Patent ID: 12215376

The Sequence Listing is submitted as an ASCII text file in the form of the file named Sequence_Listing.txt, which was created on Dec. 14, 2020, and is 19,199 bytes, which is incorporated by reference herein.

DETAILED DESCRIPTION OF THE INVENTION

In describing the present invention, the following terms will be employed, and are intended to be defined as indicated below.

“and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example “A and/or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

“Comprising” is intended to mean including the stated feature. Also contemplated are options wherein the terms “consisting of” or “consisting essentially of” are used instead of “comprising”.

A “sample” as used herein may be a biological sample, such as a blood sample. Particular (e.g. clinical) samples of interest include bile, nail, nasal/bronchial lavage, bone marrow, stem cells derived from the body, bones, non-fetal products of conception, brain, breast milk, organs, pericardial fluid, buffy coat layer, platelets, cerebrospinal fluid, pleural fluid, cystic fluid, primary cell cultures, pus, saliva, skin, fetal tissue, fluid from cystic lesions, stomach contents, hair, teeth, tumour tissue, umbilical cord blood, mucus and stem cells. Particularly preferred samples include, though, joint aspirates, faeces, urine, sputum and, especially, blood (including plasma). Also contemplated herein tissue samples (e.g. a biopsy) and biological fluids other than blood, for example, a urine sample, a cervical smear, a cerebrospinal fluid sample, or a tumour or non-tumour tissue sample. It has been found that urine and cervical smears contains cells, and so may provide a suitable sample for use in accordance with the present invention. The sample may be one which has been freshly obtained from the subject (e.g. a blood draw) or may be one which has been processed and/or stored prior to making a determination (e.g. frozen, fixed or subjected to one or more purification, enrichment or extractions steps, including centrifugation). The sample may be derived from one or more of the above biological samples via a process of enrichment or amplification. A plurality of samples may be taken from a single patient, e.g. serially during a course of treatment (e.g. for an actual or suspected infection). Moreover, a plurality of samples may be taken from a plurality of patients. For the analysis of contamination, the sample may be an animal product (such as animal meat) having or suspected of having a contamination source (e.g. a microbial pathogen). A liquid sample might have a volume of between 10 μl and 100 ml, preferably between 10 μl and 50 ml, such as between 10 μl or 100 μl and 20 ml (e.g. 0.2 ml or 1 ml).

The animal host is preferably mammalian, e.g. a human, a domestic animal (e.g. a cow, sheep, horse or pig) or a companion animal (e.g. a dog or cat). Other vertebrate animal hosts are also contemplated herein, such as a bird or a fish. As used herein “host” is intended to encompass both an animal that harbours one or more non-host organisms (e.g. a microorganism) and an animal suspected of harbouring a non-host organism (e.g. suspected of being infected with a pathogen).

Cytolysin

A cytolysin (also known as a cytolytic toxin) is a protein secreted by a microorganism, plant, fungus or animal which is specifically toxic to a heterologous cell type(s), particularly promoting lysis of target cells. Preferred cytolysins are those secreted by microorganisms, particularly by bacteria, and/or those that are toxic to an animal (e.g. mammalian) cell type(s).

The cytolysin can be a cytolysin that has a detergent effect on the target cell membrane (e.g. a 26 amino acid delta toxin produced byStaphylococcus) or forms pores in the target cell membrane (e.g. Alpha hemolysin fromS. aureus, Streptolysin O fromS. pyogenes, and Perfringiolysin O produced byC. perfringens). See e.g.:

Alpha hemolysin fromS. aureus—https://www.ncbi.nlm.nih.gov/protein/BBA23710.1 (SEQ ID No. 2):

1 mktrivssvt ttlllgcilm npvanaadsd iniktgttdi gsnttvktgd lvtydkengm61 hkkvfysfid dknhnkkilv irtkgtiagq yrvyseegan ksglawpsaf kvqlqlpdne121 vaqisdyypr nsidtkeyms tltygfngnv tgddsgkigg liganvsigh tlkyvqpdfk181 tilesptdkk vgwkvifnnm vnqnwgpydr dswnpvygnq 1fmktrngsm kaadnfldpn241 kassllssgf spdfatvitm drkaskqqtn idviyervrd dyqlywtstn wkgtntkdkw301 tdrsseryki dwekeemtn

Streptolysin O fromS. Pyogenes—https://www.ncbi.nlm.nih.gov/protein/BAD77794.2 (SEQ ID No. 3):

1 msnkktfkky srvaglltaa liignlvtan aesnkqntas tettttseqp kpesseltie61 kagqkmddml nsndmiklap kemplesaek eekksedkkk seedhteein dkiyslnyne121 levlaknget ienfvpkegv kkadkfivie rkkkninttp vdisiidsvt drtypaalql181 ankgftenkp davvtkrnpq kihidlpgmg dkatvevndp tyanvstaid nlvnqwhdny241 sggntlpart qytesmvysk sqieaalnvn skildgtlgi dfksiskgek kvmiaaykqi301 fytvsanlpn npadvfdksv tfkdlqrkgv sneapplfvs nvaygrtvfv kletssksnd361 veaafsaalk gtdvktngky sdilenssft avvlggdaae hnkvvtkdfd virnvikdna421 tfsrknpayp isytsvflkn nkiagvnnrt eyvettstey tsgkinlshq gayvaqyeil481 wdeinyddkg kevitkrrwd nnwysktspf stviplgans rnirimarec tglawewwrk541 viderdvkls keinvnisgs tlspygsity k

Preferably, the cytolysin is a cytolysin that digests a cell membrane component, (e.g. phospholipids, i.e. is a phospholipase). An example is Sphingomylinease (also known as beta-toxin) fromS. aureus, see e.g. https://www.ncbi.nlm.nih.gov/protein/CAA43885.1 (SEQ ID No. 4):

1 mmvkktksns lkkvatlala nlllvgaltd nsakaeskkd dtdlklvshn vymlstvlyp61 nwgqykradl igqssyiknn dvvifneafd ngasdkllsn vkkeypyqtp vlgrsqsgwd121 ktegsysstv aedggvaivs kypikekigh vfksgcgfdn dsnkgfvytk iekngknvhv181 igthtqseds rcgaghdrki raeqmkeisd fvkkknipkd etvyiggdln vnkgtpefkd241 mlknlnvndv lyaghnstwd pqsnsiakyn ypngkpehld yiftdkdhkq pkqlvnevvt301 ekpkpwdvya fpyyyvyndf sdhypikays k

The phospholipase can be a phospholipase A, B, C or D, such as PLD fromStreptomyces, see e.g. https://www.ncbi.nlm.nih.gov/protein/BAL15170.1 (Streptomyces vinaceus) (SEQ ID No. 5):

1 mhrhtpslrr psahlpsala vraavpaall alfaavpasa apaagsgadp aphldaveqt61 lrqvspgleg qvwertagnv ldastpggad wllqtpgcwg ddkctarpgt eqllskmtqn121 isqatrtvdi stlapfpnga fqdaivsglk tsaargnklk vrvlvgaapv yhlnvlpsky181 rdelvaklga darnvdlnva smttsktafs wnhskllvvd gqsvitggin dwkddyleta241 hpvadvdlal rgpaaasagr yldelwswtc qnksniasvw fassngaacm pamakdtapa301 apapapgdvp avavgglgvg ikrndpsssf rpalpsapdt kcvvglhdnt nadrdydtvn361 peesalrtli ssanrhieis qqdvnatcpp lprydirvyd alaarmaagv kvrivvsdpa421 nrgavgsggy sqikslseis dtlrdrlalv tgdqgaakat mcsnlqlatf rssqsptwad481 ghpyaqhhkv vsvddsafyi gsknlypawl qdfgyvvesp aaaaqlnarl lapqwqysra541 tatidheral cqs

Preferably the phospholipase is a phospholipase C (PLC) (i.e. a phospholipase that cleaves before the phosphate, releasing diacylglycerol and a phosphate-containing head group). Preferably the PLC is a bacterial PLC, selected from any of the following groups:Group 1—Zinc metallophospholipasesGroup 2—Sphingomyelinases (e.g. sphingomyelinase C)Group 3—PhosphatidylinositolGroup 4—Pseudomonad PLC

A Group 1 PLC is preferred, particularly PLC fromClostridium perfringens, see e.g. https://www.ncbi.nlm.nih.gov/protein/EDT77687.1 (SEQ ID No.1):

1 mkrkickali caalatslwa gastkvyawd gkidgtgtha mivtqgvsil endmsknepe61 svrknleilk enmhelqlgs typdydknay dlyqdhfwdp dtdnnfskdn swylaysipd121 tgesqirkfs alaryewqrg nykqatfylg eamhyfgdid tpyhpanvta vdsaghvkfe181 tfaeerkegy kintagcktn edfyadilkn kdfnawskey argfaktgks iyyshasmsh241 swddwdyaak vtlansqkgt agyiyrflhd vsegndpsvg knvkelvayi stsgekdagt301 ddymyfgikt kdgktqewem dnpgndfmtg skdtytfklk denlkiddiq nmwirkrkyt361 afpdaykpen ikviangkvv vdkdinewis gnstynik

This cytolysin provides for highly effective lysis of animal host cells in the present technology, despite reports in the literature that purifiedC. perfringensPLC when used alone has no cytotoxic activity against leukocytes.

The cytolysin can be a wild-type cytolysin or an active variant (produced e.g. by recombinant DNA technology). An active variant of a cytolysin is a variant of a cytolysin that retains the ability to lyse a target cell, demonstrating e.g. at least 10%, preferably at least 25%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95% of the activity of the wild-type protein in any assay where lytic activity against a target cell can be shown for the wild-type protein.

“An active variant thereof” includes within its scope a fragment of the wild-type protein. In preferred embodiments, a fragment of the wild-type protein is selected that is at least 10% of the length of the wild-type protein sequence, preferably at least 20%, preferably at least 30%, preferably at least 40%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90% and most preferably at least 95% of the length of the wild-type protein sequence.

“An active variant thereof” also includes within its scope a protein sequence that has homology with the wild-type protein sequence, such as at least 50% identity, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97%, and most preferably at least 99% identity, for example over the full wild-type sequence or over a region of contiguous amino acid residues representing 10% of the length of the wild-type protein sequence, preferably at least 20%, preferably at least 30%, preferably at least 40%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90% and most preferably at least 95% of the length of the wild-type protein sequence. Methods of measuring protein homology are well known in the art and it will be understood by those of skill in the art that in the present context, homology is calculated on the basis of amino acid identity (sometimes referred to as “hard homology”).

The homologous active cytolysin variant typically differs from the wild-type protein sequence by substitution, insertion or deletion, for example from 1, 2, 3, 4, 5 to 8 or more substitutions, deletions or insertions. The substitutions are preferably ‘conservative’, that is to say that an amino acid may be substituted with a similar amino acid, whereby similar amino acids share one of the following groups: aromatic residues (F/H/W/Y), non-polar aliphatic residues (G/A/P/I/L/V), polar-uncharged aliphatics (C/S/T/M/N/Q) and polar-charged aliphatics (D/E/K/R). Preferred sub-groups comprise: G/A/P; I/L/V; C/S/T/M; N/Q; D/E; and K/R.

The cytolysin or active variant (as described above) may have any number of amino acid residues added to the N-terminus and/or the C-terminus provided that the protein retains lytic activity.

Preferably, no more than 300 amino acid residues are added to either or both ends, more preferably no more than 200 amino acid residues, preferably no more than 150 amino acid residues, preferably no more than 100 amino acid residues, preferably no more than 80, 60 or 40 amino acid residues, most preferably no more than 20 or 10 or 5 amino acid residues.

Preferably, the sample is subject to mixing after the cytolysin has been added.

Preferably, to promote cytolysin activity, particular buffering conditions and/or incubation temperature might be provided for any one selected cytolysin. Cytolysin incubation can take place at e.g. between 5° C. and 50° C., such as between 15° C. and 45° C. (e.g. 37° C.), and for between 1 min and 120 min, preferably between 1 min and 60 min, more preferably between 1 min and 30 min (e.g. 15 min or 20 min). For part or all of the cytolysin incubation, the sample is preferably subject to mixing/shaking, at e.g. between 1 and 1500 rpm, preferably between 1 and 1000 rpm (e.g. at 500 rpm or 1000 rpm).

Preferably, the cytolysin is used in the sample at a concentration of at least 0.1 mg/ml, such as between 0.1 mg/ml and 100 mg/ml, preferably between 0.1 mg/ml and 100 mg/ml, preferably between 0.1 mg/ml and 50 mg/ml (e.g. at 4 mg/ml).

Nuclease

If a nuclease (e.g. a DNase) is used in the present methodology, the nuclease (e.g DNase) can be an endonuclease or an exonuclease (or a combination thereof can be provided), preferably an endonuclease.

Preferred nucleases (particularly where the biological sample is a blood sample) include HL-SAN nuclease (heat labile salt activated nuclease, supplied by Arcticzymes) and MolDNase (endonuclease active in the presence of chaotropic agents and/or surfactants, supplied by Molzym), and active variants are also contemplated, essentially as discussed above in relation to the cytolysin.

Preferably, the sample is subject to mixing after the nuclease has been added.

Preferably, to promote nuclease activity, particular buffering conditions and/or incubation temperature might be provided for any one selected nuclease. Nuclease incubation can take place at e.g. between 5° C. and 50° C., such as between 15° C. and 45° C. (e.g. 37° C.), and for between 1 min and 120 min, preferably between 1 min and 60 min, more preferably between 1 min and 30 min (e.g. 15 min). In particularly preferred embodiments, the DNase buffer is added to the sample, containing the cytolysin, and incubated (e.g. as described above) before pelleting. The pellet is then resuspended in DNase buffer and the nuclease (e.g. DNase0 itself is added (ahead of further incubation).

In embodiments where a nuclease is employed to deplete released nucleic acid (e.g. nuclear DNA) and is also employed to deplete mtNA following mitochondrial permeabilisation (e.g. with a detergent such as saponin), preferably, the same nuclease used in each case (i.e. to digest released host nuclear DNA and to digest mtNA released after, e.g., detergent treatment). However, it is specifically contemplated herein that the nuclease for nuclear DNA depletion and mtNA depletion may differ. In certain embodiments, a single nuclease treatment step is performed after both cell lysis and mitochondrial permeabilisation have been carried out.

Host Nucleic Acid Depletion

Host nucleic acid depletion, such as depletion of nuclear DNA and/or RNA (e.g. mRNA) depletion may have been carried out prior to host mitochondrial depletion and/or host mtNA depletion in accordance with the first aspect of the invention or may be carried out as part of the method in accordance with the second aspect of the invention.

Lysis of one or more host cells present in the sample is effected. This may involve addition of a cytolysin or a detergent that causes (selective) lysis of the host cells, releasing host nucleic acid such that it can be (partially or completely) depleted. Nucleic acid within a non-host cell or particle (e.g. pathogen) is essentially left intact (i.e. has not been significantly removed from the sample or digested) and identifiable, such that it can be subsequently collected and analysed and, in particular, identified (by e.g. sequencing or targeted PCR). A nucleic acid is identifiable e.g. if its sequence and/or biological origin can be ascertained. Preferably, therefore, the cytolysin is added to the sample and allowed to act for a period of time such that sufficient host cell lysis can occur.

The method of depleting host nucleic acid contemplates both physical depletion and (in the context of the present technology) virtual depletion (of nucleic acid released from host cells within the sample). Physical depletion can involve e.g. digesting the nucleic acid (i.e. breaking down nucleic acid polymers to e.g. base monomers) or removing nucleic acid from the sample (e.g. by any nucleic acid capture method known to the skilled person, such as deploying nucleic acid-binding magnetic beads in the sample to bind DNA and/or RNA, which can subsequently be removed or harvested from the sample).

Virtual depletion involves rendering (released) nucleic acid unidentifiable (via, in particular, targeted PCR or, most preferably, sequencing). For DNA, this means rendering the DNA non-amplifiable (e.g. by PCR) and/or (preferably) non-sequenceable. For RNA, this means rendering the RNA non-amplifiable, non-reverse-transcribable and/or (preferably) non-sequenceable. A preferred process for such rendering (particularly for DNA) involves adding a photoreactive nucleic acid-binding dye, such as propidium monoazide (PMA) or ethidium monoazide (EMA), to the sample and inducing photoreaction.

Most preferably, however, the method of depletion is via digestion of nucleic acid, most preferably via enzymatic digestion. Preferably, a nuclease is added to the sample and allowed to act for a period of time such that sufficient nucleic acid digestion can occur. Preferably, therefore, a nuclease (e.g. a deoxyribonuclease (DNase) and/or a

ribonuclease (RNase)) is added to the sample (and preferably allowed to act for a period of time such that sufficient DNA/RNA digestion can occur). The nuclease can have both DNase and RNase activity (e.g. HL-SAN DNase). Depletion of host DNA is important if analysis of non-host (e.g. pathogen) DNA is to be carried out. Depletion of host RNA is important if analysis of non-host (e.g. pathogen) RNA is to be carried out, and indeed can facilitate the optimisation of DNA analysis (e.g. DNA sequencing).

In such embodiments, the method preferably further comprises the subsequent step of neutralising the (or each) nuclease (i.e. decreasing or substantially eliminating the activity of the nuclease). The skilled person will recognise a range of neutralisation options, to be selected for each depletion protocol. This might include heat inactivation or, preferably, buffer exchange (i.e. the removal of a buffer in which the nuclease is active and/or replacement with or addition of a buffer in which the nuclease is substantially inactive). Optionally, the temperature of the sample (at any/all stage(s) at/before extraction of remaining nucleic acid from the sample) is maintained at 65° C. or less, 50° C. or less, preferably 45° C. or less, preferably 40° C. or less, to optimise subsequent release of nucleic acid from the pathogen (particularly from bacterial cells).

Host Mitochondrial Depletion

Depletion of host mitochondrial nucleic acids (mtNA) further enriches the non-host nucleic acid fraction of the sample and further enhances detection of non-host DNA and/or RNA by reducing the host NA “background”.

Host mtNA depletion comprises direct depletion of host mitochondria, for example, using techniques that capture or otherwise isolate substantially intact host mitochondria from the sample. One example of this approach is the use of a specific binding agent (e.g. antibody) that selectively binds an outer mitochondrial membrane protein, e.g. Mitochondrial import receptor subunit TOM22 homolog (TOM22).

Alternatively or additionally, host mtNA depletion may comprise adding a detergent (e.g. saponin, digitonin, Triton X-100, nonidet, NP-40, Tween-20 or filipin) to the sample to render host mitochondria more permeable and thereby facilitate the action of a nuclease to digest mtNA. As shown in Example 7 and Table 11, this approach resulted in a very high degree of enrichment of pathogen DNA due to efficient depletion of both human nuclear DNA and human mtDNA.

Alternatively or additionally, host mtNA depletion may comprise extraction of whole nucleic acids and further, targeted removal of the host mtNA from the pool of released or extracted nucleic acid. The human mtDNA genome is around 16 kb and so is amenable to capture and targeted removal techniques that may be infeasible for removal of the entire human nuclear genome. Therefore the present inventors specifically contemplate that, having depleted at least the host nuclear DNA, the mtDNA may be depleted from a mixed pool containing both host mtDNA and non-host nucleic acid by use of a library capture approach and/or a a RNA-guided DNA endonuclease enzyme, such as Cas9, -targeted approach.

Library capture of mitochondrial DNA may comprise a step of contacting the released nucleic acid with a panel of DNA and/or RNA and/or PNA baits (e.g. immobilised oligonucleotide probes) that target (i.e. hybridise to) sequences in the host mitochondrial genome. In this way mtDNA is captured and removed from the sample providing for relative enrichment of pathogen or other microbial or viral nucleic acid. Commercial capture libraries for mtDNA are available (e.g. xGen® Human mtDNA Research Panel v1.0 from IDT, and myBaits® Mito—Target Capture kit from Arbor Biosciences). Although these libraries are designed to enrich the sample in mtDNA, in our approach the use of this type of capture panel may be used for depleting mtDNA. Instead of working with captured DNA, our method, in certain embodiments, works with the non-captured content.

In a RNA-guided DNA endonuclease enzyme like CRISPR associated protein 9 (Cas9)-mediated approach to mtDNA depletion, binding of host mitochondrial DNA uses a guide RNA (e.g. sgRNA) that targets Cas9 to a host mitochondrial DNA target sequence. In particular embodiments, the Cas9 may be inactive. In these embodiments, the inactive Cas9 is targeted to the mtDNA via appropriate guide RNA and the Cas9-mtDNA complex is selectively removed by virtue of a Cas9-specific binding agent.

In an alternative embodiment, an RNA-guided DNA endonuclease enzyme such as CRISPR associated protein 9 (Cas9)-mediated approach may be employed for mtDNA depletion, cleavage of host mitochondrial DNA is achieved using a single guide RNA (sgRNA) that targets a host mitochondrial DNA target sequence. In particular embodiments, the extracted nucleic acid (NA) is fragmented and specific sequences (adaptors) are added to the ends (i.e. 5′ and 3′ ends) of the fragmented NAs by ligation or using a transposase. In these embodiments, the Cas9 is targeted to the mtDNA via appropriate sgRNA and the mtDNA sequence is cleaved. The resulting NAs are amplified by for example PCR using primers hybridizing to the adaptor sequences. The cleaved NAs lack an adaptor at both ends, and so are not amplified and, therefore, the mtDNA proportion is reduced. An example of this approach for depleting target sequences has been described by Gu et al.,Genome Biology(2016) 17:41

Further Steps

In preferred embodiments, the method further comprises the step of (optionally extracting) analysing remaining (preferably non-host) nucleic acid from the sample (or aliquot thereof). Part or all of the remaining nucleic acid (particularly non-host nucleic acid) will be intact and identifiable. The non-host nucleic acid will have been enriched relative to the original sample by complete or partial depletion of host nucleic acid, including host nuclear DNA and host mtNA.

Typically, the extraction process, where used, will involve a centrifugation step to collect, in particular, non-host cells/particles (e.g. pathogens) (virus particles and/or, in particular, bacterial and/or non-animal (e.g. non-mammalian) (e.g. unicellular) eukaryotic cells, such as fungi), from which the nucleic acid can be obtained. The bacterial or other microbial cells may be, but need not be, pathogenic. In particular, analysis of non-pathogenic microbiota is specifically contemplated herein. The human microbiota include bacteria, archaea, fungi, protists and viruses. Exemplary microbiota include those identified in the Human Microbiome Project (see, e.g., The Human Microbiome Project Consortium,Nature,2012, Vol. 486, pp. 215-221). Centrifugation conditions can be selected such that bacterial and non-animal cells, but not virus particles, are pelleted, or such that virus particles are pelleted in addition to bacterial and non-animal cells. If the former, standard virus detection tests could be performed on the supernatant.

Nucleic acid can be obtained from the pathogen(s) or other microorganisms using methods known in the art, and might involve the addition of a lysis buffer, a lytic enzyme(s) (degrading or abrogating cell membranes, cell walls and/or viral capsids), and/or a protease, e.g. proteinase K. Preferred lytic enzymes include lysozyme, mutanolysin, lysostaphin, chitinase and lyticase.

Optionally, the extracted nucleic acid (or aliquot thereof) is subject to a purification process, such as one known in the art. During purification of DNA, RNase is optionally used to facilitate the optimisation of subsequent DNA sequencing. However, RNase is omitted from any purification step if non-host (e.g. pathogen) RNA extraction is of interest (for e.g. subsequent RNA sequencing) (and a DNase might be used to assist with purification).

In preferred embodiments, extracted nucleic acid (or aliquot thereof) is subject to an amplification process, such as whole genome amplification, to increase the copy number of the nucleic acid, particularly where the biological sample is a blood sample.

For RNA, this might involve direct amplification or conversion of RNA to cDNA, followed by amplification of cDNA.

In preferred embodiments, the method further comprises the step of conducting a nucleic acid amplification test (e.g. targeted PCR amplification process, isothermal amplification, nucleic acid sequence-based amplification (NASBA)) on the extracted nucleic acid (RNA, DNA or cDNA) (or aliquot thereof) or, preferably, conducting a sequencing process on the extracted nucleic acid (or aliquot thereof), such as (e.g. short or long read) DNA or RNA sequencing, using e.g. nanopore or Illumina® sequencing.

In the preceding embodiments, nucleic acid (particularly host nucleic acid) previously rendered unidentifiable will not be amplified by any amplification process and/or (in particular) sequenced by any sequencing process.

In comparison with methods of the prior art (e.g. the MolYsis® technique, which deploys chaotropic agents to lyse host cells prior to host nucleic acid digestion), the method of the present invention facilitates highly improved depletion of host nucleic acid (particularly DNA and/or RNA, including mtDNA and/or mtRNA), while leaving non-host (e.g. pathogen, particularly bacterial) nucleic acid intact (and identifiable), leading to highly improved non-host (e.g. pathogen) nucleic acid enrichment, sufficient for subsequent sequencing-based (e.g. next-generation sequencing [NGS] based) (e.g. pathogen) diagnostics. A key factor in this advance has been the ability to achieve e.g. a 5×104or greater, such as 105or greater (e.g. 106or greater), fold depletion of host DNA (and host mtDNA) from within biological sample from a mammalian host, and these are preferable outcome features of the present technology (as is a fold depletion of 10 or greater, 102or greater, 103or greater, 5×103or greater, or 104or greater). It is particularly preferred that host nucleic acid (e.g. DNA and mtDNA) is undetectable or substantially depleted (e.g. undetectable or detectable only at a low level via qPCR) following deployment of the method of the invention. In more general terms, the selective depletion of host nucleic acid, including host mtNA enables enrichment of non-host nucleic acid, and hence improved identification of non-host organisms. This technology is thus applicable to fields other than medical microbiology, such as biological research, veterinary medicine/diagnostic, and agriculture/food safety.

The following is presented by way of example and is not to be construed as a limitation to the scope of the claims.

EXAMPLES

Materials and Methods

Saponin was obtained from Tokyo Chemical Industry (catalog No. S0019; CAS number 8047-15-2). Sapogenein content ca. 10%, pH 4.0-7.0 (at 5% aqueous solution).

Phospholipase C (PLC) was obtained from Sigma with the reference P7633-500UN: Phospholipase C fromClostridium perfringens(C. welchii) Type I, lyophilized powder, 10-50 units/mg protein; CAS Number: 9001-86-9; EC Number: 232-638-2.

Protocol for Human NA Depletion by PLC+Nuclease+Anti-TOM22 MACS Kit

1. Spike whole blood (200 μl) withEscherichia coli, Candida albicansorStaphylococcus aureusat desired concentration. Non-spiked blood is used as control2. Add 20 μl of PLC (0.04 mg/μl)3. Vortex 10 seconds4. Incubate 15 min at 37° C. at 1000 rpm5. Add 200 μl of HL-SAN buffer (5M NaCl, 100 mM MgCl2)6. Add 10 μl of HL-SAN nuclease (25 U/μl)7. Vortex 10 seconds8. Incubate 15 min at 37° C. at 1000 rpm9. Add 1.4 ml of PBS10. Centrifuge 10 min at 12,000 g and discard supernatant11. Add 700 μl pf PBS and resuspend the pellet by pipetting up and down12. Add additional 700 μl of PBS13. Centrifuge 10 min at 12,000 g and discard supernatant14. Add 200 μl pf PBS and resuspend the pellet by pipetting up and down
Mitochondrial Immunodepletion by Anti-Tom22 Ab

To deplete mitochondria, the Isolation kit human (MACS Miltenyi Biotec) was used following the adapted protocol as described below.1. Add 750 μl of ice-cold 1× separation buffer (included in the kit) to the resuspended pellet and mix2. Add 50 μl of microbeads anti-ToM223. Mix well and incubate 1 hour 4° C. with gentle agitation4. Just before finishing the incubation, prepare a MS column by loading 500 μl of 1× separation buffer5. Load the mixture of sample plus beads into the column (sited in the MiniMAC separator) and collect the eluate. This fraction is the enriched sample6. (Optionally) Wash the column with 1 ml of separation buffer7. (Optionally) Add 1 ml to the column, retire the column from the separator and extract the content by flushing the column with the emboli. Collecting the content in a separate tube (mitochondrial fraction)8. Extract nucleic acid (NA) with EasyMAg extraction robot (Biomerieux) standard protocol; select “blood” as matrix and elute in 40 μl of elution buffer
Evaluation of Mitochondrial Depletion by Cytometer

To check depletion efficiency, all the fractions obtained during the immunodepletion (before extracting them) were analyzed by FACS. Only one population was present, so the number of events in a determined time were compared among the fractions.

Evaluation of Mitochondrial Depletion by qPCR

To check depletion efficiency, qPCR was carried out using specific Taqman probes (Hs00537670_s1 and Hs02596867_s1) for human DNA and mtDNA, respectively, as well as for each pathogen tested. Ct were compared.

Protocol for Human NA Depletion by PLC+Nuclease+Saponin+Nuclease

1. Spike whole blood (200 μl) withEscherichia coli, Candida albicansorStaphylococcus aureusat desired concentration. Non-spiked blood is used as control2. Add 20 μl of PLC (0.04 mg/μl)3. Vortex 10 seconds4. Incubate 15 min at 37° C. at 1000 rpm5. Add 200 μl of HL-SAN buffer (5M NaCl, 100 mM MgCl2)6. Add 10 μl of HL-SAN nuclease (25 U/μl)7. Vortex 10 seconds8. Incubate 15 min at 37° C. at 1000 rpm9. Add 1.4 ml of PBS10. Centrifuge 10 min at 12,000 g and discard supernatant11. Add 700 μl pf PBS and resuspend the pellet by pipetting up and down12. Add additional 700 μl of PBS13. Centrifuge 10 min at 12,000 g and discard supernatant14. Add 200 μl pf PBS and resuspend the pellet by pipetting up and down15. Add 200 μl of 1% Saponin16. Vortex 10 seconds17. Incubate 10 min at RT18. Add 350 μl of water and wait 30 seconds19. Add 12 μl of 5M NaCl20. Centrifuge 5 min at 12,000 g and discard supernatant21. Add 200 μl of PBS and resuspend the pellet by pipetting up and down22. Add 200 μl of HL-SAN buffer (5M NaCl, 100 mM MgCl2)23. Add 10 μl of HL-SAN nuclease (25 U/μl)24. Vortex 10 seconds25. Incubate 15 min at 37° C. at 1000 rpm26. Add 1.4 ml of PBS27. Centrifuge 10 min at 12,000 g and discard supernatant28. Add 700 μl pf PBS and resuspend the pellet by pipetting up and down29. Add additional 700 μl of PBS30. Centrifuge 10 min at 12,000 g and discard supernatant31. Resuspend in 200 μl of PBS32. Extract NA with EasyMAg extraction robot (Biomerieux) standard protocol; select “blood” as matrix and elute in 40 μl of elution buffer33. Amplify 4 μl of extracted DNA by Whole genome amplification (WGA) using Repli-g single cell kit (Qiagen) following manufacturer's instructions34. Purify whole genome amplified samples with Ampure XP beads (Agencourt) at ratio 1:1 and elute with 40 μl of water35. Quantify the dsDNA in the samples with Qubit fluorometer
Library Preparation: MinION1. Treat 1 μg of dsDNA with T7 endonuclease (NEB):Add 2 μl of 10× reaction bufferAdd 1 μl of T7 endonucleaseAdd water until 20 μlMix by flicking and spinIncubate 15 min at 37° C.2. Purify T7 treated samples with Ampure XP beads (Agencourt) at ratio 1:1.8 (sample: beads) and elute with 24 μl of water3. Prepare the T7-treated DNA for being sequenced with the SQK-RLB001: Rapid Low Input by PCR Barcoding kit (ONT) following manufacturer's instructions4. Load the sample (or pool of samples if several samples have been prepared) in a 9.4 or 9.5 flow cell inserted in the MinION following ONT instructions5. Connect the MinION to the MinKNOW software and start the run selecting SQK-RLB001 kit and “live” basecalling6. The resulting fastqs are analyzed by the EPI2ME software using WIMP program (ONT) and the % of target reads are calculated comparing the specific target reads with Total reads (including classified+non classified reads). To calculate the % of human reads, both WIMP software and alignment of the fastq to Hg38 using Minimap program were carried out. The % of reads aligned to mitochondria were calculated by comparing ChrM mapped reads to the Hg38 mapped reads.
Library Preparation: MiSeq1. Calculate the volume to start with 1.5 ng of dsDNA in 5 μl. Prepare the library using Nextera XT kit (Illumines®) following manufacturer's instructions2. Quantify the library and dilute it to 4 nM. Denature 5 μl of 4 nM library or pool of libraries if more than 1 sample has been prepared.3. Load the library in the MiSeq cartridge (i.e. 300 cycles v2) and start the run following Illumina® instructions4. The resulting fastq is trimmed to eliminate adapters, short reads (<40 nt) and low quality reads and nucleotides. The trimmed fastq is aligned to the specific target reference and to the Hg38 (to calculate human and mitochondrial content) using Bowtie2. The number of mapped reads is calculated by loading the resulting .bam file in the Qualimap program. The % of target reads is calculated by comparing mapped reads to the total.
Protocol for Human NA Depletion by PLC+Nuclease+Extraction+Library Capture1. Spike whole blood (200 μl) withEscherichia coli, Candida albicansorStaphylococcus aureusat desired concentration. Non-spiked blood is used as control2. Add 20 μl of PLC (0.04 mg/μl)3. Vortex 10 seconds4. Incubate 15 min at 37° C. at 1000 rpm5. Add 200 μl of HL-SAN buffer (5M NaCl, 100 mM MgCl2)6. Add 10 μl of HL-SAN nuclease (25 U/μl)7. Vortex 10 seconds8. Incubate 15 min at 37° C. at 1000 rpm9. Add 1.4 ml of PBS10. Centrifuge 10 min at 12,000 g and discard supernatant11. Add 700 μl pf PBS and resuspend the pellet by pipetting up and down12. Add additional 700 μl of PBS13. Centrifuge 10 min at 12,000 g and discard supernatant14. Add 200 μl pf PBS and resuspend the pellet by pipetting up and down15. Extract NA with EasyMAg extraction robot (Biomerieux) standard protocol; select “blood” as matrix and elute in 40 μl of elution buffer16. Amplify 4 μl of extracted DNA by Whole genome amplification (WGA) using Repli-g single cell kit (Qiagen) following manufacturer's instructions17. Purify whole genome amplified samples with Ampure XP beads (Agencourt) at ratio 1:1 and elute with 40 μl of water18. Quantify the dsDNA in the samples with Qubit fluorometer19. Prepare a library to be sequenced in MiSeq by using the kit Nextera (Illumine). According to the quantification, start with 50 ng of dsDNA and follow manufacturer instructions.20. Once the library is prepared, quantify it by Qubit21. Library capture could be carried out by using myBaits® Mito-Target Capture kit from Arbor Biosciences or by using xGen® Human mtDNA Research Panel v1.0 from IDT, by following manufacturer's instructions, taking into account that the library has been prepared with Nextera kit and select the corresponding manufacturer protocol. In the case of xGen the Cot-1 DNA addition should be omitted.22. For both library captures the starting amount of DNA is 500 ng and the method has to be disrupted after the binding of hybridization mixture with magnetic beads. Once the hybridization is finished, magnetic beads (Streptavidine beads in case of xGen) are added to the hybridization reaction, mixed and incubated together to allow the binding of magnetic beads to capture probes (in case of xGEN the probes are labelled with biotin and bind to the streptavidine of the magnetic beads).23. The mixture is placed in a magnetic rack and the beads are pelleted. The supernatant is collected.24. The collected supernatant is purified to eliminate the blocking probes and all the reagents of the sample by Ampure XP beads (Agencourt) at ratio 1:1.5 (sample:beads) and elute with 25 μl of water.25. Amplify the purified DNA by using P5 and P7 primers (Illumina)26. Purify the PCR with Ampure XP beads (Agencourt) at ratio 1:1.8 (sample:beads) and elute with 25 μl of water.27. Quantify the library and dilute it to 4 nM. Denature 5 μl of 4 nM library or pool of libraries if more than 1 sample has been prepared.28. Load the library in the MiSeq cartridge (i.e. 300 cycles v2) and start the run following Illumina® instructions29. The resulting fastq is trimmed to eliminate adapters, short reads (<40 nt) and low quality reads and nucleotides. The trimmed fastq is aligned to the specific target reference and to the Hg38 (to calculate human and mitochondrial content) using Bowtie2. The number of mapped reads is calculated by loading the resulting .bam file in the Qualimap program. The % of target reads is calculated by comparing mapped reads to the total.
Protocol for Human NA Depletion by PLC+Nuclease+Extraction+Cas9 Cleavage1. This protocol is similar to the previous one till step 20.2. Once the library is prepared and quantified by Qubit, the library is treated with Cas9. For example with Cas9 Nuclease,S. pyogenes(M0386) from New England Biolabs (NEB) using sgRNAs containing specific mtDNA region.3. The treated sample is amplified using P5 and P7 primers (Illumina)4. Purify the PCR with Ampure XP beads (Agencourt) at ratio 1:1.8 (sample:beads) and elute with 25 μl of water.5. Quantify the library and dilute it to 4 nM. Denature 5 μl of 4 nM library or pool of libraries if more than 1 sample has been prepared.6. Load the library in the MiSeq cartridge (i.e. 300 cycles v2) and start the run following Illumina® instructions7. The resulting fastq is trimmed to eliminate adapters, short reads (<40 nt) and low quality reads and nucleotides. The trimmed fastq is aligned to the specific target reference and to the Hg38 (to calculate human and mitochondrial content) using Bowtie2. The number of mapped reads is calculated by loading the resulting .bam file in the Qualimap program. The % of target reads is calculated by comparing mapped reads to the total.
Protocol for Mitochondria and/or mtNA Enrichment1. Starting sample could be whole blood (200 μl)2. Add 20 μl of PLC (0.04 mg/μl)3. Vortex 10 seconds4. Incubate 15 min at 37° C. at 1000 rpm5. Add 200 μl of HL-SAN buffer (5M NaCl, 100 mM MgCl2)6. Add 10 μl of HL-SAN nuclease (25 U/μl)7. Vortex 10 seconds8. Incubate 15 min at 37° C. at 1000 rpm9. Add 1.4 ml of PBS10. Centrifuge 10 min at 12,000 g and discard supernatant11. Add 700 μl pf PBS and resuspend the pellet by pipetting up and down12. Add additional 700 μl of PBS13. Centrifuge 10 min at 12,000 g and discard supernatant14. Add 200 μl pf PBS and resuspend the pellet by pipetting up and down15. Intact mitochondria are ready to be used30. To obtain mtDNA and/or mtRNA, extract the NA from the sample with any nucleic acid extraction method. E.g. Extract NA with EasyMAg extraction robot (Biomerieux) standard protocol; select “blood” as matrix and elute in 30 μl of elution buffer

Example 1—Identification of Human mtDNA as the Main DNA Present in a Blood Sample after Human NA Depletion Using Phospholipase C (PLC) and Nuclease-Based Depletion

Different pathogens (bacteria and fungi) at different concentrations were spiked into human blood. Blood was first treated with phospholipase C (PLC) to selectively lyse human cells and then with a nuclease to eliminate human NA, samples centrifuged, supernatant discarded and the remaining NA (nucleic acids) present in the sample were extracted by different methods, amplified by whole genome amplification and sequenced by whole genome next generation sequencing (NGS) (Miseq, Illumina and Minion, ONT). Bioinformatics analysis was done to assess if we were able to detect the target pathogen and to assess the percentage of the total reads corresponding to that target and also to human DNA.

We discovered that the majority of sequenced reads correspond to human DNA and, specifically, to mitochondrial DNA (mtDNA) (Table 1).

TABLE 1Percentage sequencing reads of different sourcesmtDNAreadsfromhumanEnrich-Extrac-PathogentotalNGSmenttionreadsHumanreadsSamplePathogensequencermethodmethod(%)reads (%)(%)1200E. coliMiseqPLC +enzymes0.0599.888.7ul(250nucleasemix +BloodCFU/ml)Magnapure(Roche)2200A. nigerMiseqPLC +enzymes0.8498.889.0ul(10,000nucleasemix +BloodCFU/ml)Magnapure(Roche)3200E. coliMiseqPLC +enzymes0.6697.993.9ul(16,400nucleasemix +BloodCFU/ml)easyMAg(Bio-merieux)MinIONPLC +enzymes1.087.998.9nucleasemix +(Mini-(Mini-easyMAgmap)map)(Bio-1.577.0merieux)(WIMP)(WIMP)4200C.MiseqPLC +enzymes0.000499.295.9ulalbicansnucleasemix +Blood(960easyMAgCFU/ml)(Bio-merieux)5200A. nigerMiseqPLC +enzymes0.0698.895.0ul(10,000nucleasemix +BloodCFU/ml)easyMAg(Bio-merieux)6200E. coliMiseqPLC +easyMAg0.5999.197.1ul(49,000nuclease(Bio-BloodCFU/ml)merieux)7200C.MiseqPLC +enzymes0.0199.396.8ulalbicansnucleasemix +Blood(8,350easyMAgCFU/ml)(Bio-merieux)8200C.MiseqPLC +easyMAg0.1199.797.0ulalbicansnuclease(Bio-Blood(8,350merieux)CFU/ml)9200A. nigerMiseqPLC +Bead1.2397.996.5ul(10,000nucleasebeating +BloodCFU/ml)easyMAg(Bio-merieux)200A. nigerMinIONPLC +Bead4.485.699.1ul(10,000nucleasebeating +(Mini-(Mini-BloodCFU/ml)easyMAgmap)map)(Bio-3.175.6merieux)(WIMP)(WIMP)

We performed bioinformatics analysis to see if the whole mtDNA was present in the samples after PLC+DNAse treatment or if only fragments of mtDNA. We found that the whole mitochondrial genome (about 16 kb) was present and well-covered (seeFIG.1). Thus, we hypothesized that PLC did not degrade mitochondria and possibly entire mitochondria were present in the sample. As an alternative hypothesis it could also be possible that the DNAse is more efficient degrading non-circular DNA and since the mtDNA is circular (as is the bacterial DNA) the mtDNA is less sensitive to degradation by DNAse.

Example 2—Identification of Mitochondrial External Membrane Proteins in a Blood Sample after Human NA Depletion Using PLC+Nuclease

To check if not only mtDNA but also the whole mitochondria organelle was present in the sample after PLC+nuclease treatment, Western blot against Mitochondrial import receptor subunit TOM22 homolog (TOM22; UniProt Q9NS69 (UniProt sequence last modified: 23 Jan. 2007), a core component of the mitochondria outer membrane protein translocation pore, was carried out. Whole blood was treated by PLC+DNAse treatment. The supernatant discarded after first centrifugation, the final sample and the original whole blood were analyzed. Briefly, 20 ug of protein of each sample were denatured and loaded in a gel to carry out Western blot using anti-TOM22 antibody.

The obtained results confirm that TOM22 was present in the sample after human DNA depletion using PLC+nuclease (seeFIG.2), indicating that PLC treatment does not degrade mitochondrial external membrane and, thus, the mtDNA remains in the sample after PLC+nuclease treatment. The whole blood shows no signal due to the low percentage of TOM22 protein compared to the whole protein contain. Albumin is so abundant that gives non-specific signal in the whole blood and supernatant fractions.

Example 3—Confirming Presence of Mitochondria by Cytometry

We also confirmed the presence of entire mitochondria by Cytometry. We analyzed a PLC treated sample by Forward as side scattering in a FACSCalibur equipment and saw a unique population with a size/complexity compatible with mitochondria (FIG.3).

Example 4—Depletion of Human mtNA Using a Mitochondrial Isolation Method Based on Anti-Mitochondrial Antibodies

We eliminated the human mtNA that remained in the sample after treatment of blood with PLC and nuclease by eliminating the mitochondria organelles.

We used a commercial mitochondria isolation method based on antibodies (Miltenyi Biotech kit). Specifically, after PLC and nucleasese treatment, mitochondria were magnetically labeled with Anti-TOM22 nanobeads, human, which bind to TOM22. The sample was loaded onto a column and placed in a magnetic separator. After washing, magnetically labeled mitochondria were retained on the column and the rest of the sample was eluted. To check the presence of mitochondria after the depletion step, we processed the sample with the flow cytometer FACScalibur. Elution buffer and anti-TOM22 beads were used as negative controls and a sample treated with PLC+nuclease without mitochondrial depletion as positive control. As shown in Table 2 andFIG.4, the majority of mitochondria were retained in the column and only a small fraction remained in the eluted sample, which was then the sample utilized (see further below).

TABLE 2Column retention of mitochondriaCode in theCountingCytometerTreatmentSampleEventstimeplot figureNAElution buffer451 min 15 s—(Control)NAAnti-TOM22 beads301min002(Control)PLC +PLC + nuclease1000045s003nucleasesetreated samplePLC +Eluate174045s004nucleasese +Column retained885045s007mitochondriafraction1 isolation(anti-TOM22)sample 1PLC +Eluate162045s008nucleasese +Column retained1000036s011mitochondriafraction1 isolation(anti-TOM22)sample 2

Depletion of human mtDNA after mitochondria removal was evaluated also by qPCR, after extracting the NA from the samples. It was observed that mtDNA decreased between 6.5-9.1 times in samples treated with PLC+nuclease+mitochondrial removal compared to samples in which only PLC+nuclease treatment was performed (Table 3).

TABLE 3Depletion of human mtDNA assessed by qPCRHuman mtDNAdecreasetimescompared toMicro-PLC +organismTreatmentTargetCtnucleaseE. coliPLC + NucleasemtDNA18.4—(53,733PLC + Nuclease +mtDNA21.16.5CFU/ml)mitochondria depletionE. coliPLC + NucleasemtDNA18.6—(11,573PLC + Nuclease +mtDNA21.68.0CFU/ml)mitochondria depletionC. albicansPLC + NucleasemtDNA18.5—(49,443PLC + Nuclease +mtDNA21.68.6CFU/ml)mitochondria depletionC. albicansPLC + NucleasemtDNA18.5—(10,649PLC + Nuclease +mtDNA21.79.1CFU/ml)mitochondria depletion

It was checked by qPCR whether microorganisms remain in the eluate or were eliminated when mitochondria were depleted with the antibody-based method. Sample treated only with PLC+nucleasese was used as control. It was found thatE. coliwas not affected by the method, remaining in the eluate (Table 4). A decrease inC. albicanswas detected. SinceCandida albicansappears at high Cts, we have carried out whole genome amplification and then qPCR to better detect the pathogen and the elutedCandidawas comparable to the original so it was not retained by the column (data not shown).

TABLE 4Pathogen DNA as assessed by qPCR following indicatedtreatmentsPathogen DNAdecreasetimescompared toMicro-PLC +organismTreatmentTargetCtnucleaseE. coliPLC + NucleaseE. coli26.8—(53,733PLC + Nuclease +E. coli26.61.1CFU/ml)mitochondria depletionE. coliPLC + NucleaseE. coli29.3—(11,573PLC + Nuclease +E. coli29.30.0CFU/ml)mitochondria depletionC. albicansPLC + NucleaseC.35.0—(49,443albicansCFU/ml)PLC + Nuclease +C.35.5*1.4mitochondria depletionalbicansC. albicansPLC + NucleaseC.35.2*—(10,649albicansCFU/ml)PLC + Nuclease +C.36.8*3.0mitochondria depletionalbicans*High Ct outside reliable quantitative range of the PCR, indicating potentially unreliable results.

Example 5—Depletion of Human mtNA Using a Mitochondrial Isolation Method Based in Lysis of Mitochondria with Saponin

In an attempt to improve upon the PLC+nuclease method for depletion of host NA, the present inventors opted to add a second lysis step utilizing saponin to lyse the mitochondria not lysed by PLC. This method was then compared to the anti-TOM22 column based method described in Example 4 above.

Aliquots of 200 μl whole blood were treated with PLC+nuclease followed by PBS washes (centrifugation plus supernatant discard), pellet resuspended in 200 μl of PBS, pooled and redistributed in aliquots of 100 μl. Each suspension of 100 μl was treated with, respectively:1. Saponin 5%: addition of 200 μl of Saponin 5% followed by nuclease treatment and PBS washes.2. Saponin 1%: addition of 200 μl of Saponin 1% followed by nuclease treatment and PBS washes.3. Column Milteny: Incubation of the sample with beads covered by anti-TOM22 antibodies, column loading and eluate recovered (sample 3A). Also recovered the retained mitochondria after column washes (sample 3B).

Nucleic acids from all the samples were extracted and checked by qPCR with a Taqman® probe specific for mtDNA. The results are shown in Table 5.

TABLE 5qPCR assessment of mtDNA after indicated treatmentsDecreasetimesCtcomparedSampleTargetCtreferenceto controlTreatment (s)ControlMitoc15.7615.84—Blood + PLC(PLC)-1ControlMitoc15.92—Blood + PLC(PLC)-2Sample 1Mitoc22.60108.44Blood + PLC + 2°Saponin (5%)Sample 2Mitoc22.68114.64Blood + PLC + 2°Saponin (1%)Sample 3AMitoc20.3723.11Blood + PLC +Column MS MACSSample 3BMitoc18.928.46Blood + PLC +Column MSMACS − Mitoc

Example 6—Pathogen DNA Relative Enrichment by Using PLC+Nuclease Followed by Saponin+Nuclease

Since the second lysis step based on saponin was found to provide significant depletion of mtDNA, it was decided to test with samples spiked with bacteria and fungi.

Ten aliquots of 200 μl whole blood were spiked with bacteria (E. coliandS. aureus) and fungi (Candida) and were then treated with PLC+nuclease followed by PBS washes (centrifugation plus supernatant discard), resuspended in 200 μl PBS and divided in 2 aliquots of 100 μl. One aliquot was designated as control and the other was treated with 200 μl of 1% saponin+nuclease+PBS washes.

All the samples (10 PLC+10 PLC+saponin) were analyzed by qPCR for human and mitochondrial DNA. The samples treated with PLC are shown with a B suffix (e.g. “Sample 1B”); those treated with PLC+saponin are shown with a C suffix (e.g. “Sample 1C”).

The results obtained demonstrate significant reduction of mitochondrial DNA content (approximately 50-fold reduction) and a more efficient genomic DNA (gDNA) (i.e. nuclear DNA) depletion (see Tables 6 and 7). The qPCR for each of the microorganisms shows no significant reduction of microbial DNA content (see Tables 8, 9 and 10).

From the 10 samples, we selected 5 of them treated with PLC+saponin, including 2 samples of each bacteria and oneCandidaand carried out next generation sequencing (NGS) analysis by Minion and also by MiSeq. The results are shown in Table 11. The percentage of sequence reads from human DNA (nuclear and mitochondria) was very significantly depleted by the PLC+nuclease+Saponin+nuclease treatment, which led to a very significant relative enrichment of the pathogen sequence reads (over 90% in the case ofS. aureussample 4 by Minion WIMP sequencing—see Table 11).

The obtained data indicates that the method is also suitable for antibiotic resistance detection by known molecular mechanism (for example, the presence of genes and/or mutations conferring resistance to specific antibiotics). A representative example is shown inFIG.5.

TABLE 6Data for human DNA content analysisDecreasetimescompared toWHOLEMicro-CtBLOODSampleorganismTreatmentTargetCtreferencecontrolSample 1—ControlHuman25.5025.25Sample 3E. coliControlHuman25.01(13,733cfu/ml)Sample 1B—PLCHuman36.983376Sample 1C—PLC +HumanUn-NotSaponindetermineddetectableSample 3BE. coliPLCHuman36.53248(13,733cfu/ml)Sample 3CE. coliPLC +HumanUn-Not(13,733Saponindetermineddetectablecfu/ml)Sample 4BE. coliPLCHuman36.091830(1,373cfu/ml)Sample 4CE. coliPLC +HumanUn-Not(1,373Saponindetermineddetectablecfu/ml)Sample 5BC. albicansPLCHumanUn-Not(12,000determineddetectablecfu/ml)Sample 5CC. albicansPLC +HumanUn-Not(12,000Saponindetermineddetectablecfu/ml)Sample 6BC. albicansPLCHuman37.073601(1,200cfu/ml)Sample 6CC. albicansPLC +HumanUn-Not(1,200Saponindetermineddetectablecfu/ml)Sample 7BC. albicansPLCHuman38.358770(120 cfu/ml)Sample 7CC. albicansPLC +HumanUn-Not(120 cfu/ml)SaponindetermineddetectableSample 8BS. aureusPLCHuman36.222001(586,833cfu/ml)Sample 8CS. aureusPLC +HumanUn-Not(586,833Saponindetermineddetectablecfu/ml)Sample 9BS. aureusPLCHuman36.833054(58,683cfu/ml)Sample 9CS. aureusPLC +HumanUn-Not(58,683Saponindetermineddetectablecfu/ml)SampleS. aureusPLCHuman36.88315610B(5,868cfu/ml)SampleS. aureusPLC +Human38.44931610C(5,868Saponincfu/ml)CNWaterHumanUn-Notdetermineddetectable

TABLE 7Data for mtDNA content analysisDecreasetimescompared toMicro-CtcorrespondingSampleorganismTreatmentTargetCtreferencePLC controlSample 1—ControlMitoc19.8419.83Sample 3E. coliControlMitoc19.81(13,733cfu/ml)Sample 1B—PLCMitoc17.71Sample 1C—PLC +Mitoc23.3750.5SaponinSample 3BE. coliPLCMitoc17.44(13,733cfu/ml)Sample 3CE. coliPLC +(13,733SaponinMitoc23.1643.7cfu/ml)Sample 4BE. coliPLCMitoc17.57(1,373cfu/ml)Sample 4CE. coliPLC +Mitoc23.0741.0(1,373Saponincfu/ml)Sample 5BC. albicansPLCMitoc17.74(12,000cfu/ml)Sample 5CC. albicansPLC +Mitoc23.0941.6(12,000Saponincfu/ml)Sample 6BC. albicansPLCMitoc17.30(1,200cfu/ml)Sample 6CC. albicansPLC +Mitoc23.1242.2(1,200Saponincfu/ml)Sample 7BC. albicansPLCMitoc17.52(120 cfu/ml)Sample 7CC. albicansPLC +Mitoc24.80135.5(120 cfu/ml)SaponinSample 8BS. aureusPLCMitoc17.53(586,833cfu/ml)Sample 8CS. aureusPLC +Mitoc23.2345.7(586,833Saponincfu/ml)Sample 9BS. aureusPLCMitoc17.18(58,683cfu/ml)Sample 9CS. aureusPLC +Mitoc23.0740.9(58,683Saponincfu/ml)SampleS. aureusPLCMitoc16.9710B(5,868cfu/ml)SampleS. aureusPLC +Mitoc23.0440.210C(5,868Saponincfu/ml)CNWaterMitocUn-Notdetermineddetectable

TABLE 8Data for microorganismE. coliSampleMicroorganismTreatmentTargetCtSample 3E. coliControlE coli32.98(13,733 cfu/ml)Sample 3BE. coliPLCE coli30.93(13,733 cfu/ml)Sample 3CE. coliPLC + SaponinE coli31.96(13,733 cfu/ml)Sample 4E. coliControlE coli37.44(1,373 cfu/ml)Sample 4BE. coliPLCE coli34.41(1,373 cfu/ml)Sample 4CE. coliPLC + SaponinE coli35.82(1,373 cfu/ml)SampleEE. coliOriginalE coli31.20coli105(13,733 cfu/ml)cultureWaterE coliUndetermined

TABLE 9Data for microorganismC. albicansSampleMicroorganismTreatmentTargetCtSample 5C. albicansControlC albicans33.37(12,000cfu/ml)Sample 5BC. albicansPLCC albicans35.97(12,000cfu/ml)Sample 5CC. albicansPLC +C albicans35.02(12,000Saponincfu/ml)Sample 6C. albicansControlC albicansUndetermined(1,200cfu/ml)Sample 6BC. albicansPLCC albicansUndetermined(1,200cfu/ml)Sample 6CC. albicansPLC +C albicans36.58(1,200Saponincfu/ml)Sample 7C. albicansControlC albicansUndetermined(120 cfu/ml)Sample 7BC. albicansPLCC albicansUndetermined(120 cfu/ml)Sample 7CC. albicansPLC +C albicansUndetermined(120 cfu/ml)SaponinSampleC. albicansOriginalC albicans36.66Calbicans105(1,200culturecfu/ml)WaterC albicansUndetermined

TABLE 10Data for microorganismS. aureusSampleMicroorganismTreatmentTargetCtSample 8S. aureusControlS. aureus34.33(586,833cfu/ml)Sample 8BS. aureusPLCS. aureus33.85(586,833cfu/ml)Sample 8CS. aureusPLC +S. aureus30.27(586,833Saponincfu/ml)Sample 9S. aureusControlS. aureus38.69(58,683cfu/ml)Sample 9BS. aureusPLCS. aureus34.79(58,683cfu/ml)Sample 9CS. aureusPLC +S. aureus34.32(58,683Saponincfu/ml)Sample 10S. aureusControlS. aureusUndetermined*(5,868 cfu/ml)Sample 10BS. aureusPLCS. aureus38.58(5,868 cfu/ml)Sample 10CS. aureusPLC +S. aureusUndetermined*(5,868 cfu/ml)SaponinCNWaterS. aureusUndetermined*The spiked quantity is in the detection limit

TABLE 11Data from NGS analysismtDNAreadsTargetfromPatho-humanEnrich-Extrac-NGSgenHumantotalPatho-menttionse-readsreadsreadsSamplegenmethodmethodquencer(%)(%)(%)1200 ulE. coliPLC +easyMAgMiseq86.59%0.30%56%Blood(13,733Nu-(Bio-Minion84.0%0.1%NDcfu/ml)clease +merieux)WIMPSaponin +Nuclease2200 ulE. coliPLC +easyMAgMiseq53.85%0.90%76%Blood(1,373Nu-(Bio-Minion47.4%0.5%NDcfu/ml)clease +merieux)WIMPSaponin +Nuclease3200 ulC.PLC +easyMAgMiseq23.3%3.8%9%BloodalbicansNu-(Bio-Minion28.8%2.0%ND(1,200clease +merieux)WIMPcfu/ml)Saponin +Nuclease4200 ulSPLC +easyMAgMiseq87.5%0.40%37%BloodaureusNu-(Bio-Minion94.1%0.1%ND(58,683clease +merieux)WIMPcfu/ml)Saponin +Nuclease5200 ulSPLC +easyMAgMiseq20.6%7.30%5%BloodaureusNu-(Bio-Minion38.3%6.2%ND(5,868clease +merieux)WIMPcfu/ml)Saponin +Nuclease

Example 7—Comparison of Human Nucleic Acid Depletion by Using PLC+Nuclease Followed by Saponin+Nuclease Vs. Only PLC+Nuclease Method and Vs. Only Saponin+Nuclease

We next carried out an experiment with 2 samples (E. coliandS. aureus) treated in parallel with:1. PLC+nuclease;2. Saponin+Nuclease3. PLC+Nuclease+Saponin+Nuclease (in accordance with the method of the invention).

One ml of whole blood was spiked withE. coliand other 1 ml of whole blood was spiked withS. aureusand aliquots of 200 μl were made. Each aliquot was treated in accordance with one of the above two methods, as was a non-spiked whole blood control.

We carried out qPCR to detect human gDNA and mtDNA. The major human and mitochondrial decrease was observed for the PLC+Saponin method (data not shown).

The extracted DNA was amplified and a library was prepared with Nextera XT and, a run of MiSeq includingE. coliandS. aureustreated with the 3 different methods was performed. The results are shown in Table 12. The results demonstrate that method 3 above (PLC+Nuclease+Saponin+Nuclease) achieved the greatest relative enrichment of Pathogen sequence reads for both of the pathogen species tested.

TABLE 12NGS data for human DNA and pathogen DNAfor the identified enrichment methodsmtDNAreadsfromhumanPathogenHumantotalEnrichmentExtractionNGSreadsreadsreadsSamplePathogenmethodmethodsequencer(%)(%)(%)200 ulE. coliPLC +easyMAgMiseq6.8592.0786.17Blood(25,383Nuclease(Biomerieux)cfu/ml)200 ulE. coliSaponin +easyMAgMiseq6.7892.2649.32Blood(25,383Nuclease(Biomerieux)cfu/ml)200 ulE. coliPLC +easyMAgMiseq87.750.9756.14Blood(25,383Nuclease +(Biomerieux)cfu/ml)Saponin +Nuclease200 ulS. aureusPLC +easyMAgMiseq0.7398.8388.74Blood(11,083Nuclease(Biomerieux)cfu/ml)200 ulS. aureusSaponin +easyMAgMiseq0.1199.4592.23Blood(11,083Nuclease(Biomerieux)cfu/ml)200 ulS. aureusPLC +easyMAgMiseq50.6921.8138.34Blood(11,083Nuclease +(Biomerieux)cfu/ml)Saponin +Nuclease

Example 8—Minimal Spontaneous Lysis of Cells During Isolation and Storage of Human Whole Blood Samples

Spontaneous lysis of red cells (autohemolysis) is believed to be low in the absence of specific treatment to lyse cells. For example, Young et al.,Blood(1956), Vol. 11, pp. 977-997, report a rate of autohemolysis in red cells below 0.5% when the sample was stored for 48 hrs without further manipulation.

The present inventors analyzed the spontaneous lysis during an 7-day period for 3 whole blood samples. Identical aliquots of the whole samples were stored at room temperature (RT) and at 4° C. At concrete times (Day 0 to Day 7) plasma samples were prepared following two different centrifugation speeds: standard for plasma preparation, 900×g, obtaining an acellular fraction containing released mitochondria (since they do not precipitate at this speed) and higher, 10,000×g were the majority of mitochondria are precipitated and, therefore, the content of this fraction is acellular and with no mitochondria. The level of hemolysis of red cells (despite not having DNA, their rupture is considered the indicator of lysis since the erythrocytes are the most fragile blood cell type), the gDNA content and also the mtDNA content in the plasma were measured to determine whether both data change during the time, showing the lysis of the cells and mitochondria during the storage.

The setup of the experiment was as follows:

Storage Time024487296120144168hhhhhhhhWhole bloodX4° C.PlasmaXXXXX900 × gPlasmaXXXXX10000 × gRTPlasmaXXXXXX900 × g3 different whole blood samples250 μl aliquots for each condition (stored at RT or 4° C. during 7 days).X: sampling 80 μl:20 μl to analyze hemolysis by measuring 412 nm Absorbance after diluting 1:50 in water (1:5000 in case of whole blood because it is too much concentrated and the Absorbance is saturated using 1:50)and 60 μl frozen for further DNA extraction. 50 μl were extracted using easyMAG (Biomereux) and eluted in 25 μl. Four μl were used to check gDNA by qPCR using an specific taqman assay for gDNA, and other 4 μl were used to analysed mtDNA content also by qPCR with a specific Taqman assayThe % of lysis has been obtained comparing the initial measure of each parameter within equivalent fractions at time 0. It has been also calculated the plasma fraction content for each parameter with total whole blood measures.

Results

Measures correspond to whole blood of 2 tubes collected from 3 different individuals: 6 samples for each measure point.

The table below shows % of lysis based on the measures for the 3 studied parameters compared to whole blood measures (first column: % vs Total) and to each corresponding fraction at point 0 (Second column: % increase vs same fraction time 0; i.e. % of measure for plasma at time 72 h RT of the one of plasma at time 0). The % of lysis due to storage is the corresponding to the second column, since the first is only included to show that there is already some content of hemoglobin, mtDNA and gDNA in the plasma fraction with no treatment of time of storage.

As shown in the table, typical sample preparation and storage (e.g. 4° C. for up to 48 hours), results in essentially no lysis (all measured parameters approximately zero). Even after 96 and 168 hours at standard storage (4° C.), lysis remains low at around 2% and 4%, respectively, looking to gDNA, even lower looking at hemolysis and about 7.5% looking at mtDNA (less confidence due to its high desvest). Even in the case of having the blood at RT during 48 h the lysis is almost 0. No long storage at RT is carried out in the routine of whole blood handling.

The present inventors therefore conclude that spontaneous lysis, i.e. in the absence of a treatment to cause lysis, is low under typical conditions.

HEMOLYSIS MEASUREmtDNA MEASURE% increase% increase% increase% increasevs samevs samevs samevs sameCentrif% vs Totalfraction t0% vs Totalfraction t1% vs Totalfraction t2% vs Totalfraction t3TimeTempxgAverageAverageDevestDevestAverageAverageDevestDevest0—9000.32%0.05%4.5%4.4%0.0%24 h4° C.9000.35%0.03%0.10%0.11%48 h4° C.9000.43%0.11%0.16%0.18%4.2%−0.4%2.2%5.6%72 h4° C.9000.39%0.07%0.09%0.08%96 h4° C.9000.56%0.24%0.07%0.09%12.3%7.8%15.7%13.5%168 h4° C.9000.55%0.23%0.10%0.08%12.1%7.5%11.6%13.7%24 hRT9000.28%−0.04%0.12%0.14%48 hRT9000.23%−0.09%0.04%0.07%4.8%0.3%2.8%4.7%72 hRT9000.32%0.00%0.12%0.13%96 hRT9000.45%0.14%0.14%0.14%12.7%8.2%6.8%3.3%168 hRT9000.84%0.52%0.24%0.28%7.1%2.5%4.5%3.7%0—100000.31%0.06%0.1%0.0%0.1%0.0%24 h4° C.1000048 h4° C.100000.36%0.05%0.05%0.09%1.9%1.8%3.5%3.5%72 h4° C.100000.64%0.33%0.16%0.18%96 h4° C.100000.44%0.13%0.09%0.07%1.3%1.2%0.9%0.9%168 h4° C.100000.43%0.12%0.06%0.08%2.7%2.6%2.7%2.7%gDNA MEASURE% increase% increasevs samevs same% vs Totalfraction t4% vs Totalfraction t5TimeAverageAverageDevestDevest00.7%0.0%0.8%0.0%24h48h0.3%−0.4%0.2%0.8%72h96h2.8%2.1%5.5%4.8%168h4.6%3.9%3.0%2.9%24h48h1.6%0.9%1.2%1.4%72h96h6.4%5.7%4.0%3.3%168h28.5%27.9%18.7%17.9%00.1%0.0%0.1%0.0%24h48h0.4%0.3%0.5%0.4%72h96h0.4%0.3%0.2%0.1%168h0.8%0.7%0.5%0.5%

All references cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

The specific embodiments described herein are offered by way of example, not by way of limitation. Any sub-titles herein are included for convenience only, and are not to be construed as limiting the disclosure in any way.