective agents and spectrophotometer assays measuring cell density. A plating assay typically involves plating an aliquot of enrichment media onto an agar plate comprising nutrients to sustain growth of the pathogen of interest. The inoculated agar plates are then incubated for a specified temperature and time and growth of pathogen colonies assessed. In some embodiments, the agar plates are incubated at about 25° C. to about 42° C. for about 12 hours to about 48 hours. In aspects of this embodiments, the agar plates are incubated at about 37° C. for about 14 hours to about 16 hours. This assessment may be qualitative by simply assessing the presence or absence of pathogen colonies, or quantitative, where the number of pathogen colonies are counted. Besides nutrients, an agar plate may contain a chromogenic compound that stains or otherwise provides a visual signal that identifies the colony as a pathogen colony. In addition, an agar plate may contain a compound that selects for pathogen colony growth by either supplying a compound that facilitates or is required for pathogen growth or inhibits the growth of contaminating microorganisms. A spectrophotometer assays measuring cell density typically involves measuring the cell density of an aliquot taken from enrichment media using a spectrophotometer at a specific wavelength.
In one embodiment, detection presence or absence of a pathogen of interest occurs using a sensor-based detection method. Sensors can be classified according to the type of energy being transfer, such as, e.g., thermal, electromagnetic, mechanical, and electrochemical.
In an aspect of this embodiment, a sensor-based detection method is a biosensor-based detection method. A biosensor is a type of analytical device incorporating a biological material, a biologically derived material or a biomimic intimately associated with or integrated within a physicochemical transducer or transducing microsystem. A biosensor converts the modification of the physical or chemical properties of a biomatrix (e.g., enzyme, antibodies, receptors, organelles, microorganisms) into an electric or other kinds of signal whose amplitude depends on the concentration of defined analytes in the solution.
Non-limiting examples of a biosensor include an enzymatic biosensor, a DNA sensor, and an immunosensor. Enzyme-based biosensor require the immobilization of an enzyme onto an electrode surface for the quantification of an analyte. DNA-based biosensor require the immobilization of non-complimentary DNA strand of a target sequence onto an electrode surface for the quantification of an analyte. Immuno-based biosensor require the immobilization of an antibody onto an electrode surface for the quantification of an analyte.
A biosensor comprises a biorecognition element, a signal transducer, and a detector. The biorecognition element includes antibodies, peptides, nucleic acids, or enzymes and is the portion of the sensor that initially binds to or interacts with the analyte (e.g., a pathogen). In many cases this is associated with a conformational change, substrate cleavage, or enzymatic reaction that transduces the biorecognition event into a signal that may be detected via several modalities. A biosensor comprises two electrodes (reference and a working electrodes) or three electrodes (reference, working, and counter electrode).
A reference electrode includes liquid and solid-state reference electrodes and is an electrode having a known stable potential to which all other electrode potentials are referenced. A reference electrode can be manufactured by film deposition, electroplating and screen printing. Non-limiting examples of a reference electrode include silver-silver chloride electrode, calomel electrode, hydrogen electrode, mercury-mercury oxide electrode, mercury-mercurous sulfate electrode, copper-copper sulfate electrode, and palladium hydride electrode.
A biosensor typically uses the power of electrochemical techniques for biological processes by quantitatively producing an electrical signal that relates to the concentration of a biological analyte. Such electrochemical biosensors can be classified as potentiometric, amperometric, voltametric and impedimetric/conductimetric based upon the analytical principles of operation. Potentiometric electrochemical sensors measure an equilibrium potential difference between a working electrode and a reference electrode at zero current flow. Voltammetric electrochemical sensors measure the current as a function of varying potential is applied between a working electrode and a reference electrode. Amperometric electrochemical sensors measure the current as a function of constant potential is applied between a working electrode and a reference electrode. Impedimetric/conductimetric electrochemical sensors measure the changes of electrical properties between a working electrode and a reference electrode. Electrochemical detection using a biosensor may be a direct measurement of the change in electrical properties or an indirect measurement using an auxiliary reaction which involves a marking (redox active) compound for signal generation.
Aspect of the present specification disclose, in part, a detection solution. When a biosensor is a glucose-based biosensor, a detection solution disclosed herein typically includes a buffered solution comprises magnesium chloride, p-aminofenil phosphate, and glucose. Optionally, such a detection solution may comprise a surfactant as disclosed herein.
Any buffer may be used, with the proviso that the resulting buffered solution is useful to practice the methods disclosed herein. A buffered solution can be varied as appropriate by one skilled in the art and generally depends, in part, on the pH value desired for the mobile phase, the protein being eluted, and the conductivity values being employed. Therefore, aspects of this embodiment may optionally include, e.g., 2-(N-morpholino) ethanesulfonic acid (MES), N-(2-acetamido)iminodiacetic acid (ADA), dimethylarsinic acid (Cacodylate), piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES), N-(2-Acetamido)-2-aminoethanesulfonic acid (ACES), cholamine chloride, N,N′-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), 3-(N-morpholino) propanesulfonic acid (MOPS), 2-{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid (TES), N-(2-hydroxyethyl) piperazine-N′-(2-ethanesulfonic acid) (HEPES), piperazine-N,N′-bis(2-hydroxypropanesulfonic acid) (POPSO), N-tris(hydroxymethyl) methylglycine (Tricine), tris(hydroxymethyl)methylamine (Tris), acetamidoglycine, N,N-bis(2-hydroxyethyl)glycine (Bicine), N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), 3-[(1,1-dimethyl-2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (AMPSO), 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO), and 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS); acetate buffers, such as, e.g., magnesium acetate, potassium acetate, and Tris acetate; borate buffers; citrate buffers; phosphate buffers, such as, e.g., potassium phosphate buffers and sodium phosphate buffers; saline buffers, such as, e.g., phosphate-buffered saline (PBS), HEPES-buffered saline (HBS), and Tris-buffered saline (TBS), saline sodium citrate (SSC); universal buffers, such as, e.g., buffers comprising citric acid and potassium phosphate, Britton-Robinson buffer, Carmody buffer and the like, or any combination thereof. Non-limiting examples of how to make and use specific buffers are described in, e.g., MOLECULAR CLONING, A LABORATORY MANUAL (Joseph Sambrook & David W. Russell eds., Cold Spring Harbor Laboratory Press, 3rd ed. 2001) and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Frederick M. Ausubel et al., eds. John Wiley & Sons, 2004).
Any concentration of magnesium chloride may be used in a detection solution, with the proviso that the concentration is useful to practice the methods disclosed herein. In aspects of this embodiment, a concentration of magnesium chloride used in a detection solution may be, e.g., about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, or about 100 mM. In other aspects of this embodiment, a concentration of magnesium chloride used in a detection solution may be, e.g., at least 5 mM, at least 10 mM, at least 15 mM, at least 20 mM, at least 25 mM, at least 30 mM, at least 35 mM, at least 40 mM, at least 45 mM, at least 50 mM, at least 55 mM, at least 60 mM, at least 65 mM, at least 70 mM, at least 75 mM, at least 80 mM, at least 85 mM, at least 90 mM, at least 95 mM, or at least 100 mM. In yet other aspects of this embodiment, a concentration of magnesium chloride used in a detection solution may be, e.g., at most 5 mM, at most 10 mM, at most 15 mM, at most 20 mM, at most 25 mM, at most 30 mM, at most 35 mM, at most 40 mM, at most 45 mM, at most 50 mM, at most 55 mM, at most 60 mM, at most 65 mM, at most 70 mM, at most 75 mM, at most 80 mM, at most 85 mM, at most 90 mM, at most 95 mM, or at most 100 mM. In still other aspects of this embodiment, a concentration of magnesium chloride used in a detection solution may be, e.g., about 5 mM to about 10 mM, about 5 mM to about 20 mM, about 5 mM to about 30 mM, about 5 mM to about 40 mM, about 5 mM to about 50 mM, about 5 mM to about 60 mM, about 5 mM to about 70 mM, about 5 mM to about 80 mM, about 5 mM to about 90 mM, about 5 mM to about 100 mM, about 10 mM to about 20 mM, about 10 mM to about 30 mM, about 10 mM to about 40 mM, about 10 mM to about 50 mM, about 10 mM to about 60 mM, about 10 mM to about 70 mM, about 10 mM to about 80 mM, about 10 mM to about 90 mM, about 10 mM to about 100 mM, about 20 mM to about 30 mM, about 20 mM to about 40 mM, about 20 mM to about 50 mM, about 20 mM to about 60 mM, about 20 mM to about 70 mM, about 20 mM to about 80 mM, about 20 mM to about 90 mM, or about 20 mM to about 100 mM.
Any concentration of p-aminofenil phosphate may be used in a detection solution, with the proviso that the concentration is useful to practice the methods disclosed herein. In aspects of this embodiment, a concentration of p-aminofenil phosphate used in a detection solution may be, e.g., about 0.1 mM, about 0.2 mM, about 0.3 mM, about 0.4 mM, about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, about 1.0 mM, about 1.1 mM, about 1.2 mM, about 1.3 mM, about 1.4 mM, about 1.5 mM, about 1.6 mM, about 1.7 mM, about 1.8 mM, about 1.9 mM, about 2.0 mM, about 2.1 mM, about 2.2 mM, about 2.3 mM, about 2.4 mM, or about 2.5 mM. In other aspects of this embodiment, a concentration of p-aminofenil phosphate used in a detection solution may be, e.g., at least 0.1 mM, at least 0.2 mM, at least 0.3 mM, at least 0.4 mM, at least 0.5 mM, at least 0.6 mM, at least 0.7 mM, at least 0.8 mM, at least 0.9 mM, at least 1.0 mM, at least 1.1 mM, at least 1.2 mM, at least 1.3 mM, at least 1.4 mM, at least 1.5 mM, at least 1.6 mM, at least 1.7 mM, at least 1.8 mM, at least 1.9 mM, at least 2.0 mM, at least 2.1 mM, at least 2.2 mM, at least 2.3 mM, at least 2.4 mM, or at least 2.5 mM. In yet other aspects of this embodiment, a concentration of p-aminofenil phosphate used in a detection solution may be, e.g., at most 0.1 mM, at most 0.2 mM, at most 0.3 mM, at most 0.4 mM, at most 0.5 mM, at most 0.6 mM, at most 0.7 mM, at most 0.8 mM, at most 0.9 mM, at most 1.0 mM, at most 1.1 mM, at most 1.2 mM, at most 1.3 mM, at most 1.4 mM, at most 1.5 mM, at most 1.6 mM, at most 1.7 mM, at most 1.8 mM, at most 1.9 mM, at most 2.0 mM, at most 2.1 mM, at most 2.2 mM, at most 2.3 mM, at most 2.4 mM, or at most 2.5 mM. In still other aspects of this embodiment, a concentration of p-aminofenil phosphate used in a detection solution may be, e.g., about 0.1 mM to about 0.5 mM, about 0.1 mM to about 1.0 mM, about 0.1 mM to about 1.5 mM, about 0.1 mM to about 2.0 mM, about 0.1 mM to about 2.5 mM, about 0.3 mM to about 0.5 mM, about 0.3 mM to about 1.0 mM, about 0.3 mM to about 1.5 mM, about 0.3 mM to about 2.0 mM, about 0.3 mM to about 2.5 mM, about 0.5 mM to about 1.0 mM, about 0.5 mM to about 1.5 mM, about 0.5 mM to about 2.0 mM, about 0.5 mM to about 2.5 mM, about 0.7 mM to about 1.0 mM, about 0.7 mM to about 1.5 mM, about 0.7 mM to about 2.0 mM, about 0.7 mM to about 2.5 mM, about 1.0 mM to about 1.5 mM, about 1.0 mM to about 2.0 mM, about 1.0 mM to about 2.5 mM, about 1.5 mM to about 2.0 mM, about 1.5 mM to about 2.5 mM, or about 2.0 mM to about 2.5 mM.
Any concentration of glucose may be used in a detection solution, with the proviso that the concentration is useful to practice the methods disclosed herein. In aspects of this embodiment, a concentration of glucose used in a detection solution may be, e.g., about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, or about 50 mM. In other aspects of this embodiment, a concentration of glucose used in a detection solution may be, e.g., at least 1 mM, at least 2 mM, at least 3 mM, at least 4 mM, at least 5 mM, at least 6 mM, at least 7 mM, at least 8 mM, at least 9 mM, at least 10 mM, at least 15 mM, at least 20 mM, at least 25 mM, at least 30 mM, at least 35 mM, at least 40 mM, at least 45 mM, or at least 50 mM. In yet other aspects of this embodiment, a concentration of glucose used in a detection solution may be, e.g., at most 1 mM, at most 2 mM, at most 3 mM, at most 4 mM, at most 5 mM, at most 6 mM, at most 7 mM, at most 8 mM, at most 9 mM, at most 10 mM, at most 15 mM, at most 20 mM, at most 25 mM, at most 30 mM, at most 35 mM, at most 40 mM, at most 45 mM, or at most 50 mM. In still other aspects of this embodiment, a concentration of glucose used in a detection solution may be, e.g., about 1 mM to about 10 mM, about 1 mM to about 15 mM, about 1 mM to about 20 mM, about 1 mM to about 30 mM, about 1 mM to about 40 mM, about 1 mM to about 50 mM, about 5 mM to about 10 mM, about 5 mM to about 15 mM, about 5 mM to about 20 mM, about 5 mM to about 30 mM, about 5 mM to about 40 mM, about 5 mM to about 50 mM, about 10 mM to about 15 mM, about 10 mM to about 20 mM, about 10 mM to about 30 mM, about 10 mM to about 40 mM, or about 10 mM to about 50 mM.
Any concentration of surfactant may be used in a detection solution, with the proviso that the concentration is useful to practice the methods disclosed herein. Surfactants disclosed herein at the concentrations disclosed herein may be used in preparing a detection solution. In aspects of this embodiment, a detection solution comprises between 0.1% (v/v) and 10% (v/v) detergent B-Per, or a surfactant of polyoxyethylene glycol sorbitan alkyl esters family (TWEEN® family) or polyoxyethylene glycol octylphenol ethers family (TRITON® family).
In an aspect of this embodiment, a detection solution may be 200 mM Phosphate buffer (pH 5.5) detection solution. In an aspect of this embodiment, 200 mM Phosphate buffer (pH 5.5) detection solution may comprise 0.0021 g/mL Sodium Hydrogen Phosphate, 0.0289 g/mL Sodium Dihydrogen Phosphate, 0.0020 g/mL Magnesium chloride, 0.0018 g/mL Glucose, and 0.0002 g/mL p-aminophenyl phosphate.
In an aspect of this embodiment, a detection solution may be 200 mM Phosphate buffer (pH 5.7) detection solution. In an aspect of this embodiment, 200 mM Phosphate buffer (pH 5.7) detection solution may comprise 0.0174 g/mL Potassium Hydrogen Phosphate, 0.0204 g/mL Potassium Phosphate (Phtalate), 0.0020 g/mL Magnesium chloride, 0.0018 g/mL Glucose, and 0.0002 g/mL p-aminophenyl phosphate.
In an aspect of this embodiment, a detection solution may be 200 mM Phosphate buffer (pH 7.0) detection solution. In an aspect of this embodiment, 200 mM Phosphate buffer (pH 7.0) detection solution may comprise 0.0156 g/mL Sodium Hydrogen Phosphate, 0.0142 g/mL Sodium Dihydrogen Phosphate, 0.0020 g/mL Magnesium chloride, 0.0018 g/mL Glucose, and 0.0002 g/mL p-aminophenyl phosphate.
In an aspect of this embodiment, a detection solution may be 160 mM Phosphate-citrate buffer (pH 5.5) detection solution. In an aspect of this embodiment, 160 mM Phosphate-citrate buffer (pH 5.5) detection solution may comprise 0.0161 g/mL Sodium Hydrogen Phosphate, 0.0090 g/mL Citric Acid, 0.0020 g/mL Magnesium chloride, 0.0018 g/mL Glucose, and 0.0002 g/mL p-aminophenyl phosphate.
In an aspect of this embodiment, a detection solution may be 200 mM Acetate buffer (pH 5.7) detection solution. In an aspect of this embodiment, 200 mM Acetate buffer (pH 5.7) detection solution may comprise 1.15 μL Acetic Acid, 0.0015 g/mL Sodium Acetate, 0.0020 g/mL Magnesium chloride, 0.0018 g/mL Glucose, and 0.0002 g/mL p-aminophenyl phosphate.
In an aspect of this embodiment, a detection solution may be 200 mM Tris buffer (pH 9.8) detection solution. In an aspect of this embodiment, 200 mM Tris buffer (pH 9.8) detection solution may comprise 0.0243 g/mL TRIS, 0.0020 g/mL Magnesium chloride, 0.0018 g/mL Glucose, and 0.0002 g/mL p-aminophenyl phosphate.
Aspect of the present specification disclose, in part, incubation of a detection solution. Incubation of a detection solution is performed under temperature and time parameters that facilitate detection of a signal. Incubation a detection solution may be performed under agitation/rotation.
Any temperature may be used during incubation of a detection solution, with the proviso that the temperature is useful to practice the methods disclosed herein. In aspects of this embodiment, a temperature used to incubate a detection solution may be, e.g., about 15° C., about 16° C., about 17° C., about 18° C., about 19° C., about 20° C., about 21° C., about 22° C., about 23° C., about 24° C., or about 25° C. In other aspects of this embodiment, a temperature used to incubate a detection solution may be, e.g., at least 15° C., at least 16° C., at least 17° C., at least 18° C., at least 19° C., at least 20° C., at least 21° C., at least 22° C., at least 23° C., at least 24° C., or at least 25° C. In yet other aspects of this embodiment, a temperature used to incubate a detection solution may be, e.g., at most 15° C., at most 16° C., at most 17° C., at most 18° C., at most 19° C., at most 20° C., at most 21° C., at most 22° C., at most 23° C., at most 24° C., or at most 25° C. In still other aspects of this embodiment, a temperature used to incubate immunmagnetic particles with enrichment media comprising the pathogen of interest may be, e.g., about 15° C. to about 19° C., about 16° C. to about 20° C., about 17° C. to about 21° C., about 18° C. to about 22° C., about 19° C. to about 23° C., about 20° C. to about 24° C., about 21° C. to about 25° C., about 22° C. to about 26° C., about 23° C. to about 27° C., about 24° C. to about 28° C., or about 25° C. to about 29° C.
Any time may be used during incubation of a detection solution, with the proviso that the time is useful to practice the methods disclosed herein. In aspects of this embodiment, a time used to incubate a detection solution may be, e.g., about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 70 minutes, about 80 minutes, about 90 minutes, about 100 minutes, about 110 minutes, about 120 minutes, about 130 minutes, about 140 minutes, or about 150 minutes. In other aspects of this embodiment, a time used to incubate a detection solution may be, e.g., at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, at least 60 minutes, at least 70 minutes, at least 80 minutes, at least 90 minutes, at least 100 minutes, at least 110 minutes, at least 120 minutes, at least 130 minutes, at least 140 minutes, or at least 150 minutes. In yet other aspects of this embodiment, a time used to a detection solution may be, e.g., at most 5 minutes, at most 10 minutes, at most 15 minutes, at most 20 minutes, at most 30 minutes, at most 40 minutes, at most 50 minutes, at most 60 minutes, at most 70 minutes, at most 80 minutes, at most 90 minutes, at most 100 minutes, at most 110 minutes, at most 120 minutes, at most 130 minutes, at most 140 minutes, or at most 150 minutes.
In yet other aspects of this embodiment, a time used to a detection solution may be, e.g., about 5 minutes to about 20 minutes, about 5 minutes to about 30 minutes, about 5 minutes to about 40 minutes, about 5 minutes to about 50 minutes, about 5 minutes to about 60 minutes, about 5 minutes to about 70 minutes, about 5 minutes to about 80 minutes, about 5 minutes to about 90 minutes, about 5 minutes to about 100 minutes, about 5 minutes to about 110 minutes, about 5 minutes to about 120 minutes, about 5 minutes to about 130 minutes, about 5 minutes to about 140 minutes, about 5 minutes to about 150 minutes, about 10 minutes to about 20 minutes, about 10 minutes to about 30 minutes, about 10 minutes to about 40 minutes, about 10 minutes to about 50 minutes, about 10 minutes to about 60 minutes, about 10 minutes to about 70 minutes, about 10 minutes to about 80 minutes, about 10 minutes to about 90 minutes, about 10 minutes to about 100 minutes, about 10 minutes to about 110 minutes, about 10 minutes to about 120 minutes, about 10 minutes to about 130 minutes, about 10 minutes to about 140 minutes, about 10 minutes to about 150 minutes, about 20 minutes to about 30 minutes, about 20 minutes to about 40 minutes, about 20 minutes to about 50 minutes, about 20 minutes to about 60 minutes, about 20 minutes to about 70 minutes, about 20 minutes to about 80 minutes, about 20 minutes to about 90 minutes, about 20 minutes to about 100 minutes, about 20 minutes to about 110 minutes, about 20 minutes to about 120 minutes, about 20 minutes to about 130 minutes, about 20 minutes to about 140 minutes, about 20 minutes to about 150 minutes, about 30 minutes to about 40 minutes, about 30 minutes to about 50 minutes, about 30 minutes to about 60 minutes, about 30 minutes to about 70 minutes, about 30 minutes to about 80 minutes, about 30 minutes to about 90 minutes, about 30 minutes to about 100 minutes, about 30 minutes to about 110 minutes, about 30 minutes to about 120 minutes, about 30 minutes to about 130 minutes, about 30 minutes to about 140 minutes, about 30 minutes to about 150 minutes, about 60 minutes to about 70 minutes, about 60 minutes to about 80 minutes, about 60 minutes to about 90 minutes, about 60 minutes to about 100 minutes, about 60 minutes to about 110 minutes, about 60 minutes to about 120 minutes, about 60 minutes to about 130 minutes, about 60 minutes to about 140 minutes, about 60 minutes to about 150 minutes, about 90 minutes to about 100 minutes, about 90 minutes to about 110 minutes, about 90 minutes to about 120 minutes, about 90 minutes to about 130 minutes, about 90 minutes to about 140 minutes, about 90 minutes to about 150 minutes, about 120 minutes to about 130 minutes, about 120 minutes to about 140 minutes, or about 120 minutes to about 150 minutes.
In aspects of this embodiment, a detection solution may be incubated at a temperature of, e.g., about 15° C., about 16° C., about 17° C., about 18° C., about 19° C., about 20° C., about 21° C., about 22° C., about 23° C., about 24° C., or about 25° C. for a time of, e.g., about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 70 minutes, about 80 minutes, about 90 minutes, about 100 minutes, about 110 minutes, about 120 minutes, about 130 minutes, about 140 minutes, or about 150 minutes.
In other aspects of this embodiment, a detection solution may be incubated at a temperature of, e.g., at least 15° C., at least 16° C., at least 17° C., at least 18° C., at least 19° C., at least 20° C., at least 21° C., at least 22° C., at least 23° C., at least 24° C., or at least 25° C. for a time of, e.g., at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, at least 60 minutes, at least 70 minutes, at least 80 minutes, at least 90 minutes, at least 100 minutes, at least 110 minutes, at least 120 minutes, at least 130 minutes, at least 140 minutes, or at least 150 minutes.
In yet other aspects of this embodiment, a detection solution may be incubated at a temperature of, e.g., at most 15° C., at most 16° C., at most 17° C., at most 18° C., at most 19° C., at most 20° C., at most 21° C., at most 22° C., at most 23° C., at most 24° C., or at most 25° C., for a time of, e.g., at most 5 minutes, at most 10 minutes, at most 15 minutes, at most 20 minutes, at most 30 minutes, at most 40 minutes, at most 50 minutes, at most 60 minutes, at most 70 minutes, at most 80 minutes, at most 90 minutes, at most 100 minutes, at most 110 minutes, at most 120 minutes, at most 130 minutes, at most 140 minutes, or at most 150 minutes.
In still other aspects of this embodiment, a detection solution may be incubated at a temperature of, e.g., about 15° C. to about 19° C., about 16° C. to about 20° C., about 17° C. to about 21° C., about 18° C. to about 22° C., about 19° C. to about 23° C., about 20° C. to about 24° C., about 21° C. to about 25° C., about 22° C. to about 26° C., about 23° C. to about 27° C., about 24° C. to about 28° C., or about 25° C. to about 29° C. for a time of, e.g., about 5 minutes to about 20 minutes, about 5 minutes to about 30 minutes, about 5 minutes to about 40 minutes, about 5 minutes to about 50 minutes, about 5 minutes to about 60 minutes, about 5 minutes to about 70 minutes, about 5 minutes to about 80 minutes, about 5 minutes to about 90 minutes, about 5 minutes to about 100 minutes, about 5 minutes to about 110 minutes, about 5 minutes to about 120 minutes, about 5 minutes to about 130 minutes, about 5 minutes to about 140 minutes, about 5 minutes to about 150 minutes, about 10 minutes to about 20 minutes, about 10 minutes to about 30 minutes, about 10 minutes to about 40 minutes, about 10 minutes to about 50 minutes, about 10 minutes to about 60 minutes, about 10 minutes to about 70 minutes, about 10 minutes to about 80 minutes, about 10 minutes to about 90 minutes, about 10 minutes to about 100 minutes, about 10 minutes to about 110 minutes, about 10 minutes to about 120 minutes, about 10 minutes to about 130 minutes, about 10 minutes to about 140 minutes, about 10 minutes to about 150 minutes, about 20 minutes to about 30 minutes, about 20 minutes to about 40 minutes, about 20 minutes to about 50 minutes, about 20 minutes to about 60 minutes, about 20 minutes to about 70 minutes, about 20 minutes to about 80 minutes, about 20 minutes to about 90 minutes, about 20 minutes to about 100 minutes, about 20 minutes to about 110 minutes, about 20 minutes to about 120 minutes, about 20 minutes to about 130 minutes, about 20 minutes to about 140 minutes, about 20 minutes to about 150 minutes, about 30 minutes to about 40 minutes, about 30 minutes to about 50 minutes, about 30 minutes to about 60 minutes, about 30 minutes to about 70 minutes, about 30 minutes to about 80 minutes, about 30 minutes to about 90 minutes, about 30 minutes to about 100 minutes, about 30 minutes to about 110 minutes, about 30 minutes to about 120 minutes, about 30 minutes to about 130 minutes, about 30 minutes to about 140 minutes, about 30 minutes to about 150 minutes, about 60 minutes to about 70 minutes, about 60 minutes to about 80 minutes, about 60 minutes to about 90 minutes, about 60 minutes to about 100 minutes, about 60 minutes to about 110 minutes, about 60 minutes to about 120 minutes, about 60 minutes to about 130 minutes, about 60 minutes to about 140 minutes, about 60 minutes to about 150 minutes, about 90 minutes to about 100 minutes, about 90 minutes to about 110 minutes, about 90 minutes to about 120 minutes, about 90 minutes to about 130 minutes, about 90 minutes to about 140 minutes, about 90 minutes to about 150 minutes, about 120 minutes to about 130 minutes, about 120 minutes to about 140 minutes, or about 120 minutes to about 150 minutes.
After incubation of a detection solution, an aliquot is removed for analysis of a detectable electrochemical signal. In aspects of this embodiment, a volume of an aliquot to be analyzed for a detectable electrochemical signal may be, e.g., about 1 μL, about 2 μL, about 3 μL, about 4 μL, about 5 μL, about 6 μL, about 7 μL, about 8 μL, about 9 μL, about 10 μL, about 11 μL, about 12 μL, about 13 μL, about 14 μL, about 15 μL, about 16 μL, about 17 μL, about 18 μL, about 19 μL, or about 20 μL. In other aspects of this embodiment, a volume of an aliquot to be analyzed for a detectable electrochemical signal may be, e.g., at least 1 μL, at least 2 μL, at least 3 μL, at least 4 μL, at least 5 μL, at least 6 μL, at least 7 μL, at least 8 μL, at least 9 μL, at least 10 μL, at least 11 μL, at least 12 μL, at least 13 μL, at least 14 μL, at least 15 μL, at least 16 μL, at least 17 μL, at least 18 μL, at least 19 μL, or at least 20 μL. In yet other aspects of this embodiment, a volume of an aliquot to be analyzed for a detectable electrochemical signal may be, e.g., at most 1 μL, at most 2 μL, at most 3 μL, at most 4 μL, at most 5 μL, at most 6 μL, at most 7 μL, at most 8 μL, at most 9 μL, at most 10 μL, at most 11 μL, at most 12 μL, at most 13 μL, at most 14 μL, at most 15 μL, at most 16 μL, at most 17 μL, at most 18 μL, at most 19 μL, or at most 20 μL. In yet other aspects of this embodiment, a volume of an aliquot to be analyzed for a detectable electrochemical signal may be, e.g., about 1 μL to about 5 μL, about 1 μL to about 10 μL, about 1 μL to about 15 μL, about 1 μL to about 20 μL, 2 μL to about 5 μL, about 2 μL to about 10 μL, about 2 μL to about 15 μL, about 2 μL to about 20 μL, about 5 μL to about 10 μL, about 5 μL to about 15 μL, about 5 μL to about 20 μL, about 10 μL to about 15 μL, about 10 μL to about 20 μL, or about 15 μL to about 20 μL.
An electrochemical signal can be analyzed using an instrument which is capable of measuring and/or analyzing potentiometric, voltammetric, amperometric and/or impedance/conductance parameters. Typically, such instrumentation is operated using computer-controlled software. A non-limiting example is PalmSens3, a potentiostat, galvanostat, and impedance analyzer and PSTrace, its accompanying software (PalmSens BV, Utrecht, Netherlands).
In other embodiments, a method disclosed herein can be performed to completion in, e.g., about 18 hours, about 20 hours, about 22 hours, about 24 hours, about 26 hours, about 28 hours, about 30 hours, about 32 hours, about 34 hours, or about 36 hours. In yet other embodiments, a method comprising a pre-enrichment step disclosed herein and an enrichment step disclosed herein can be performed to completion in, e.g., about 18 hours, about 20 hours, about 22 hours, about 24 hours, about 26 hours, about 28 hours, about 30 hours, about 32 hours, about 34 hours, or about 36 hours. In still other embodiments, a method comprising a pre-enrichment step disclosed herein, an enrichment step disclosed herein and a detection step disclosed herein can be performed to completion in, e.g., about 18 hours, about 20 hours, about 22 hours, about 24 hours, about 26 hours, about 28 hours, about 30 hours, about 32 hours, about 34 hours, or about 36 hours. In other embodiments, a method comprising a pre-enrichment step disclosed herein, an enrichment step disclosed herein, a purification step disclosed herein, and a detection step disclosed herein can be performed to completion in, e.g., about 18 hours, about 20 hours, about 22 hours, about 24 hours, about 26 hours, about 28 hours, about 30 hours, about 32 hours, about 34 hours, or about 36 hours.
In other embodiments, a method disclosed herein can be performed to completion in, e.g., less than 18 hours, less than 20 hours, less than 22 hours, less than 24 hours, less than 26 hours, less than 28 hours, less than 30 hours, less than 32 hours, less than 34 hours, or less than 36 hours. In yet other embodiments, a method comprising a pre-enrichment step disclosed herein and an enrichment step disclosed herein can be performed to completion in, e.g., less than 18 hours, less than 20 hours, less than 22 hours, less than 24 hours, less than 26 hours, less than 28 hours, less than 30 hours, less than 32 hours, less than 34 hours, or less than 36 hours. In still other embodiments, a method comprising a pre-enrichment step disclosed herein, an enrichment step disclosed herein and a detection step disclosed herein can be performed to completion in, e.g., less than 18 hours, less than 20 hours, less than 22 hours, less than 24 hours, less than 26 hours, less than 28 hours, less than 30 hours, less than 32 hours, less than 34 hours, or less than 36 hours. In other embodiments, a method comprising a pre-enrichment step disclosed herein, an enrichment step disclosed herein, a purification step disclosed herein, and a detection step disclosed herein can be performed to completion in, e.g., less than 18 hours, less than 20 hours, less than 22 hours, less than 24 hours, less than 26 hours, less than 28 hours, less than 30 hours, less than 32 hours, less than 34 hours, or less than 36 hours.
In other embodiments, a method disclosed herein can be performed to completion in, e.g., about 18 hours to about 20 hours, about 18 hours to about 22 hours, about 18 hours to about 24 hours, about 18 hours to about 26 hours, about 18 hours to about 28 hours, about 18 hours to about 30 hours, about 18 hours to about 32 hours, about 18 hours to about 34 hours, about 18 hours to about 36 hours, about 20 hours to about 22 hours, about 20 hours to about 24 hours, about 20 hours to about 26 hours, about 20 hours to about 28 hours, about 20 hours to about 30 hours, about 20 hours to about 32 hours, about 20 hours to about 34 hours, about 20 hours to about 36 hours, about 22 hours to about 24 hours, about 22 hours to about 26 hours, about 22 hours to about 28 hours, about 22 hours to about 30 hours, about 22 hours to about 32 hours, about 22 hours to about 34 hours, about 22 hours to about 36 hours, about 24 hours to about 26 hours, about 24 hours to about 28 hours, about 24 hours to about 30 hours, about 24 hours to about 32 hours, about 24 hours to about 34 hours, about 24 hours to about 36 hours, about 26 hours to about 28 hours, about 26 hours to about 30 hours, about 26 hours to about 32 hours, about 26 hours to about 34 hours, about 26 hours to about 36 hours, about 28 hours to about 30 hours, about 28 hours to about 32 hours, about 28 hours to about 34 hours, about 28 hours to about 36 hours, about 30 hours to about 32 hours, about 30 hours to about 34 hours, about 30 hours to about 36 hours, about 32 hours to about 34 hours, about 32 hours to about 36 hours, or about 34 hours to about 36 hours.
In yet other embodiments, a method comprising a pre-enrichment step disclosed herein and an enrichment step disclosed herein can be performed to completion in, e.g., about 18 hours to about 20 hours, about 18 hours to about 22 hours, about 18 hours to about 24 hours, about 18 hours to about 26 hours, about 18 hours to about 28 hours, about 18 hours to about 30 hours, about 18 hours to about 32 hours, about 18 hours to about 34 hours, about 18 hours to about 36 hours, about 20 hours to about 22 hours, about 20 hours to about 24 hours, about 20 hours to about 26 hours, about 20 hours to about 28 hours, about 20 hours to about 30 hours, about 20 hours to about 32 hours, about 20 hours to about 34 hours, about 20 hours to about 36 hours, about 22 hours to about 24 hours, about 22 hours to about 26 hours, about 22 hours to about 28 hours, about 22 hours to about 30 hours, about 22 hours to about 32 hours, about 22 hours to about 34 hours, about 22 hours to about 36 hours, about 24 hours to about 26 hours, about 24 hours to about 28 hours, about 24 hours to about 30 hours, about 24 hours to about 32 hours, about 24 hours to about 34 hours, about 24 hours to about 36 hours, about 26 hours to about 28 hours, about 26 hours to about 30 hours, about 26 hours to about 32 hours, about 26 hours to about 34 hours, about 26 hours to about 36 hours, about 28 hours to about 30 hours, about 28 hours to about 32 hours, about 28 hours to about 34 hours, about 28 hours to about 36 hours, about 30 hours to about 32 hours, about 30 hours to about 34 hours, about 30 hours to about 36 hours, about 32 hours to about 34 hours, about 32 hours to about 36 hours, or about 34 hours to about 36 hours.
In still other embodiments, a method comprising a pre-enrichment step disclosed herein, an enrichment step disclosed herein and a detection step disclosed herein can be performed to completion in, e.g., about 18 hours to about 20 hours, about 18 hours to about 22 hours, about 18 hours to about 24 hours, about 18 hours to about 26 hours, about 18 hours to about 28 hours, about 18 hours to about 30 hours, about 18 hours to about 32 hours, about 18 hours to about 34 hours, about 18 hours to about 36 hours, about 20 hours to about 22 hours, about 20 hours to about 24 hours, about 20 hours to about 26 hours, about 20 hours to about 28 hours, about 20 hours to about 30 hours, about 20 hours to about 32 hours, about 20 hours to about 34 hours, about 20 hours to about 36 hours, about 22 hours to about 24 hours, about 22 hours to about 26 hours, about 22 hours to about 28 hours, about 22 hours to about 30 hours, about 22 hours to about 32 hours, about 22 hours to about 34 hours, about 22 hours to about 36 hours, about 24 hours to about 26 hours, about 24 hours to about 28 hours, about 24 hours to about 30 hours, about 24 hours to about 32 hours, about 24 hours to about 34 hours, about 24 hours to about 36 hours, about 26 hours to about 28 hours, about 26 hours to about 30 hours, about 26 hours to about 32 hours, about 26 hours to about 34 hours, about 26 hours to about 36 hours, about 28 hours to about 30 hours, about 28 hours to about 32 hours, about 28 hours to about 34 hours, about 28 hours to about 36 hours, about 30 hours to about 32 hours, about 30 hours to about 34 hours, about 30 hours to about 36 hours, about 32 hours to about 34 hours, about 32 hours to about 36 hours, or about 34 hours to about 36 hours.
In other embodiments, a method comprising a pre-enrichment step disclosed herein, an enrichment step disclosed herein, a purification step disclosed herein, and a detection step disclosed herein can be performed to completion in, e.g., about 18 hours to about 20 hours, about 18 hours to about 22 hours, about 18 hours to about 24 hours, about 18 hours to about 26 hours, about 18 hours to about 28 hours, about 18 hours to about 30 hours, about 18 hours to about 32 hours, about 18 hours to about 34 hours, about 18 hours to about 36 hours, about 20 hours to about 22 hours, about 20 hours to about 24 hours, about 20 hours to about 26 hours, about 20 hours to about 28 hours, about 20 hours to about 30 hours, about 20 hours to about 32 hours, about 20 hours to about 34 hours, about 20 hours to about 36 hours, about 22 hours to about 24 hours, about 22 hours to about 26 hours, about 22 hours to about 28 hours, about 22 hours to about 30 hours, about 22 hours to about 32 hours, about 22 hours to about 34 hours, about 22 hours to about 36 hours, about 24 hours to about 26 hours, about 24 hours to about 28 hours, about 24 hours to about 30 hours, about 24 hours to about 32 hours, about 24 hours to about 34 hours, about 24 hours to about 36 hours, about 26 hours to about 28 hours, about 26 hours to about 30 hours, about 26 hours to about 32 hours, about 26 hours to about 34 hours, about 26 hours to about 36 hours, about 28 hours to about 30 hours, about 28 hours to about 32 hours, about 28 hours to about 34 hours, about 28 hours to about 36 hours, about 30 hours to about 32 hours, about 30 hours to about 34 hours, about 30 hours to about 36 hours, about 32 hours to about 34 hours, about 32 hours to about 36 hours, or about 34 hours to about 36 hours.
Aspects of the present specification disclose, in part, a pathogen analysis kit to carry out the method of detecting a pathogen disclosed herein. A pathogen analysis kit disclosed herein contains components necessary for the detection of the pathogen of interest. In some aspects, a pathogen analysis kit disclosed herein components necessary for the detection of a single pathogen of interest. In some aspects, a pathogen analysis kit disclosed herein components necessary for the detection of a plurality of pathogens of interest.
In some embodiment, a pathogen analysis kit disclosed herein typically comprises a pre-enrichment media and an enrichment media. In some embodiment, a pathogen analysis kit disclosed herein typically comprises a pre-enrichment media, an enrichment media, and a detection solution. In some embodiment, a pathogen analysis kit disclosed herein typically comprises a pre-enrichment media, an enrichment media, a detection solution and an electrochemical biosensor.
In some embodiment, a pathogen analysis kit disclosed herein typically comprises a pre-enrichment media, an enrichment media, and immunmagnetic particles capable of binding to a pathogen of interest. In some embodiment, a pathogen analysis kit disclosed herein typically comprises a pre-enrichment media, an enrichment media, immunmagnetic particles capable of binding to a pathogen of interest, and a magnetic source used to capture the immunoparticles. In some embodiment, a pathogen analysis kit disclosed herein typically comprises a pre-enrichment media, an enrichment media, immunmagnetic particles capable of binding to a pathogen of interest, and a detection solution. In some embodiment, a pathogen analysis kit disclosed herein typically comprises a pre-enrichment media, an enrichment media, immunmagnetic particles capable of binding to a pathogen of interest, a magnetic source used to capture the immunoparticles, and a detection solution. In some embodiment, a pathogen analysis kit disclosed herein typically comprises a pre-enrichment media, an enrichment media, immunmagnetic particles capable of binding to a pathogen of interest, a detection solution, and an electrochemical biosensor. In some embodiment, a pathogen analysis kit disclosed herein typically comprises a pre-enrichment media, an enrichment media, immunmagnetic particles capable of binding to a pathogen of interest, a detection solution, a magnetic source used to capture the immunoparticles, and an electrochemical biosensor.
A pathogen analysis kit disclosed herein may further comprise an instrument which is capable of measuring and/or analyzing potentiometric, voltammetric, amperometric and/or impedance/conductance parameters.
A pathogen analysis kit disclosed herein may further comprise a suitable container, for example, a vessel, vials, tubes, mini- or microfuge tubes, test tube, flask, bottle, syringe or other container. Where an additional component or agent is provided, the kit can contain one or more additional containers into which this agent or component may be placed. Kits herein will also typically include a means for containing the agent (e.g. a vessel), composition and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.
A pathogen analysis kit disclosed herein may further comprise a labels or inserts. Labels or inserts include “printed matter,” e.g., paper or cardboard, or separate or affixed to a component, a kit or packing material (e.g., a box), or attached to an ampule, tube or vial containing a kit component. Labels or inserts can additionally include a computer readable medium, such as a disk (e.g., hard disk, flash memory), optical disk such as CD- or DVD-ROM/RAM, DVD, MP3, magnetic tape, or an electrical storage media such as RAM and ROM or hybrids of these such as magnetic/optical storage media, FLASH media or memory type cards. Labels or inserts may include identifying information of one or more components therein, amounts used for one or more components therein, step by step instructions of how to perform a method of detecting a pathogen of interest. Labels or inserts can include information identifying manufacturer information, lot numbers, manufacturer location and date and patent information.
Aspects of the present specification may also be described as follows:

    1. A method of detecting a pathogen in a sample the method comprising the steps: a) incubation of the sample in a pre-enrichment comprising a low growth nutrient component, a growth inhibiting agent, and a surfactant with bacteriostatic or bactericidal action, wherein the incubation at 37° C. for about 6 hours to about 24 hours; b) incubating an aliquot of pre-enrichment media from step (a) in an enrichment media, the enrichment media comprising a high growth nutrient component and ferric ammonium citrate, wherein the incubation at about 15° C. to about 50° C. for about 2 hours to about 24 hours; and c) detecting the presence of absence of a pathogen by analyzing an aliquot of enrichment media from step (b).
    2. The method according to embodiment 1, wherein the low growth nutrient component is a peptone.
    3. The method according to embodiment 2, wherein the peptone is a peptone from an animal source or a peptone from a plant source.
    4. The method according to embodiment 3, wherein the peptone from an animal source is an acid casein peptone, a bacteriological peptone, a beef extract powder, a casein peptone, a casein cc peptone, a gelatin peptone, a meat peptone, a polypeptone proteose peptone, or a proteose peptone No 3.
    5. The method according to embodiment 3, wherein the peptone from a plant source is a malt extract, a soya peptone, or a yeast extract.
    6. The method according to any one of embodiments 1-5, wherein the surfactant is at a concentration of about 0.001 (v/v) to about 10.0% (v/v).
    7. The method according to any one of embodiments 1-6, wherein the surfactant is an ionic surfactant, a zwitterionic (amphoteric) surfactant, or a non-ionic surfactant.
    8. The method according to embodiments 7, wherein the ionic surfactant is an anion surfactant or cationic surfactant.
    9. The method according to embodiments 8, wherein the anionic surfactant is an alkyl sulfate, an alkyl ether sulfate, a docusate, a sulfonate fluorosurfactant, an alkyl benzene sulfonate, an alkyl aryl ether phosphate, an alkyl ether phosphate, an; alkyl carboxylate, a sodium lauroyl sarcosinate, or a carboxylate fluorosurfactant.
    10. The method according to embodiments 8, wherein the cationic surfactant is an alkyltrimethylammonium salt, cetylpyridinium chloride (CPC), polyethoxylated tallow amine (POEA), benzalkonium chloride (BAC), benzethonium chloride (BZT), 5-bromo-5-nitro-1,3-dioxane, dimethyldioctadecylammonium chloride, dioctadecyldimethylammonium bromide (DODAB), a pH-dependent primary amine, a pH-dependent secondary amine, or a pH-dependent tertiary amine.
    11. The method according to embodiments 7, wherein the zwitterionic surfactant is 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), a sultaine, a, betaine, or a lecithin.
    12. The method according to embodiments 7, wherein the non-ionic surfactant is a polyoxyethylene glycol sorbitan alkyl ester, a poloxamer, an alkyl phenol polyglycol ether, a polyethylene glycol alkyl aryl ether, a polyoxyethylene glycol alkyl ether, 2-dodecoxyethanol, a polyoxyethylene glycol octyl phenol ether, a nonylphenol ethoxylate, a polyoxyethylene glycol alkylphenol ether, a phenoxypolyethoxylethanol, a glucoside alkyl ether, a maltoside alkyl ether, a thioglucoside alkyl ether, a digitonin, a glycerol alkyl ester, an alkyl aryl polyether sulfate, an alcohol sulfonate, a sorbitan alkyl ester, a cocamide ethanolamine, sucrose monolaurate, dodecyl dimethylamine oxide, or sodium cholate.
    13. The method according to any one of embodiments 1-6, where the surfactant is tetradecyl sodium dodecyl sulfate, octadecyl sulphate, or 7-ethyl-2-methyl-4-undecyl sodium sulphate.
    14. The method according to any one of embodiments 1-13, where the growth inhibiting agent is at a concentration of about 0.001 (v/v) to about 10.0% (v/v).
    15. The method according to any one of embodiments 1-14, wherein the growth inhibiting agent is a triarylmethane dye.
    16. The method according to embodiment 15, where the triarylmethane dye is a methyl violet dye, a fuchsine dye, a phenol dye, or a malachite green dye.
    17. The method according to embodiment 16, where the methyl violet dye is methyl violet 2B, methyl violet 6B, or methyl violet 10B.
    18. The method according to embodiment 16, where the fuchsine dye is pararosaniline, fuchsine, new fuchsine, fuchsin basic violet, or fuchine acid.
    19. The method according to embodiment 16, where the phenol dye is phenol red, chlorophenol red, or cresol red.
    20. The method according to embodiment 16, where the malachite green dye is malachite green or brilliant green.
    21. The method according to embodiment 15, where the triarylmethane dye is aluminon, aniline Blue WS, aurin, aurintricarboxylic acid, brilliant blue FCF, brilliant green, bromocresol green, bromocresol purple, bromophenol blue, bromopyrogallol red, bromothymol blue, bromsulphthalein, chlorophenol red, coomassie brilliant blue, cresol red, crystal violet, crystal violet lactone, ethyl green, fast green FCF, fluoran, fuchsine, fuchsine acid, fuchsin basic violet, gentian, green S, light green SF yellowish, malachite green, methyl blue, methyl violet, new fuchsine, pararosaniline, patent blue V, phenol red, phenolphthalein, rose bengal, thymolphthalein, victoria blue BO, water blue, xylene cyanol, or xylenol orange.
    22. The method according to any one of embodiments 1-22, wherein the pre-enrichment media further comprises a growth enhancing agent.
    23. The method according to embodiment 22, wherein the growth enhancing agent is a siderophore.
    24. The method according to embodiment 23, wherein the siderophore is Aerobactin, Alcaligin, Azotobactin, Bacillibactin, Desferrioxamine B, Desferrioxamine E, Enterobactin, Ferrichrome, Ferrioxiamina-B, Ferrioxiamina-E, Fusarinine C, Mycobactin, Ornibactin, Petrobactin, Pyoverdine, Pyochelin, Salmochelin, Staphyloferring A, Vibriobactin, or Yersiniabactin.
    25. The method according to any one of embodiments 1-24, wherein the aliquot of pre-enrichment media in step (b) is about 1/50 to about 1/500 a volume of an enrichment media used in step (b).
    26. The method according to any one of embodiments 1-25, wherein the high growth nutrient component is a peptone.
    27. The method according to embodiment 26, wherein the peptone is a peptone from an animal source or a peptone from a plant source.
    28. The method according to embodiment 27, wherein the peptone from an animal source is an acid casein peptone, a bacteriological peptone, a beef extract powder, a casein peptone, a casein cc peptone, a gelatin peptone, a meat peptone, a polypeptone proteose peptone, or a proteose peptone No 3.
    29. The method according to embodiment 27, wherein the peptone from a plant source is a malt extract, a soya peptone, or a yeast extract.
    30. The method according to any one of embodiments 1-29, wherein the high growth nutrient component is at a concentration of about 1.0 (v/v) to about 4.0% (v/v).
    31. The method according to any one of embodiments 1-29, wherein the ammonium ferric citrate is at a concentration of about 0.1 mg/mL to about 15 mg/mL.
    32. The method according to any one of embodiments 1-31, wherein the enrichment media further comprises a growth enhancing agent.
    33. The method according to embodiment 32, wherein the growth enhancing agent is a siderophore.
    34. The method according to embodiment 33, wherein the siderophore is Aerobactin, Alcaligin, Azotobactin, Bacillibactin, Desferrioxamine B, Desferrioxamine E, Enterobactin, Ferrichrome, Ferrioxiamina-B, Ferrioxiamina-E, Fusarinine C, Mycobactin, Ornibactin, Petrobactin, Pyoverdine, Pyochelin, Salmochelin, Staphyloferring A, Vibriobactin, or Yersiniabactin.
    35. The method according to any one of embodiments 1-34, wherein detection step (c) is performed using a sensor-based detection method, a nucleic acid-based detection method, a protein-based detection method, an activity-based detection method, or a growth-based detection method.
    36. The method according to embodiment 35, wherein the sensor-based detection method is an electrochemical detection method.
    37. The method according to embodiment 36, wherein the electrochemical detection method comprises an enzymatic biosensor, a DNA sensor, or an immunosensor.
    38. The method according to embodiment 36, wherein the electrochemical detection method measures potentiometric parameters, amperometric parameters, voltametric parameters, impedimetric/conductimetric parameters, or any combination thereof.
    39. The method according to embodiment 35, wherein the nucleic acid-based detection method comprises a DNA-based detection method or an RNA-based detection method.
    40. The method according to embodiment 39, wherein the DNA-based detection method comprises a Southern blot analysis, a PCR-based assay, a sequence analysis, an immuno-based detection assay, or a hybridization assays using FRET, polarization or other fluorescent, chemiluminescent or bioiluminescent detection.
    41. The method according to embodiment 40, wherein the PCR-based assay comprises a real-time PCR-based assay.
    42. The method according to embodiment 39, wherein the RNA-based detection method comprises a Northern blot analysis, a RT-PCR-based assay, a RNA sequence analysis, an immuno-based detection assay, or a hybridization assays using FRET, polarization or other fluorescent, chemiluminescent or bioiluminescent detection.
    43. The method according to embodiment 35, wherein the protein-based detection method is a gel-based detection method, an immuno-based detection method or a protein-interaction-based method.
    44. The method according to embodiment 43, wherein the immuno-based detection method comprises a Western blot analysis, an ELISA, or an immunoprecipitation assay.
    45. The method according to embodiment 35, wherein the activity-based detection method comprises an enzymatic activity assay or an assay based on protein function.
    46. The method according to embodiment 45, wherein the enzymatic activity assay uses a spetrophotometric detection method measures a disappearance of a substrate or a formation of a product.
    47. The method according to embodiment 35, wherein the growth-based detection method comprises a plating assays measuring colony formation or spectrophotometer assays measuring cell density.
    48. The method according to any one of embodiments 1-47, wherein the pathogen is a prion, a virus, a bacterium, a fungus, a protozoan, a helimenth, or a parasite.
    49. The method according to embodiment 48, wherein the virus belongs to the family Adenoviridae, Picornaviridae, Herpesviridae, Hepadnaviridae, Flaviviridae, Retroviridae, Orthomyxoviridae, Paramyxoviridae, Papovaviridae, Polyomavirus, Rhabdoviridae, or Togaviridae.
    50. The method according to embodiment 48, wherein the bacteria belongs to the genus Bacillus, Bordetella, Borrelia, Brucella, Campylobacter, Chlamydia, Chlamydophila, Clostridium, Corynebacterium, Enterobacter, Enterococcus, Escherichia, Francisella, Haemophilus, Helicobacter, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Treponema, Vibrio, or Yersinia. 
    51. The method according to embodiment 48, wherein the fungus belongs to the genus Asperfillus, Canidia, Cryptococcus, Histoplasma, Pneumocystis, or Stachybotrys. 
    52. The method according to embodiment 48, wherein the protozoan belongs to the genus Acanthamoeba, Balamuthia, Cryptosporidium, Dientamoeba, Endolimax, Entamoeba, Giardia, Iodamoeba, Leishmania, Naegleria, Plasmodium, Sappinia, Toxoplasma, Trichomonas, or Trypanosoma. 
    53. A pathogen analysis kit comprising a pre-enrichment media and an enrichment media.
    54. The pathogen analysis kit according to embodiment 53, further comprising a detection solution.
    55. The pathogen analysis kit according to embodiment 53 or embodiment 54, further and an electrochemical biosensor.
    56. The pathogen analysis kit according to any one of embodiments 53-55, further comprising immunmagnetic particles capable of binding to a pathogen of interest.
    57. The pathogen analysis kit according to any one of embodiments 53-56, further comprising a magnetic source used to capture the immunoparticles.
    58. The pathogen analysis kit according to any one of embodiments 53-57, further comprising an instrument which is capable of measuring and/or analyzing potentiometric, voltammetric, amperometric and/or impedance/conductance parameters.
    59. The pathogen analysis kit according to any one of embodiments 53-58, further comprising a suitable container.
    60. The pathogen analysis kit according to any one of embodiments 53-59, further comprising a label or an insert.


EXAMPLES
The following non-limiting examples are provided for illustrative purposes only in order to facilitate a more complete understanding of the disclosed subject matter. These examples should not be construed to limit any of the embodiments described in the present specification, including those pertaining to the methods disclosed, the type of detection employed or the type of pathogen that can be detected using the methods disclosed herein.
Example 1
Preparation of Sensors and Electrochemical Readings
Enzyme-based electrochemical sensors were manufactured using a screen printing process by depositing a series of layers of different material on a substrate material. Initially, a layer of compound paste comprising silver is deposited directly onto a polyester substrate (MYLAR® A (500 gauge); DuPont E. I. De Nemours & Co., Wilmington, Del.). A layer of silver/silver chloride, which functions as both the counter electrode and reference electrode, is then deposited over the compound paste on one side to form a wall. The third layer comprised carbon graphite (Gwent Electronic Materials Ltd., Pontypool, UK) which functions as the working electrode is deposited on the other side to form an opposing wall. A channel is thus formed inbetween the silver/silver chloride wall and the carbon graphite wall. A layer of insulating paste (Gwent Electronic Materials Ltd., Pontypool, UK) is then deposited over the silver/silver chloride and carbon graphite layers and a pressure-sensitive adhesive (KIWO, Inc., Seabrook, Tex.) is then deposited over this insulating paste layer. A sensor is then modified by adding a biological layer within the channel by incubating the sensor in an atmosphere saturated with 5% glutaraldehyde to 5% for 5 minutes at room temperature in the presence of an enzyme solution comprising glucose dehydrogenase (GDH), which is then dried for 30 minutes at 37° C. Finally, a layer of polyester (MELINEX®; DuPont E. I. De Nemours & Co., Wilmington, Del.) is placed on top of the insulating paste layer to cover the sensor and enclose the channel. The sensor is then heated at 60° C. and pressure applied using a pressure roller to set the polyester layer. The sensor is stored in a dry place at 4° C. until needed.
Presence of the desired pathogen was detected by measuring the electrochemical signal produced as a result of a redox reaction associated with the conversion of glucose to gluconic acid. In this detection procedure, an aliquot of the enriched pathogen culture was added to a detection solution comprising p-aminophenyl phosphate (PAPH) and glucose. Alternatively, alfanaphtylphosphate may be used instead of PAPH. A sensor as described above was inserted into this solution and electrochemical signals were detected amperometrically under an applied potential of about 200 mV using a PalmSens3 (a potentiostat, galvanostat, and impedance analyzer) and its accompanying software PSTrace (PalmSens BV, Utrecht, Netherlands).
Briefly, the mechanism underlying the generation of an electrochemical signal is as follows. Pathogens detected using this procedure synthesize the enzyme alkaline phosphatase (ALP), which is subsequently released into the culture media. Addition of an enriched pathogen culture comprising ALP results in the hydrolysis PAPH into PAP. GDH, immobilized in the biological layer of the sensor, catalyzes the conversion of glucose into gluconic acid, which is associated with a redox reaction that oxidizes PAP into PIQ. The electrochemical signals detected by the sensor occurs when it measures the electrons generated when PIQ is reduced back to PAP.
Example 2
Relationship Between Measured Electrochemical Signal, Bacterial Population and Incubation Time
Generation of an electrochemical signal and its relationship with the population size of bacteria was determined by measuring the current generated by populations comprising different concentrations of bacteria. A series of 50 μL detection solutions, each comprising 500 mM acetate buffer (pH 5.7), 10 mM magnesium chloride, 1.0 mM PAPH and 10 mM glucose, was set up. Each detection solution was then mixed with a 1 mL solution of phospho-buffered saline comprising bacteria (Salmonella typhimurium) at one of the following concentrations: 1×102 cfu/mL, 1×103 cfu/mL, 1×104 cfu/mL, 1×105 cfu/mL, 1×106 cfu/mL, 1×107 cfu/mL, or 1×108 cfu/mL. The detection solution was then incubated at 37° C. for 30 minutes. The electrochemical signal for each solution was detected amperometrically under an applied potential of about 200 mV using a PalmSens3 as described above. As shown in FIG. 1, a bacterial concentration of about 1×104 cfu/mL produced a detectable signal of about 0.5 μA. In addition, a linear concentration-response curve was observed in a range of about 1×105 cfu/mL to about 1×107 cfu/mL. These results indicate that and average bacterial concentration of about 1×104 cfu/mL can be detected using the electrochemical detection method disclosed herein.
Generation of an electrochemical signal and its relationship with the incubation time was determined by measuring the current generated by a bacterial population incubated over time. A 225 mL culture media comprising buffered peptone and water was inoculated with 10 cfu/mL of bacteria (S. typhimurium) and incubation at 37° C. over time. A 100 μL aliquot of this inoculated media was taken every two hours and added to a 50 μL detection solution comprising 500 mM acetate buffer (pH 5.7), 10 mM magnesium chloride, 1.0 mM PAPH and 10 mM glucose. The detection solution was then incubated at 37° C. for 30 minutes. The electrochemical signal for each solution was detected amperometrically under an applied potential of about 200 mV using a PalmSens3 as described above. As shown in FIG. 2, a detectable signal of about 0.5 μA was detected after 4 hours of incubation. A linear time-response curve was observed in a range of about 6 hours to about 8 hours. These results indicate that and average anodic current of >0.5 μA is indicative of the presence of bacteria.
Example 3
Determination of the Presence of Salmonella in Pure Culture
Generation of an electrochemical signal by a bacteria from a pure sample was determined by measuring the current generated by a defined bacterial population of two different concentrations. A 225 mL pre-enrichment culture media comprising buffered peptone and 0.4% (w/v) Tergitol-4 was inoculated with either 20 cfu or 200 cfu of bacteria (S. typhimurium), mixed vigorously for 10 seconds, and incubation at 37° C. for about 14 to about 17 hours.
After this pre-enrichment step, 1 mL of the pre-enrichment culture was taken for each bacterial concentration and a 10 μL aliquot was added to 990 μL of an enrichment culture media comprising 2.72% (w/v) of Rappaport-Vassiliadis Soya (pH 5.2) comprising 4.5 g/L soya peptone, 7.2 g/L sodium chloride, 1.26 g/L potassium dihydrogen phosphate, 0.18 mg/L dipotassium phosphate, 13.6 mg/L magnesium chloride and 0.036 g/L malachite green (Cultimed), supplemented with 0.62 mg/mL of ammonium ferric citrate (Sigma-Aldrich Corp., St. Louis, Mo.). The enrichment culture for both concentrations were incubated for about 5 to about 7 hours at 41.5° C. with circular stirring. After this enrichment step, 20 μL of a solution 3.0×108 anti-Salmonella immunomagnetic particles/mL (DYNABEADS® Anti-Salmonella, Life Technologies, Inc., Carlsbad, Calif.) was added to the enrichment cultures and incubated for 30 minutes at room temperature with agitation circular. After this incubation time, the enrichment cultures were contacted with a magnet for 3 minutes in order to localize the anti-Salmonella immunomagnetic particles within the container tube and the supernatant was extracted using 1 mL micropipette. The supernatant was then added to 1 mL of 100 mM phosphate buffer, mixed for 10 seconds, contacted with a magnet for 3 minutes and the washed supernatant was extracted using 1 mL micropipette. The processed supernatant was then tested to detect the presence of the bacteria using two different assays: 1) a plating assay; and 2) an electrochemical detection assay disclosed herein.
To detect the presence of bacteria using a plating assay, a 100 μL aliquot of the processed supernatant was mixed into 500 μL of 100 mM buffer phosphate (pH 7.4) and this mixture was plated on agar comprising a selective chromogenic agent (Chromosalm MICROKIT®, Laboratorios MICROKIT, S.L.). The agar plates were then incubated at 37° C. for about 15 hours and identifying of colored bacterial colonies determined. As shown in Table 1, growth of chromogenic bacterial colonies was observed from a supernatant sample derived from both the 20 cfu and 200 cfu of pure inoculum cultures. These results indicate that the growth methods disclosed herein in conjunction with a plating assay can effectively detect the presence of a pathogen.







TABLE 1







Detection of Salmonella in pure inoculum sample










Plating Method
Electrochemical method












Ex-

Pure
Pure
Pure
Pure


peri-

inoculum
inoculum
inoculum
inoculum


ment
n
20 cfu
200 cfu
20 cfu
200 cfu





1
4
Growth
Growth
2.37 ± 0.52 μA
2.45 ± 0.58 μA









To detect the presence of bacteria using a electrochemical detection assay, a 100 μL aliquot of the processed supernatant was added to a 50 μL detection solution comprising 500 mM acetate buffer (pH 5.7), 10 mM magnesium chloride, 1.0 mM PAPH and 10 mM glucose. The detection solution was then incubated at 37° C. for 30 minutes. The electrochemical signal for each solution was detected amperometrically under an applied potential of about 200 mV verse Ag/AgCl for 30 seconds using a PalmSens3 as described above. As shown in Table 1, a current above 2 μA was detected from a supernatant sample derived from both the 20 cfu and 200 cfu of pure inoculum cultures. These results indicate that the growth methods disclosed herein in conjunction with an electrochemical detection assay disclosed herein can effectively detect the presence of a pathogen.
Example 4
Determination of the Presence of Salmonella in Fecal Sample
Generation of an electrochemical signal by a bacteria from a sample material was determined by measuring the current generated from fecal material. Twenty-five gram samples of fecal material, each suspected to contain bacteria (S. typhimurium), were added into a 225 mL pre-enrichment culture media comprising buffered peptone and 0.4% Tergitol-4 and incubation at 37° C. for about 14 to about 15 hours. Fecal material samples proven not to contain bacteria (S. typhimurium) were also tested as a negative control. The procedures regarding pre-enrichment, enrichment, and immunoseparation for Experiment 1 in Table 2 were all performed as described above in Example 3. In addition, detection of the presence of the bacteria using a plating assay and an electrochemical detection assay disclosed herein were performed as described above in Example 3. For experiments 2 and 3 in Table 1, the immunoseparation step was not performed. In these experiments, after this enrichment step, a 1 mL aliquot of enriched culture medium was centrifuged and supernatant was extracted using 1 mL micropipette. The supernatant was then added to 1 mL of 100 mM phosphate buffer, mixed for 10 seconds, centrifuged, and the washed supernatant was extracted using 1 mL micropipette. The processed supernatant was then tested to detect the presence of the bacteria using a plating assay and an electrochemical detection assay disclosed herein as described above in Example 3.
As shown in Table 2, growth of chromogenic bacterial colonies was observed from a supernatant sample derived from fecal samples contaminated with bacteria (S. typhimurium) while fecal samples not contaminated with bacteria (S. typhimurium) did not exhibit any chromogenic bacterial colonies. In addition, currents above 2 μA were detected from supernatant samples derived from fecal samples contaminated with bacteria (S. typhimurium) (Table 2). However, as shown in Table 2, currents below 0.5 μA were measured from fecal samples not contaminated with bacteria (S. typhimurium). These results indicate that a fecal sample contaminated with a pathogen can be identified and distinguished from a fecal sample not contaminated with a pathogen using the methods disclosed herein.







TABLE 2







Detection of Salmonella in fecal sample










Ex-





peri-

Plating Assay
Electrochemical Detection Assay












ment
n
Fecal (+)
Fecal (−)
Fecal (+)
Fecal (−)





1
4
Growth
No Growth
1.97 ± 0.62 μA
0.30 ± 0.11 μA


2
4
Growth
No Growth
2.36 ± 0.20 μA
0.25 ± 0.08 μA


3
4
Growth
No Growth
3.33 ± 0.53 μA
0.36 ± 0.08 μA









Example 5
Determination of the Presence of Salmonella in Chicken Skin Sample
Generation of an electrochemical signal by a bacteria from a sample material was determined by measuring the current generated from skin samples from chickens. Twenty-five gram samples of chicken skin, each suspected to contain bacteria (S. typhimurium), were added into a 225 mL pre-enrichment culture media comprising buffered peptone, 0.4% (v/v) brilliant green, 0.4% (w/v) Tergitol-4, mixed vigorously for 10 seconds, and then incubation at 37° C. for about 14 to about 15 hours. Chicken skin samples proven not to contain bacteria (S. typhimurium) were also tested as a negative control. The procedures regarding pre-enrichment, enrichment, and immunoseparation for Experiment 1 in Table 2 were all performed as described above in Example 3. In addition, detection of the presence of the bacteria using a plating assay and an electrochemical detection assay disclosed herein were performed as described above in Example 3. For Experiment 2 in Table 1, the immunoseparation step was not performed. In this experiment, after this enrichment step, a 1 mL aliquot of enriched culture medium was centrifuged and supernatant was extracted using 1 mL micropipette. The supernatant was then added to 1 mL of 100 mM phosphate buffer, mixed for 10 seconds, centrifuged, and the washed supernatant was extracted using 1 mL micropipette. The processed supernatant was then tested to detect the presence of the bacteria using a plating assay and an electrochemical detection assay disclosed herein as described above in Example 3.
As shown in Table 3, growth of chromogenic bacterial colonies was observed from a supernatant sample derived from chicken skin samples contaminated with bacteria (S. typhimurium) while chicken skin samples not contaminated with bacteria (S. typhimurium) did not exhibit any chromogenic bacterial colonies. In addition, currents above 2 μA were detected from supernatant samples derived from chicken skin samples contaminated with bacteria (S. typhimurium) (Table 2). However, as shown in Table 3, currents below 0.5 μA were measured from chicken skin samples not contaminated with bacteria (S. typhimurium). These results indicate that a chicken skin sample contaminated with a pathogen can be identified and distinguished from a chicken skin sample not contaminated with a pathogen using the methods disclosed herein.







TABLE 3







Detection of Salmonella in chicken skin sample










Ex-





peri-

Plating Assay
Electrochemical Detection Assay












ment
n
Skin (+)
Skin (−)
Skin (+)
Skin (−)





1
4
Growth
No Growth
1.84 ± 0.61 μA
0.24 ± 0.10 μA


2
4
Growth
No Growth
2.35 ± 0.49 μA
0.35 ± 0.22 μA









Example 6
Determination of the Presence of Salmonella in Foodstuff Sample
Generation of an electrochemical signal by a bacteria from a sample material was determined by measuring the current generated from foodstuff samples of corn. Twenty-five gram samples of chicken skin, each suspected to contain bacteria (S. typhimurium), were added into a 225 mL pre-enrichment culture media comprising buffered peptone, 0.4% (v/v) brilliant green, 0.4% (w/v) Tergitol-4, mixed vigorously for 10 seconds, and then incubation at 37° C. for about 14 to about 15 hours. Foodstuff samples proven not to contain bacteria (S. typhimurium) were also tested as a negative control. The procedures regarding pre-enrichment and enrichment were all performed as described above in Example 3, however, the immunoseparation step was not performed. In this experiment, after this enrichment step, a 1 mL aliquot of enriched culture medium was centrifuged and supernatant was extracted using 1 mL micropipette. The supernatant was then added to 1 mL of 100 mM phosphate buffer, mixed for 10 seconds, centrifuged, and the washed supernatant was extracted using 1 mL micropipette. The processed supernatant was then tested to detect the presence of the bacteria using a plating assay and an electrochemical detection assay disclosed herein as described above in Example 3.
As shown in Table 4, growth of chromogenic bacterial colonies was observed from a supernatant sample derived from foodstuff samples contaminated with bacteria (S. typhimurium) while foodstuff samples not contaminated with bacteria (S. typhimurium) did not exhibit any chromogenic bacterial colonies. In addition, currents above 2 μA were detected from supernatant samples derived from foodstuff samples contaminated with bacteria (S. typhimurium) (Table 2). However, as shown in Table 4, currents below 0.5 μA were measured from foodstuff samples not contaminated with bacteria (S. typhimurium). These results indicate that a foodstuff sample contaminated with a pathogen can be identified and distinguished from a foodstuff sample not contaminated with a pathogen using the methods disclosed herein.







TABLE 4







Detection of Salmonella in foodstuff sample










Ex-





peri-

Plating Assay
Electrochemical Detection Assay












ment
n
Foodstuff (+)
Foodstuff (−)
Foodstuff (+)
Foodstuff (−)





1
4
Growth
No Growth
2.38 ± 0.62 μA
0.62 ± 0.06 μA









Example 7
Determination of the Presence of Salmonella Using Spectrophotometer Assay
Generation of an electrochemical signal by a bacteria from a sample material was determined by measuring enzymatic activity using a spectrophotometer. Twenty-five gram samples of fecal material, each suspected to contain bacteria (S. typhimurium), were added into a 225 mL pre-enrichment culture media comprising buffered peptone and 0.4% Tergitol-4 and incubation at 37° C. for about 16 hours. Fecal material samples proven not to contain bacteria (S. typhimurium) were also tested as a negative control. The procedures regarding enrichment and immunopurification were performed as described above in Example 3, except that an aliquot of culture media was removed from the enrichment culture at each hour for the first eight hours post-inoculation and then again at 24 hours post-inoculation. The immunomagnetic particles were resuspended in 50 μL of 200 mM phosphate buffer (pH 5.7) and incubated at room temperature overnight. The incubated sample was then contacted with a magnet for 3 minutes in order to localize the immunomagnetic particles within the container tube and the supernatant was extracted using 1 mL micropipette. The 50 μL aliquot of supernatant was then added to a well from a 96-well plate containing 50 μL of substrate solution comprising 5.4 mM p-Nitrophenol phosphate. A standard solution was also prepared and comprised 0.05 mM p-Nitrophenol in 0.5 N sodium hydroxide. Control wells were also established. Negative control wells included 50 μL of 200 mM phosphate buffer (pH 5.7) and 50 μL of substrate solution. Positive control wells included 2 μL of 0.5-3 units/mg of acid phosphatase, 48 μL of 200 mM phosphate buffer (pH 5.7) and 50 μL of substrate solution. The plates were incubated at 37° C. for 30 minutes and then a 200 μL stop solution of 0.5 N sodium hydroxide. In the presence of alkaline phosphatase, the p-Nitrophenol phosphate will be converted into p-Nitrophenol, resulting in a color change. The absorption at 405 nm was measured for each well using a spectrophotometer. The amount of alkaline phosphatase activity for each aliquot was determined based on the standard curve generated from the standard solution.
As shown in FIG. 3, a linear time-response curve was observed for the contaminated fecal samples for the first 6 hours with the signal then plateauing for the remainder of the time course. The blank and negative control showed no enzyme activity during the entire time course. These results indicate that the presence of bacteria can be detecting using a spectrophotometric measurements.
Example 8
Determination of the Presence of Salmonella Using PCR Assay
Generation of an electrochemical signal by a bacteria from a sample material was determined by measuring enzymatic activity using a spectrophotometer. Twenty-five gram samples of fecal material, each suspected to contain bacteria (S. typhimurium), were added into a 225 mL pre-enrichment culture media comprising buffered peptone and 0.4% Tergitol-4 and incubation at 37° C. for about 16 hours. Fecal material samples proven not to contain bacteria (S. typhimurium) were also tested as a negative control. The procedures regarding enrichment and immunopurification were performed as described above in Example 3. The immunomagnetic particles were resuspended in 200 μL of DNAase-free water and then heated for 30 minutes at 98° C. to lyse the cells. After centrifugation at 6000 rpm for 10 minutes at 4° C., the supernatant was transferred to a new tube. A 5 μL aliquot was added to a 15 μL PCR reaction mixture containing 10 μM of a forward and reverse primer specific for and diagnostic of S. typhimurium. PCR was performed as follows: 1 cycle at 95° C. for 10 minutes and up to 40 cycles of 95° C. for 30 seconds and 60° C. for 1 minute. The level of target DNA amplified during each cycle was measured by detecting fluorescence and recording the RFU. Alternatively, the assay was performed without an immunopurification step. In this case, after the incubation period for the enrichment step was completed, the culture was centrifugation at 6000 rpm for 10 minutes at 4° C., the supernatant was transferred to a new tube, and a 5 μL aliquot used for the PCR reaction.
As shown in FIG. 4A-C, detection of target DNA was initially observed after 20 cycles of amplification. Amplification of target DNA exhibited a sigmoidal curve that typically began plateauing by cycle 30. The negative control showed no detectable level of target DNA amplification during the entire time course. These results indicate that the presence of bacteria can be detecting using a PCR-based assay.
Example 9
Determination of the Presence of Other Organisms
Generation of an electrochemical signal by different bacteria was determined using three different detection solutions at various bacterial concentrations. A series of 50 μL detection solutions was established for three different pH values as follows: 1) 200 mM acetate buffer (pH 5.7), 10 mM magnesium chloride, 1.0 mM PAPH and 10 mM glucose; 2) 200 mM phosphate buffer (pH 7.0), 10 mM magnesium chloride, 1.0 mM PAPH and 10 mM glucose; or 3) 200 mM TRIS buffer (pH 9.8), 10 mM magnesium chloride, 1.0 mM PAPH and 10 mM glucose. Each detection solution was then mixed with a 1 mL solution of phospho-buffered saline comprising a bacteria at one of the following concentrations: 1×102 cfu/mL, 1×103 cfu/mL, 1×104 cfu/mL, 1×105 cfu/mL, 1×106 cfu/mL, 1×107 cfu/mL, or 1×108 cfu/mL. The bacteria tested were as follows: Campylobacter jejuni Cluster 1, Campylobacter jejuni ST45, Campylobacter coli, Escherichia coli, Salmonella typhimurium, Pseudomonas sp., Pseudomonas aureginosa, Enterobacter cloacae, MRSA, Proteus sp., Bacillus thuringiensis, Citrobacter sp., Shigella sp., Staphylococccus aureus, Klebsiella pneumoniae, and Enterococcus faecalis. After the bacteria was added, these detection solution was mixed for 10 seconds and then incubated at 37° C. for 30 minutes. Detection of the presence of the bacteria using an electrochemical detection assay disclosed herein were performed as described above in Example 3.
As shown in FIGS. 5-7, a bacterial concentration of about 1×104 cfu/mL produced a detectable signal of about 0.5 μA for all bacteria tested, although pH of the detection solution had an influence on the sensitivity of detection for certain bacterial species. In addition, a linear concentration-response curve was observed in a range of about 1×105 cfu/mL to about 1×107 cfu/mL. These results indicate that and average bacterial concentration of about 1×104 cfu/mL can be detected using the electrochemical detection method disclosed herein.







TABLE 5







Electrochemical detection of other pathogens












Organism
Intensity
Assay
pH 5.7
pH 7.0
pH 9.8





C. jejuni Cluster 1
>1 μA
cfu/mL
—
—
2.11 × 105




μA
—
—
1.46 ± 0.35



<1 μA
cfu/mL
4.94 × 106
1.00 × 107
2.11 × 104




μA
0.27 ± 0.05
0.70 ± 0.08
0.89 ± 0.07


C. jejuni ST45
>1 μA
cfu/mL
—
—
2.34 × 105




μA
—
—
1.54 ± 0.17



<1 μA
cfu/mL
8.33 × 105
2.34 × 106
2.34 × 104




μA
0.29 ± 0.06
0.18 ± 0.04
0.53 ± 0.07


C. coli
>1 μA
cfu/mL
—
—
1.53 × 105




μA
—
—
1.52 ± 0.28



<1 μA
cfu/mL
3.00 × 104
7.17 × 105
1.53 × 104




μA
0.24 ± 0.07
0.19 ± 0.04
0.62 ± 0.07


E. coli
>1 μA
cfu/mL
5.40 × 107
2.04 × 108
—




μA
3.25 ± 0.36
1.44 ± 0.45
—



<1 μA
cfu/mL
5.40 × 106
2.41 × 106
4.00 × 107




μA
0.74 ± 0.10
0.28 ± 0.00
0.73 ± 0.12


S. typhimurium
>1 μA
cfu/mL
1.72 × 106
1.65 × 106
3.39 × 107




μA
1.60 ± 0.26
1.26 ± 0.14
2.29 ± 0.42



<1 μA
cfu/mL
1.72 × 105
1.65 × 105
3.39 × 106




μA
0.41 ± 0.05
0.25 ± 0.01
0.47 ± 0.04


Pseudomonas sp.
>1 μA
cfu/mL
1.63 × 106
2.45 × 106
3.43 × 106




μA
2.46 ± 0.35
1.45 ± 0.15
1.85 ± 0.46



<1 μA
cfu/mL
1.63 × 105
2.45 × 105
3.43 × 105




μA
0.59 ± 0.03
0.47 ± 0.11
0.87 ± 0.05


P. aureginosa
>1 μA
cfu/mL
*
—
—




μA
*
—
—



<1 μA
cfu/mL
2.18 × 106
1.65 × 106
1.50 × 106




μA
0.93 ± 0.01
0.74 ± 0.01
0.77 ± 0.05


E. cloacae
>1 μA
cfu/mL
8.99 × 107
—
—




μA
1.37 ± 0.11
—
—



<1 μA
cfu/mL
8.99 × 106
9.88 × 107
1.28 × 108




μA
0.27 ± 0.02
0.25 ± 0.02
0.41 ± 0.05


MRSA
>1 μA
cfu/mL
1.01 × 106
3.64 × 106
—




μA
1.33 ± 0.29
1.19 ± 0.10
—



<1 μA
cfu/mL
1.01 × 105
3.64 × 105
3.23 × 107




μA
0.32 ± 0.04
0.33 ± 0.04
0.75 ± 0.06


Proteus sp.
>1 μA
cfu/mL
1.70 × 108
4.92 × 107
5.79 × 107




μA
4.10 ± 0.31
2.82 ± 0.16
1.36 ± 0.05



<1 μA
cfu/mL
1.70 × 107
4.92 × 106
5.79 × 105




μA
0.86 ± 0.09
0.41 ± 0.01
0.85 ± 0.05


B. thuringiensis
>1 μA
cfu/mL
2.11 × 105
3.11 × 106
—




μA
1.22 ± 0.24
2.08 ± 0.10
—



<1 μA
cfu/mL
2.11 × 104
3.11 × 105
1.93 × 106




μA
0.26 ± 0.04
0.55 ± 0.07
0.74 ± 0.06


Citrobacter sp.
>1 μA
cfu/mL
4.23 × 107
2.06 × 108
—




μA
1.27 ± 0.08
2.40 ± 0.08
—



<1 μA
cfu/mL
4.23 × 106
2.06 × 107
7.62 × 107




μA
0.27 ± 0.04
0.41 ± 0.05
0.62 ± 0.11


Shigella sp.
>1 μA
cfu/mL
8.66 × 107
9.87 × 107
—




μA
4.46 ± 0.44
1.68 ± 0.31
—



<1 μA
cfu/mL
8.66 × 106
9.87 × 106
1.60 × 108




μA
0.78 ± 0.09
0.35 ± 0.05
0.69 ± 0.15


S. aureus
>1 μA
cfu/mL
2.63 × 107
2.15 × 107
—




μA
2.99 ± 0.23
1.22 ± 0.20
—



<1 μA
cfu/mL
2.63 × 106
2.15 × 106
9.44 × 106




μA
0.56 ± 0.05
0.31 ± 0.07
0.49 ± 0.13


K. pneumoniae
>1 μA
cfu/mL
6.20 × 106
1.12 × 108
—




μA
1.74 ± 0.08
2.56 ± 0.32
—



<1 μA
cfu/mL
6.20 × 105
1.12 × 107
4.43 × 107




μA
0.41 ± 0.02
0.49 ± 0.13
0.82 ± 0.17


E. faecalis
>1 μA
cfu/mL
4.84 × 107
—
—




μA
3.82 ± 0.92
—
—



<1 μA
cfu/mL
4.84 × 106
7.33 × 107
4.72 × 107




μA
0.92 ± 0.01
0.54 ± 0.03
0.86 ± 0.08









In closing, it is to be understood that although aspects of the present specification are highlighted by referring to specific embodiments, one skilled in the art will readily appreciate that these disclosed embodiments are only illustrative of the principles of the subject matter disclosed herein. Therefore, it should be understood that the disclosed subject matter is in no way limited to a particular methodology, protocol, and/or reagent, etc., described herein. As such, various modifications or changes to or alternative configurations of the disclosed subject matter can be made in accordance with the teachings herein without departing from the spirit of the present specification. Lastly, the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. Accordingly, the present invention is not limited to that precisely as shown and described.
Certain embodiments of the present invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the present invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described embodiments in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
Groupings of alternative embodiments, elements, or steps of the present invention are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other group members disclosed herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.
Unless otherwise indicated, all numbers expressing a characteristic, item, quantity, parameter, property, term, and so forth used in the present specification and claims are to be understood as being modified in all instances by the term “about.” As used herein, the term “about” means that the characteristic, item, quantity, parameter, property, or term so qualified encompasses a range of plus or minus ten percent above and below the value of the stated characteristic, item, quantity, parameter, property, or term. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical indication should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and values setting forth the broad scope of the invention are approximations, the numerical ranges and values set forth in the specific examples are reported as precisely as possible. Any numerical range or value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Recitation of numerical ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate numerical value falling within the range. Unless otherwise indicated herein, each individual value of a numerical range is incorporated into the present specification as if it were individually recited herein.
The terms “a,” “an,” “the” and similar referents used in the context of describing the present invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the present invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the present specification should be construed as indicating any non-claimed element essential to the practice of the invention.
Specific embodiments disclosed herein may be further limited in the claims using consisting of or consisting essentially of language. When used in the claims, whether as filed or added per amendment, the transition term “consisting of” excludes any element, step, or ingredient not specified in the claims. The transition term “consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s). Embodiments of the present invention so claimed are inherently or expressly described and enabled herein.
All patents, patent publications, and other publications referenced and identified in the present specification are individually and expressly incorporated herein by reference in their entirety for the purpose of describing and disclosing, for example, the compositions and methodologies described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

## Claims

1. A method of detecting Salmonella in a sample, the method consisting essentially of:
a) incubating the sample in a liquid pre-enrichment medium consisting essentially of a peptone low growth nutrient component, a malachite green growth inhibiting agent, and a nonylphenol ethoxylate surfactant with bacteriostatic or bactericidal action, wherein the incubation is at about 35° C. to about 39° C. for about 14 to 15 hours, and wherein the sample is a food sample, a feces sample or a raw livestock sample:
b) incubating an aliquot of the pre-enrichment medium from step (a) in a separate liquid enrichment medium, the enrichment medium consisting essentially of a peptone high growth nutrient component and ferric ammonium citrate, wherein the incubation is at about 39° C. to about 43° C. for about 5-7 hours, wherein the aliquot of the pre-enrichment medium is about 1/50 to about 1/1000 a volume of the enrichment medium;
c) purifying the liquid enrichment medium to increase concentration of the Salmonella and/or decrease contaminants using an immuno-precipitation method employing antibodies for the Salmonella linked to magnetic particles; and
d) detecting the presence or absence of the Salmonella by analyzing an aliquot of the purified enrichment medium from step (c), using an electrochemical sensor-based detection method.

2. The method according to claim 1, wherein the nonylphenol ethoxylate surfactant is selected from the group consisting of Nonoxynol-4 (NP-4), Nonoxynol-6 (NP-6), Nonoxynol-7 (NP-7), Nonoxynol-8 (NP-8), Nonoxynol-9 (NP-9), Nonoxynol-10 (NP-10), Nonoxynol-11 (NP-11), Nonoxynol-12 (NP-12), Nonoxynol-13 (NP-13), Nonoxynol-15 (NP-15), Nonoxynol-30 (NP-30), Nonoxynol-40 (NP-40), Nonoxynol-50 (NP-50), Nonoxynol-55 (NP-55), and Nonoxynol-70 (NP-70).

3. The method according to claim 1, wherein the malachite green growth inhibiting agent is selected from the group consisting of malachite green dye and brilliant green dye.

4. The method according to claim 1, wherein the aliquot of pre-enrichment medium in step (b) is about 1/50 to about 1/500 of the volume of the enrichment medium used in step (b).

5. The method according to claim 1, wherein the ammonium ferric citrate is at a concentration of about 0.1 mg/m L to about 5.0 mg/m L.

6. The method according to claim 1, wherein the electrochemical sensor-based detection method is a biosensor-based detection method.

7. The method according to claim 1, wherein the Salmonella is Salmonella typhimurium or Salmonella typhi.

8. The method according to claim 1, wherein the malachite green growth inhibiting agent is at a concentration of about 0.5% (v/v) to about 3% (v/v).

9. The method according to claim 1, wherein the surfactant is at a concentration of about 0.5% (v/v) to about 5% (v/v).