Patent Publication Number: US-2015087540-A1

Title: Composition for Diagnosing Sepsis, and Method and Kit Therefor

Description:
TECHNICAL FIELD 
     The present invention relates to a composition for diagnosing sepsis, and a method and a kit therefor. 
     BACKGROUND ART 
     Sepsis is a serious disease having a death rate of 23% to 46% depending on the time of detection of sepsis. Since sepsis significantly increases a financial burden, it is a big problem in health policy (Dellinger R P: Cardiovascular management of septic shock. Crit. Care Med 2003; 31: 946-55; Osborn T M, Tracy J K, Dunne J R, Pasquale \M, Napolitano L M: Crit. Care Med 2004; 32: 2234-40; Angus D C, Linde-Zwirble W T. Lidicker J, Clermont G, Carcillo J, Pinsky M R: Crit. Care Med 2001; 29: 1303-10). 
     Sepsis is the leading cause of death in non-cardiac intensive care units (ICUs), and in the United States, at least 750,000 cases of sepsis occurs each year, 50% of them proceed to septic shock cases, and half of the septic shock cases, i.e. 200,000 cases, die (Martin G S, Mannino D M, Eaton S, Moss M: N Engl J Med 2003; 348: 1546-54). 
     In recent decades, the incidence of septic shock is gradually on the rise, but its death rate has little changed or slightly decreased (Friedman G, Silva E, Vincent J L: Crit. Care Med 1998; 26: 2078-86). 
     Sepsis has complicated and various clinical signs since infection of a pathogenic microbe affects various functional systems of the body, such as the immune system, the coagulation system, the neurohormone system, etc. of a host, which results in a response of the whole body related thereto. Therefore, both a response degree of the host and a characteristic of the causative organism of the infection have significant effects on prognosis of sepsis. 
     Since the mid-1980s, gram-positive bacteria are mostly regarded as the cause of sepsis as compared with conventional gram-negative bacteria, and since 1990s, the incidence of sepsis caused by fungi has also been on the rise (Martin O S, Mannino D M, Eaton 5, Moss M: TN Engl J Med 2003; 348: 1546-54). It is deemed that such a change in strain causing sepsis is caused by an increase in elderly patients with associated diseases, improved and active medical and surgical treatment as compared with the past, an increase in diseases caused by human immunodeficiency virus, and expression of resistant bacteria having a resistance to conventional antibiotics. 
     Recently, a lot of yeast like fungi known as being non-pathogenic has emerged as critical opportunistic pathogens of immunodepressed patients. Opportunistic fungal infections often occur as complications in various medical and surgical inpatients such as a malignant tumor patient, an acquired immunodeficiency syndrome (AIDS) patient, a critical surgery patient, a severe burn patient, a hone-marrow or organ transplant patient, a patient undergoing intravascular catheter placement, a patient administered with antibiotics for a long time, and a patient undergoing chemotherapy (Kiehn T E, Edwards F F, Armstrong D. Am J Clin Pathol. 1980; 73:518-21; Komshian S V, Uwaydah A K, Sobel J D, Crane L R. Rev Infect Dis. 1989; 11:379-90). In addition to  Candidal albican  known as a common causative organism,  Candida tropicalis  and  Candida parapsilosis  among  Candida  spp. Have been on the rise in recent years (Goldani L Z and Mario P S. J. Infect. 2003; 46:155-60; Fraser V J, Jones M, Dunkel J, Storfer S, Medoff G, Dunagan W C. Clin Infect Dis. 1992; 15: 414-21), and it has been increasingly reported that as a result of preventive administration of fluconazole to hone-marrow transplant patients or the like, there occur opportunistic fungal infections caused by  Candida krusei  and  Candida glabrata  having a resistant to the fluconazole (Baran J Jr, Muckatira B, Khatib R. Scand J Infect Dis. 2001; 33:137-9; Diekema D J, Messer S A, Brueggemann A B, Coffman S L, Doern G V, Herwaldt L A, et al. J clin Microbiol. 2002; 40:1298-302; Collin B, Clancy C J, Nguyen M H. Drug Resist Updat. 1999; 2: 9-14). 
     For the past 70 years since 1940 when penicillin was first introduced to the clinical medicine, many antibiotics have been developed and have been used clinically, thereby having made a critical contribution toward saving numerous patients&#39; lives from infections. However, along with use of antibiotics, antibiotic resistances of bacteria have been rapidly expressed and more rapidly progressed than development of antibiotics. Therefore, in only 70 years, curative effects of most of the antibiotics have been decreased all over the world. Considering the incidence and clinical importance of bacterial infections, antibiotic resistance can be said as a crisis in world&#39;s health care systems. 
     Major bacteria problematic regarding antibiotic resistance are  Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter  spp. (Boucher H W, Talbot G H, Bradley J S, Edwards J E, Gilbert D, Rice L B, Scheld M, Spellberg B, Bartlett J. Badbugs, Clin Infect Dis 2009; 48: 1-12). 
     Particularly, as gram-positive bacteria, for example,  Staphylococcus aureus  has a representative resistance to methicillin (including community-acquired methicillin-resistant  Staphylococcus aureus  (MRSA)) and vancomycin,  Enterococcus faecium  has a representative resistance to vancomycin, and  Streptococcus pneumoniae  as a major community-acquired bacterium has a representative resistance to macrolide and multiple resistance. 
     MRSA has become the most critical nosocomial microbe in hospitals in the world. In particular, it is reported that in Korea, Japan, Taiwan, Hong Kong, Singapore, and Sri Lanka and in some hospitals in the United States, 50% or more of  Staphylococcus aureus  separated from pathogens are MRSA (Grundmann H, Aires-de-Sousa M, Boyce J, Tiemersma E. Lancet 2006; 368; 874-85). 
     The incidence of MRSA in the domestic hospitals was 83.7% as a result of the prospective research conducted in 15 domestic hospitals in 1996 (Kim J M, Park E S, Jeong J S, et al. Am J Infect control 2000; 28:454-8). According to the multi-institutional research from 1997 to 2006, the incidence of MRSA was reported as 64 to 72%, which confirmed that the MRSA is the most common causative organism of nosocomial infections in the domestic hospitals (Chong Y, Lee K, Park Y J, Jeon D S, et al. Yonsei Med J 1998; 39: 569-77). 
       Enterococcus  was known as a major pathogen of endocarditis. However, as a use of third generation cephalosporin antibiotics has been increased since the mid-1970s,  Enterococcus  has become regarded as a major causative organism of nosocomial infections.  Enterococcus  has an inherent resistance to most of the antibiotics and can easily acquire antibiotic resistance through transfer of plasmid and transposon. Vancomycin-resistant Enterococci (VRE) were first reported in 1988 in Britain and France (Uttley A H, Collins C H, Naidoo J, George R C. Vancomycin-resistant Enterococci. Jancet 1988; 1: 57-8; Keclercq R, Duval J, Courvalin P. N Engl J Med 1988; 319: 157-61). 
     Since then, VRE showed a tendency to increase in the United States, and it was revealed that a transfer of VanA gene by means of transposon is a major mechanism thereof (Frieden T R, munsiff S S, Low D E et al. Lancet 1993; 342: 76-9). 
     In Korea, VRE infection was first reported in 1992. In Korea, the percentage of VRE among separated  E. faecium  was 4% in 1997 and has been gradually increased to 29% in 2009. According to a result of the nationwide multi-institution research, the percentage of  E. faecium  causing nosocomial infections from 2009 to 2010 was 38.9% (Park J W, Kim Y R, Shin W S, Kang M W, et al. Korean J Infect Dis 1992; 24: 133-7; Lee K, Kim M N, Kim J S et al, Yonsei Med J 2011; 52:793-802). 
     A blood culture is a very important method for testing bacteremia and a test method required for diagnosing a disease and determining a guideline for treatment and a prognosis (Aronson M I) and Bor D H. Blood culture. Ann Intern Med 1987; 106: 246-53). 
     Sepsis can be accompanied with various infectious diseases and can be caused by various bacterial species. Therefore, in order to identify a causative organism thereof, the blood culture is often used. By analyzing a result of the blood culture, studying development of changes thereof, and understanding a separated bacterial species and an antibiogram, important information for treatment of patients has been offered. Since a bacteremia patient is in a very serious condition, a fast result of the blood culture is very important in saving a life of the patient. However, the blood culture typically takes 5 days or more. If a blood culture-positive signal comes out, a subculture is carried out and then Gram staining, identification of a bacterial species, and an antimicrobial susceptibility test are carried out, which takes 10 days or more. 
     PATENT LITERATURE 
     
         
         Patent Literature 1: Korean Patent Laid-open Publication No. 10-2005-0016987 
         Patent Literature 2: Korean Patent Laid-open Publication No. 10-2008-0006617 
       
    
     DISCLOSURE 
     Technical Problem 
     In order to solve the conventional problems, an object of the present invention is to provide an information offering method for diagnosing sepsis. 
     Another object of the present invention is to provide a primer for diagnosing sepsis. 
     Still another object of the present invention is to provide a probe for diagnosing sepsis. 
     Still another object of the present invention is to provide a kit for diagnosing sepsis. 
     Technical Solution 
     In order to achieve the above objects, an exemplary embodiment of the present invention provides an information offering method for diagnosing sepsis comprising: (a) separating a DNA from a clinical specimen; (b) performing polymerase chain reaction (PCR) amplification of a 16s rRNA gene, an ITS (internal transcribed sequence) gene, a Nuc (heat-stable DNA nuclease) gene, a Cps gene ( S. pneumoniae  encoding biosyntheiss of capsular polysaccharide), a MecA gene (gene encoding methicillin resistance in saphylococci), an invA (invasion A) gene for detecting  salmonella , an ipaH (invasion plasmid antigen) for detecting  Shigella , and genetic fragments of a Van (Vancomycin resistance protein) A and a Van (Vancomycin resistance protein) B from the DNA by using respective primers; and (c) forming a PCR-reverse blot hybrid with a solid support upon which an oligomer probe for detecting the 16s rRNA gene for distinguishing gram-positive bacteria and gram-negative bacteria, an oligomer probe for detecting the ITS (internal transcribed sequence) gene for identifying a fungus, an oligomer probe for detecting the MecA gene for checking whether or not MRSA has antibiotic resistance, a probe for detecting the Nuc gene specific to  S. aureus , a probe for detecting the Cps gene specific to  S. pneumoniae , and a probe for detecting the invA gene for detecting  salmonella , a probe for detecting the ipaH for detecting  Shigella , and an oligomer probe for detecting the VanA and the VanB for checking whether or not VRE have antibiotic resistance are attached, and an amplified product obtained from the step (b). 
     In an exemplary embodiment of the present invention, preferably, a primer for amplifying a segment of the 16s rRNA gene is a primer having sequence numbers of 1 to 4, a primer for amplifying a segment of the ITS (internal transcribed sequence) gene is a primer having sequence numbers of 5 to 6, a primer for amplifying a segment of the Nuc (heat-stable DNA nuclease) gene is a primer having sequence numbers of 7 to 8, a primer for amplifying a segment of the MecA gene is a primer having sequence numbers of 9 to 10, primers for amplifying segments of the VanA and the VanB are primers having sequence numbers of 11 to 12 and sequence numbers of 13 to 14, respectively, a primer for amplifying a segment of the invA gene is a primer having sequence numbers of 15 to 16, a primer for amplifying a segment of the ipaH gene is a primer having sequence numbers of 17 to 18, and a primer for amplifying a segment of the Cps gene is a primer having sequence numbers of 19 to 20, but the present invention is not limited thereto. 
     In another exemplary embodiment of the present invention, preferably, a probe for detecting the gram-negative bacteria is a probe having sequence numbers of 22 to 30, a probe for detecting the gram-positive bacteria is a probe having sequence numbers of 31 to 37, a probe for detecting the Nuc (heat-stable DNA nuclease) gene is a probe having a sequence number of 38, a probe for detecting the MecA gene for checking whether or not MRSA has antibiotic resistance is a probe having a sequence number of 39, probes for detecting the VanA and the VanB for checking whether or not VRE have antibiotic resistance are probes having a sequence number of 40 and a sequence number of 41, respectively, and a probe for detecting the gene for identifying a fungus is a probe having sequence numbers of 42 to 47, but the present invention is not limited thereto. 
     Further, an exemplary embodiment of the present invention provides an information offering method for detecting gram-positive bacteria and gram-negative bacteria comprising: separating a DNA from a clinical specimen; and performing real-time PCR amplification using a primer pair having sequence numbers of 48 to 49 and a probe having sequence numbers of 50 to 53. 
     Furthermore, an exemplary embodiment of the present invention provides an information offering method for detecting a fungus comprising: separating a DNA from a clinical specimen; and performing real-time PCR amplification using a primer pair having sequence numbers of 54 to 55 and a probe having a sequence number of 56. 
     Moreover, an exemplary embodiment of the present invention provides a primer composition comprising: a primer having sequence numbers of 1 to 4; a primer having sequence numbers of 5 to 6 for amplifying a segment of an ITS (internal transcribed sequence) gene; a primer having sequence numbers of 7 to 8 for amplifying a segment of a Nuc (heat-stable DNA nuclease) gene; a primer having sequence numbers of 9 to 10 for amplifying a segment of a MecA gene; primers having sequence numbers of 11 to 12 and sequence numbers of 13 to 14 for amplifying segments of a VanA and a VanB, respectively; a primer having sequence numbers of 15 to 16 for amplifying a segment of an invA gene; a primer having sequence numbers of 17 to 18 for amplifying a segment of an ipaH gene; and a primer having sequence numbers of 19 to 20 for amplifying a segment of a Cps gene. 
     Besides, an exemplary embodiment of the present invention provides a probe composition comprising: a probe having sequence numbers of 22 to 30 for detecting gram-negative bacteria; a probe having sequence numbers of 31 to 37 for detecting gram-positive bacteria; a probe having a sequence number of 38 for detecting a Nuc (heat-stable DNA nuclease) gene; a probe having a sequence number of 39 for detecting a MecA gene for checking whether or not MRSA has antibiotic resistance; probes having a sequence number of 40 and a sequence number of 41 for detecting a VanA and a VanB, respectively, for checking whether or not VRE have antibiotic resistance; and a probe having sequence numbers of 42 to 47 for detecting a gene for identifying a fungus. 
     Further, an exemplary embodiment of the present invention provides a kit for diagnosing sepsis, the kit containing the primer composition and the oligomer probe composition of the present invention as active ingredients. 
     Furthermore, an exemplary embodiment of the present invention provides a composition for detecting gram-positive bacteria and gram-negative bacteria, the composition containing a primer pair having sequence numbers of 48 to 49 and a probe having sequence numbers of 50 to 53 as active ingredients.— 
     Moreover, an exemplary embodiment of the present invention provides a composition for detecting a fungus, the composition containing a primer pair having sequence numbers of 54 to 55 and a probe having a sequence number of 56 as active ingredients. 
     The primer of the present invention can be chemically synthesized by using a phosphoramidite solid support method or other well-known methods. This nucleic acid sequence can also be deformed by the known methods in the art. Non-limited examples of such deformation may include methylation, “capping”, substitution to analogues of at least one natural nucleotide, and deformation between nucleotides, for example, deformation into an uncharged connector (for example, methyl phosphonate, phosphotriester, phosphoramidate, carbamate, or the like) or a charged connector (for example, phosphorothioate, phosphorodithioate, or the like). The nucleic acid may contain at least one additional covalently bonded residue, for example, proteins (for example, nuclease, toxin, antibody, signal peptide, poly-L-lysine, or the like), an intercalator (for example, acridine, psoralene, or the like), a chelating agent (for example, a metal, a radioactive metal, iron, an oxidative metal, or the like), and an alkylating agent. The nucleic acid sequence of the present invention can also be deformed by using a marker capable of directly or indirectly providing a detectable signal. For example, the marker may include a radioactive isotope, a fluorescent molecule, biotin, and the like. 
     The term “real-time polymerase chain reaction (real-time PCR)” refers to a molecular biological polymerization method using a DNA as a template to amplify a target by using a target probe including a target primer and a marker and simultaneously quantitatively detect a signal generated from the marker of the target probe to the amplified target. 
     Effect 
     According to the present invention, Real GP-GN/Real Can using a real-time PCR method which can rapidly distinguish gram positive bacteria, gram negative bacteria, and  Candida  species and can be substituted for the above-described blood culture and REBA (Reverse blot hybridization assay) Sepsis-ID as a rapid and accurate identification method capable of distinguishing gram positive bacteria and gram negative bacteria, identifying a fungus including  Candida  spp., and identifying whether or not MRSA and VRE have antibiotic resistance at the same time have been developed, and usefulness thereof have been confirmed. 
    
    
     
       DESCRIPTION OF DRAWINGS 
         FIG. 1  shows a result of a PCR amplified product for REBA Sepsis-ID; 
         FIGS. 2 to 5  show results of specificity of REBA Sepsis-ID in a reference strain; 
         FIG. 6  shows a result of color development of REBA Sepsis-ID using a reference strain; 
         FIGS. 7 and 8  show result examples of REBA Sepsis-ID in a clinical blood culture strain; 
         FIG. 9A-D  are provided to check sensitivity in gram-positive bacteria, gram-negative bacteria, and a fungus by using a real-time PCR; and 
         FIG. 1D  shows Real GP-GN PCR negative and positive specimens in Lanes 1 to 21 as a result of REBA Sepsis-ID with respect to 21 negative and positive specimens from a blood culture. 
     
    
    
     BEST MODE 
     Hereinafter, the present invention will be described in detail with reference to non-limited examples below. The following examples are provided for illustrating the present invention. Therefore, the scope of the present invention should not be construed as being limited by the following examples. 
     Example 1 
     Materials 
     Blood culture positive bacterial species from a blood culture specimen sent to the Department of the Laboratory Medicine of Wonju Severance Christian Hospital and a specimen having an antimicrobial susceptibility test result were used. 
     For a blood culture, 5 to 10 ml of blood from a suspected bacteremia case was inoculated into each of a pair of blood culture bottles including a BACTEC Standard 10 Aerobic/F bottle and a BACTEC PLUS Anaerobic/F bottle. All the blood culture bottles were received within 24 hours and cultured within 3 hours in two kinds of automated blood culture systems, a BACTEC 9240 system (BD, Franklin Lakes, N. J., USA) and a BacT/Alert 3D system (BioMerieux, Durham, N. C., *SA), at 37° C. for 5 days. When a blood culture-positive signal came out, a subculture was carried out and then Gram staining, identification of a bacterial species, and an antimicrobial susceptibility test were carried Out. The identification of a bacterial species was carried out by a biochemical method using a Vitek II system (BioMerieux, Marcy, l&#39;Eltoile, France). As for a  Candida  species, after a germ tube test, if positive, the  Candida  species was identified as a  Candida albicans , and if negative, the  Candida  species was identified by using a ATB ID 32C (BioMerieux S A, Marcy-l&#39;Etoile, France). 
     Example 2 
     Extraction of Genomic DNA from Reference Strain and Clinical Isolate 
     A part of a culture medium cultured in a BACTEC 9240 system (Becton Dickinson Microbiology System, Sparks, Md, USA) was obtained. 100 μl of a DNA extraction solution was put into the obtained culture medium, vortexed for 1 minute, heated at 100° C. for 10 minutes, and centrifuged at room temperature at 13,000 rpm for 3 minutes so as to obtain a supernatant. A nucleic acid was separated by using this method. The separated nucleic acid was used as a template of a PCR for implementing Real GP-GN/Real Can and REBA Sepsis-ID. 
     Example 3 
     Gene Collection and Analysis in Real GP-GN/Real can and REBA Sepsis-ID 
     From Genbank of National center for biotechnology information (NCBI, http://www.ncbi.nlm.nih.gov)), as targeted PCR primers and oligonucleotide probes for accurate identification of a bacterial species of sepsis, a 16s rRNA gene for detecting gram-positive bacteria and gram-negative bacteria, an ITS (internal transcribed sequence) gene existing between 18S rRNA and 5.8S rRNA for detecting a fungus, a MecA gene for checking whether or not MRSA has antibiotic resistance, and VanA and VanB genes for checking whether or not VRE have antibiotic resistance were searched, and base sequences thereof were collected. After multi-alignment (http://multalin.toulouse.inra.fr/multalin) was carried out, two pairs of primers were designed at a base sequence site common to the targeted genes. At a reverse primer of them, biotin was attached to 5′-terminal for PCR-REBA. Then, oligonucleotide probes capable of detecting target bacterial species from pathogen-specific sites existing within the two pairs of primers were designed. At each of the oligonucleotide probes, an amine group was attached to 5′-terminal so as to form a peptide bond with a thin membrane. As for the designed primers and oligonucleotide probes, Bioneer (Daejeon, Korea) was requested to synthesize them. 
     Example 4 
     One-Tube Nested PCR 
     A PCR was carried out with the extracted genomic DNA of each strain as a template by using a commercialized Prime Taq Premix (2×) (Genet Bio, Nonsan, Korea). A composition of Prime Taq Premix (2×) included primer Taq polymerase I unit/10 μl, a 2× reaction buffer, 4 mM MgCl 2 , an enzyme stabilizer, a sediment, a loading dye, pH 9.0, 0.5 mM of each dATP, dCTP, dGTP, and dTTP. Each composition for PCR included 10 μl of Prime Taq premix (2×), 1 μl of each of a pair of 10 pmole primers, 3 μl of ultrapure water, and 5 μl of the genomic DNA of each strain to be the total reaction amount of 20 μl. During the PCR, pre-denaturation at 95° C. for 5 minutes, primary amplification at 95° C. for 30 seconds, and a reaction at 60° C. for 30 seconds were carried out repeatedly 15 times; then secondary amplification at 95° C. for 30 seconds and a reaction at 54° C. for 30 seconds were carried out repeatedly 35 times; and then full extension at 72° C. for 10 minutes was carried out. After the PCR was completed, a PCR product was electrophoresed in a 2% TBE (Tris-borate-ethylenediaminetetraacetic acid disodium salt dehydrate) agarose gene (W/V ratio) at 290 bolt for 20 minutes and dyed in ethidium bromide for 10 minutes. Then, whether or not the PCR product was amplified was checked. Herein, a 16s rRNA capable of identifying GN-GP had a sequence of 280 bp, an ITS gene positioned between 18S and 5.8S rRNAs and capable of identifying a fungus had a sequence of 250 bp, a nuc gene specific to only  S. aureus  had a sequence of 136 bp, a Spn gene gene specific to only  S. pneumoniae  had a sequence of 120 hp, a MecA gene for checking methicillin resistance had a sequence of 140 bp, and VanA and VanB genes for checking vancomycin resistance had sequences of 170 hp and 100 hp, respectively. 
     Example 5 
     Implementation of REBA Sepsis-ID 
     REBA Sepsis-ID using the amplified PCR product was carried out in experimental conditions suggested by a manufacturer according to the following experiment method. The double stranded PCR product was mixed and reacted with an equivalent amount of a denaturation solution (0.2 N NaOH, 0.2 mM EDTA) at room temperature for 5 minutes to be single stranded. During the reaction, a thin REBA Sepsis-ID membrane (M&amp;D, Korea) attached with a pathogen-specific oligonucleotide probe was diluted in 2× SSPE/0.1% SDS, put into a Membrane minitray (Bio-rad, USA), reacted at 55° C. for 30 minutes, washed two times with a WS (Washington solution) at 55° C. for 10 minutes, and reacted at room temperature for 30 minutes with an alkaline phosphatase-labeled Streptavidin conjugate (Roche, Mannheim, Germany) diluted at 1:2000 (v/v). After the reaction, the membrane was washed two times at room temperature for 1 minute with a SS (Staining solution, TBS pH 7.5), exposed for 10 minutes to NBT/BCIP (Nitro blue tetrazolium chloride and 5-Bromo-4-chloro-3-indolylphosphate, toluidine salt in 67% DMSO (v/v), Roche, Germany) as a staining solution reacting to the alkaline phosphatase, washed with DW, and then dried to determine whether or not there was detection. 
     Example 6 
     Implementation of Real GP-GN/Real Can 
     Real GP-GN/Real Can used the extracted genomic DNA of each strain as a template with addition of 10 μl of a 2× PCR premix (TOYOBO, Japan), 5 μl of a mixture of GP-GN primer/probe, and 5 μl of an extracted DNA specimen to be the total amount of 20 μl. PCR conditions included one-time pre-denaturation at 94° C. for 3 minutes, denaturation at 94° C. for 20 seconds, annealing at 60° C. for 40 seconds, extension, and a reaction using CFX 96 (Bio-Rad, USA) 40 times in total. Real GP-GN included a GP probe (HEX) for detecting gram-positive bacteria, a GN probe (FAM) for detecting gram-negative bacteria, and an internal control (IC) probe (Cy5). A measured threshold cycle (Ct) value was analyzed and determined as being positive if equal to or lower than 35 and determined as being negative if higher than 35. Further, Real Can used a Can probe (FAM) for detecting a  Candida  species, and in the same manner, a threshold cycle (Ct) value was analyzed and determined as being positive if equal to or lower than 35 and determined as being negative if higher than 35. Furthermore, when the IC probe and the GP probe were detected at the same time or when only the GP probe was detected, it was determined as gram-positive bacteria; when the IC probe and the GN probe were detected at the same time or when only the GN probe was detected, it was determined as gram-negative bacteria; and when the IP probe and the GN-GP probes were detected at the same time or when the GN-GP probes were detected at the same time, it was determined that gram-positive bacteria and gram-negative bacteria were mixed. However, when any signal from the probes including the IC probe was not detected, it was determined as failure of the test. 
     Results of Examples above were as shown below. 
     1. Result of PCR Amplification 
     PCR amplification products having a sequence of 280 hp could be observed from gram-positive bacteria such as  E. facalis, E. solitarius, E. faecium, E. malodoratus, E. saccharolytivus, E. casseliflavus, S. pneumoniae, S. agalactiae , etc. (photograph A and B of  FIG. 1 ) and gram-negative bacteria such as  E. coli, K. pneumoniae, K. oxytoca, S. liquifaciens, P. alcalifaciens, P. vulgaris, P. mirabilis, S. typhi , etc. (photograph C of  FIG. 1 ) using the 16s rRNA gene. Further, a PCR amplification product having a sequence of 250 hp could be observed from a  Candida -related DNA such as  C. albicans, C. tropicalis, C. glabrala, C. parapsilosis , etc. using the ITS gene between the 18S and 5.8S rRNAs (photograph D of  FIG. 1 ). 
     2. Pathogen Specificity Test of GN-GP Bacteria Using Reference Strain 
     PCR-REBA was carried out to check specificity of the pathogen-specific oligonucleotide probe manufactured by using the PCR amplification product through a reference strain. As a result thereof, it could be confirmed that all of 38 gram-positive strains made positive reactions with the gram-positive probe, an  Enterococcus  spp. probe ( FIG. 2 ), a  Streptococcus  probe ( FIG. 3 ), and a  Stapyhlococcus  probe ( FIG. 4 ), and all of 21 gram-negative strains made positive reactions the gram-negative probe and specific probes of respective bacterial species ( FIG. 5 ). Further, a result of the staining using a membrane strip can be seen from  FIG. 6 . 
     3. Evaluation of Usefulness of PCR-REBA Using Clinical Solid Culture Strain 
     PCR-REBA was carried out to 118 solid culture strains in total including clinically separated 41 gram-positive strains, 64 gram-negative strains, and 13 fungi (Table 1). As a result thereof, it could be confirmed that all of the 118 strains were identified by respective probes for gram-positive bacteria, gram-negative bacteria, and fungi. 
     4. Evaluation of Usefulness of PCR-REBA Using Clinical Blood Culture Positive Strain 
     As a result of evaluating usefulness of REBA Sepsis-ID using 70 gram-positive strains, 32 gram-negative strains, and 13 fungi through clinical blood culture positive strains ( FIG. 7 ), all of 13  Staphylococcus aureus  specimens, 23  S. epidermidis  specimens, 5  S. capitis  specimens, 5  S. haemolyticus  specimens, 5  S. hominis  specimens, and a  S. warneri  specimen among the 70 gram-positive strains were identified by a  Staphylococcus  spp. probe;  Streptococcus parasanguinis , each  S. salivarius  specimen was identified by a  Streptococcus  spp. probe, and it could be confirmed that all of 6  Enterococcus faecium  specimens were detected by an  Enterococcus  spp. probe. An  E. facalis  specimen was detected by an  Enterococcus  spp. probe, and it could be confirmed that a  Corynebacterium  spp. specimen, 2  Micrococcus  spp. specimens, a  Propionibacterium acnes  specimen, 5 Gram-positive rods specimens were detected by a GP probe. It was confirmed that among the 32 gram-negative strains, all of 14  Escherichia coli  specimens, 7  Klebsiella pneumonia  specimens, and each of  Salmonella  group,  Haemophilus influenzae, Pseudomonas aeruginosa  specimens were detected by the probes respectively corresponding thereto. It was confirmed that all of 2  K. oxytoca  specimens and each of  Acinetobacter lwoffii, Aeromonas  spp.,  Citrobacter koseri, Neisseria sicca, Proteus mirabilis, Sphingomonas paucimobilis  specimens were detected by a GN probe. Further, it was confirmed that among the 13 fungi specimens, all of 8  C. albicans  specimens, 2  C. parapsilosis  specimens, and a  C. glabrata  specimen were detected by the probes respectively corresponding thereto, and it was confirmed that a  Cryptococcus neoformans  and a  Saccharomyces cerevisiae  specimen were detected by Pan-fungus (Table 2). 
     5. Evaluation of Usefulness of MRSA and VRE Through Antibiotic Resistance Test Strain 
     As a result of checking whether or not the MecA gene was detected to check methicillin resistance by using the solid culture gram-positive strains, 9 of 12  S. aureus  specimens were confirmed as MRSA, and 7 of 8  Staphylococcus  spp. specimens were confirmed as MRCoNS, and, thus, detection of the MecA gene was confirmed (Table 1). Further, among the blood culture positive specimens, 10 of 13  S. aureus  specimens were confirmed as MRSA, and 32 of 39  Staphylococcus  spp. specimens were confirmed as MRCoNS, and, thus, existence of the MecA gene could be confirmed. In a test for checking vancomycin resistance regarding  Enterococcus  spp., it was confirmed that 7 of 16 solid culture strains had a resistance to the VanA gene and it was confirmed that 2 of 7 blood culture positive specimens had a resistance to the VanA gene (Table 2). 
     6. Check of Sensitivity of Real GP-GN/Real Can 
     In order to rapidly check gram-positive bacteria, gram-negative bacteria, or fungi first during a blood culture, the Real-time PCR method was used. In order to do so, sensitivity in gram-positive bacteria, gram-negative bacteria, and fungi were checked by 10 times diluting DNAs of  S. aureus  as gram-positive bacteria,  E. coli  as gram-negative bacteria, and  C. glabrata  as fungi. As a result thereof, it could be found that the gram-positive bacteria and the gram-negative bacteria showed sensitivity in a range of 100 fg to 10 fg, and the fungi showed sensitivity of 1 pg ( FIG. 8 ). 
     7. Result of Real GP-GN PCR Using Reference Strain 
     As a result of checking specificity of Real GP-GN by using 63 reference strain specimens, it could be confirmed that all of 39 gram-positive bacteria specimens were detected by a GP (HEX) probe, all of 24 gram-negative bacteria specimens were detected by a GN (FAM) probe, and all of 5  Candida  strains were detected by a Can (FAM) probe (Tables 3 and 4). 
     8. Check of Usefulness of Real GP-GN PCR Using Solid Culture Bacteria 
     As a result of checking usefulness of Real GP-GN/Real Can by using 105 solid culture bacteria specimens, it could be confirmed that all of 41 gram-positive bacteria specimens including 12  S. aureus  specimens were detected by a GP (HEX) probe with a Ct value of 17.35 to 30.46. Further, it could be confirmed that all of 64 gram-negative bacteria specimens including 16  E. coli  specimens were detected by a GN (FAM) probe with a Ct value of 12.68 to 33.54, and it could be confirmed that all of 10  Candida  specimens including 5  C. albicans  specimens were detected by a Can (FAM) probe with a Ct value of 17.61 to 30.95 (Table 5). 
     9. Check of Usefulness of Real GP-GN PCR Using Blood Culture Positive Bacteria 
     As a result of checking usefulness of Real GP-GN by using 176 positive specimens from a blood culture bottle, all of 175 gram-positive bacteria specimens were detected by a GP (HEX) probe and a Ct value at that time was in a range of 12.43 to 34, and all of 70 gram-negative bacteria specimens were detected by a GN (FAM) probe and a Ct value at that time was in a range of 6.56 to 24.08. As a result of Real GP-GN PCR of a specimen obtained from the culture and including a mixture of  S. agalactiae  and  C. koseri , Ct values of 14.61 and 10.61 were shown at GP and GN probes, respectively. Therefore, it was confirmed that even the mixed specimens could be detected accurately. Thus, it could be found that sensitivity of seroprevalence of the Real GP-GN PCR using the positive bacteria in the blood culture bottle was 99.6%. As a result of checking usefulness of Real Can by using 24 fungi specimens, 2  C. albicans  specimens and each of  C. parapsilosis  and  C. tropicalis  specimens were detected by a Can (FAM) probe and a Ct value at that time was in a range of 22.81 to 31.98 (Tables 6 to 8). 
     10. Check of Usefulness of Real GP-GN PCR Using Blood Culture Negative Bacteria 
     As a result of checking usefulness of Real GP-GN PCR by using 200 negative specimens from a blood culture bottle, 21 specimens in total including 7 gram-positive bacteria specimens by a GP (HEX) probe and 14 gram-negative bacteria specimens by a GN (FAM) probe were confirmed as being positive. Thus, it was found that specificity was 89.5% (Table 9). The positive specimens from the Real GP-GN PCR underwent PCR REBA and sequencing, and results thereof were compared ( FIG. 9A-D ). As a result of the PCR-REBA, 7 Real GP specimens were detected by a GP probe, and among 14 Real GN specimens, 12 specimens were detected by a GN probe, another specimen was detected by Pan-bacteria, and the other specimen was not detected. As a result of the sequencing, all of 7 gram-positive specimens were shown as uncultured bacteria. One of 14 gram-negative specimens was shown as an uncultured bacterium, and among the other 13 specimens, 2 Proteobacterium specimens and 2  Pseudomonas  spp. specimens were detected and the other specimens were  Janthinoba cterium  sp.,  Polynucleobacter  sp.,  Hyalangium  sp.,  Duganella  sp.,  Ochrobacterum  sp., Burkholderiales bacterium,  Methylophilus  sp., Nitrosomonadaceae, and  Enterobacter  sp. (Table 10). 
     Although the conventional culture method has been used so far to check gram-positive bacteria and gram-negative bacteria, it is possible to rapidly and accurately check gram-positive bacteria and gram-negative bacteria through Real GP-GN in 1 hour to 1 hour and 30 minutes and also possible to check bacterial species of the gram-positive bacteria and gram-negative bacteria, methicilin resistance, and vancomycin resistance through REBA Sepsis-ID in further 1 hour to 1 hour to 30 minutes, i.e. 4 hours in total. Therefore, it can be substituted for the culture method in which a culture takes 3 to 5 days and then antibiotic resistance is checked and a result thereof can be obtained in further 5 to 7 days. Further, the Real GP-GN PCR method is a useful method which can be substituted for the culture method considering that even if a culture is carried out for 3 to 5 days, there may be a negative result and the culture negative bacteria as shown in the present experiment has a seroprevalence of about 10%. 
     
       
         
           
               
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                   
                 Number of isolates 
               
            
           
           
               
               
               
            
               
                 Conventional methods 
                 REBA Sepsis-ID 
                 Antibiotics resistance 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Gram 
                 
                   Staphylococcus aureus 
                 
                 12 
                 
                   S. aureus 
                 
                 MRSA (9) 
               
               
                 positives 
                 
                   S. epidermidis 
                 
                 4 
                   Staphylococcus  sp. 
                 MRCoNS (4) 
               
               
                 (41) 
                 
                   S. haemolyticus 
                 
                 3 
                   Staphylococcus  sp. 
                 MRCoNS (3) 
               
               
                   
                 
                   S. capitis 
                 
                 1 
                   Staphylococcus  sp. 
                 MRCoNS (1) 
               
               
                   
                 
                   Streptococcus agalactiae 
                 
                 1 
                   Streptococcus  sp. 
                   
               
               
                   
                 
                   S. mitis 
                 
                 2 
                   Streptococcus  sp. 
                   
               
               
                   
                 
                   S. parasanguis 
                 
                 1 
                   Streptococcus  sp. 
                   
               
               
                   
                 
                   S. salivorius 
                 
                 1 
                   Streptococcus  sp. 
                   
               
               
                   
                 
                   S. pyogenes 
                 
                 1 
                   Streptococcus  sp. 
                   
               
               
                   
                 
                   Enterococcus faecalis 
                 
                 4 
                   Enterococcus  sp. 
                   
               
               
                   
                 
                   E. faecium 
                 
                 10 
                   Enterococcus  sp. 
                 VRE (7) 
               
               
                   
                 
                   E. mundtii 
                 
                 1 
                   Enterococcus  sp. 
                   
               
               
                   
                   Corynebacterium  spp. 
                 1 
                 Gram positives 
                   
               
               
                   
                 Enterobacteriaceae 
                   
                   
                   
               
               
                 Gram 
                 
                   Escheria coli 
                 
                 16 
                 
                   E. coli 
                 
                   
               
               
                 negatives 
                 
                   Enterobacter asburiae 
                 
                 1 
                 Gram negatives 
                   
               
               
                 (64) 
                 
                   E. cloacae 
                 
                 1 
                 Gram negatives 
                   
               
               
                   
                 
                   Klebsiella pneumoniae 
                 
                 14 
                 
                   K. pneumoniae 
                 
                   
               
               
                   
                 
                   Citrobacter freundii 
                 
                 1 
                 
                   C. freundii 
                 
                   
               
               
                   
                 
                   Morganella morgannii 
                 
                 1 
                 Gram negatives 
                   
               
               
                   
                 
                   Proteus mirabilis 
                 
                 1 
                 Gram negatives 
                   
               
               
                   
                 
                   Serratio marcescens 
                 
                 1 
                 Gram negatives 
                   
               
               
                   
                 
                   Providencia rettgeri 
                 
                 1 
                 Gram negatives 
                   
               
               
                   
                 Glucose non-fermenter 
                   
                   
                   
               
               
                   
                 
                   Acinetobacter baumannii 
                 
                 11 
                 
                   A. baumannii 
                 
                   
               
               
                   
                 
                   Pseudomonas aeruginosa 
                 
                 13 
                 
                   P. aeruginosa 
                 
                   
               
               
                   
                 Others 
                   
                   
                   
               
               
                   
                   Aeromonas  spp. 
                 1 
                 Gram negatives 
                   
               
               
                   
                 
                   Haemophilus influenzae 
                 
                 1 
                 
                   H. influenzae 
                 
                   
               
               
                   
                 
                   Moraxella catarrholis 
                 
                 1 
                 Gram negatives 
                   
               
               
                 Fungus 
                 
                   Candido albicans 
                 
                 5 
                 
                   C. albicans 
                 
                   
               
               
                 (13) 
                 
                   C. glabrata 
                 
                 1 
                 
                   C. glabrata 
                 
                   
               
               
                   
                 
                   C. parapsilosis 
                 
                 3 
                 
                   C. parapsilosis 
                 
                   
               
               
                   
                 
                   C. tropicalis 
                 
                 2 
                 
                   C. tropicalis 
                 
                   
               
               
                   
                 
                   Saccharomyces cerevisiae 
                 
                 2 
                 Fungi 
                   
               
               
                 Total 
                   
                 118 
                   
                 24 
               
               
                   
               
            
           
         
       
     
     Table 1 shows a comparison between a culture method and a PCR-REBA method in 118 solid culture strains. 
     
       
         
           
               
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Conventional methods 
                 REBA Sepsis-ID 
               
            
           
           
               
               
               
               
            
               
                 Genus and species (n) 
                 Antibiotic resistance 
                 Genus and species (n) 
                 Antibiotic resistance 
               
               
                   
               
            
           
           
               
            
               
                 Gram-positive bacteria (70) 
               
            
           
           
               
               
               
               
            
               
                   Stahylococcus aureus  (13) 
                 MRSA (10) 
                   S. aureus  (13) 
                 MRSA (10) 
               
               
                   
                 MSSA (3) 
                   
                 MSSA (3) 
               
               
                   S. epidermidis  (23) 
                 MRCoNS (20) 
                   Staphylococcus  spp. (23) 
                 MRCoNS (20) 
               
               
                   
                 MSCoNS (3) 
                   
                 MSCoNS (3) 
               
               
                   S. capitis  (5) 
                 MRCoNS (3) 
                   Staphylococcus  spp. (5) 
                 MRCoNS (3) 
               
               
                   
                 MSCons (1) 
                   
                 MSCons (1) 
               
               
                   S. haemolyticus  (5) 
                 MRCoNS (5) 
                   Staphylococcus  spp. (5) 
                 MRCoNS (5) 
               
               
                   S. hominis  (5) 
                 MRCoNS (3) 
                   Staphylococcus  spp. (5) 
                 MRCoNS (3) 
               
               
                   
                 MSCoNS (2) 
                   
                 MSCoNS (2) 
               
               
                   S. warneri  (1) 
                   
                   Staphylococcus  spp. (1) 
                   
               
               
                   Streptococcus parasanguinis  (1) 
                   
                   Streptococcus  spp. (1) 
                   
               
               
                   S. salivarius  (1) 
                   
                   Streptococcus  spp. (1) 
                   
               
               
                   Enterococcus faecium  (6) 
                 VRE (2) 
                   Enterococcus  spp. (6) 
                   
               
               
                   
                 VSE (4) 
                   
                   
               
               
                   E. faecalis  (1) 
                   
                   Enterococcus  spp. (1) 
                   
               
               
                   Corynebacterium  spp. (1) 
                   
                 Gram positive (1) 
                   
               
               
                   Micrococcus  spp. (2) 
                   
                 Gram positive (1) 
                   
               
               
                   Propionibacterium acnes  (1) 
                   
                 Gram positive (1) 
                   
               
               
                 Gram-positive rods (5) 
                   
                 Gram positive (5) 
                   
               
            
           
           
               
            
               
                 Gram-negative bacteria (32) 
               
            
           
           
               
               
               
               
            
               
                   Escherichia coli  (14) 
                   
                   E. coli  (14) 
                   
               
               
                   Klebsiella pneumoniae  (7) 
                   
                   K. pneumoniae  (7) 
                   
               
               
                   K. oxytoca  (2) 
                   
                 Gram negatives (2) 
                   
               
               
                   Salmonella  group D (1) 
                   
                   Salmonella  spp. (1) 
                   
               
               
                   Acinetobacter lwoffii  (1) 
                   
                 Gram negatives (1) 
                   
               
               
                   Aeromonas  spp. (1) 
                   
                 Gram negatives (1) 
                   
               
               
                   Citrobacter koseri  (1) 
                   
                 Gram negatives (1) 
                   
               
               
                   Haemophilus influenzae  (1) 
                   
                   H. influenzae  (1) 
                   
               
               
                   Neisseria sicca  (1) 
                   
                 Gram negatives (1) 
                   
               
               
                   Proteus mirabilis  (1) 
                   
                 Gram negatives (1) 
                   
               
               
                   Pseudomonas aeruginosa  (1) 
                   
                   P. aeruginosa  (1) 
                   
               
               
                   Splingomonas paucimobilis  (1) 
                   
                 Gram negatives (1) 
                   
               
            
           
           
               
            
               
                 Fungi (13) 
               
            
           
           
               
               
               
               
            
               
                   Candida albicans  (8) 
                   
                   C. albicans  (8) 
                   
               
               
                   C. parapsilosis  (2) 
                   
                   C. parapsilosis  (2) 
                   
               
               
                   C. glabrata  (1) 
                   
                   C. glabrata  (1) 
                   
               
               
                   Cryptococcus neoformans  (1) 
                   
                 Fungi (1) 
                   
               
               
                   Saccharomyces cerevisiae  (1) 
                   
                 Fungi (1) 
               
               
                   
               
            
           
         
       
     
     Table shows a comparison between a culture method and a PCR-REBA method in 115 blood culture positive specimens. 
     
       
         
           
               
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                   
                 Real-time PCR TaqMan 
               
               
                   
                 assay (C T  value) 
               
            
           
           
               
               
               
               
               
               
            
               
                 Genus 
                 Species 
                 *ATCC no. 
                 Real-GP ™ 
                 Real-GN ™ 
                 Real-CAN ™ 
               
               
                   
               
            
           
           
               
            
               
                 Gram positive bacteria (39) 
               
            
           
           
               
               
               
               
               
               
            
               
                   Staphylococcus  (3) 
                 
                   S. aureus 
                 
                 29213 
                 26.59 
                   s UD 
                 UD 
               
               
                   
                 
                   S. aureus 
                 
                 25923 
                 28.42 
                 UD 
                 UD 
               
               
                   
                 
                   S. xylosus 
                 
                 29971 
                 20.81 
                 UD 
                 UD 
               
               
                   Enterococcus  (17) 
                 
                   E. hirae 
                 
                 9790 
                 26.31 
                 UD 
                 UD 
               
               
                   
                 
                   E. raffinosus 
                 
                 49427 
                 28.09 
                 UD 
                 UD 
               
               
                   
                 
                   E. sulfureus 
                 
                 49903 
                 28.40 
                 UD 
                 UD 
               
               
                   
                 
                   E. durans 
                 
                 19432 
                 23.48 
                 UD 
                 UD 
               
               
                   
                 
                   E. casseliflavus 
                 
                 700327 
                 25.98 
                 UD 
                 UD 
               
               
                   
                 
                   E. faecium 
                 
                 19434 
                 26.72 
                 UD 
                 UD 
               
               
                   
                 
                   E. faecalis 
                 
                 29212 
                 25.33 
                 UD 
                 UD 
               
               
                   
                 
                   E. mundtii 
                 
                 43186 
                 29.90 
                 UD 
                 UD 
               
               
                   
                 
                   E. cecorum 
                 
                 43198 
                 21.68 
                 UD 
                 UD 
               
               
                   
                 
                   E. flavescens 
                 
                 49997 
                 22.21 
                 UD 
                 UD 
               
               
                   
                 
                   E. gallinarum 
                 
                 49573 
                 23.88 
                 UD 
                 UD 
               
               
                   
                 
                   E. faeclis 
                 
                 51299 
                 24.10 
                 UD 
                 UD 
               
               
                   
                 
                   E. solitarius 
                 
                 49428 
                 30.44 
                 UD 
                 UD 
               
               
                   
                 
                   E. faecium 
                 
                 35667 
                 24.66 
                 UD 
                 UD 
               
               
                   
                 
                   E. malodoratus 
                 
                 43197 
                 27.02 
                 UD 
                 UD 
               
               
                   
                 
                   E. saccharolyticus 
                 
                 43076 
                 23.06 
                 UD 
                 UD 
               
               
                   
                 
                   E. casseliflavus 
                 
                 25788 
                 26.28 
                 UD 
                 UD 
               
               
                   Streptococcus  (2) 
                 
                   S. puemoniae 
                 
                 49619 
                 21.11 
                 UD 
                 UD 
               
               
                   
                 
                   S. agalactiae 
                 
                 13813 
                 26.41 
                 UD 
                 UD 
               
               
                   Micrococcus  (1) 
                 
                   M. luteus 
                 
                 49732 
                 22.36 
                 UD 
                 UD 
               
               
                   Mycobacterium  (16) 
                 
                   M. avium 
                 
                 25291 
                 23.06 
                 UD 
                 UD 
               
               
                   
                 
                   M. chelonae 
                 
                 35749 
                 21.34 
                 UD 
                 UD 
               
               
                   
                 
                   M. gastri 
                 
                 15754 
                 23.9 
                 UD 
                 UD 
               
               
                   
                 
                   M. kcoisasii 
                 
                 12478 
                 22.17 
                 UD 
                 UD 
               
               
                   
                 
                   M. nonchromogenicum 
                 
                 19530 
                 18.31 
                 UD 
                 UD 
               
               
                   
                 
                   M. phlei 
                 
                 11758 
                 25.09 
                 UD 
                 UD 
               
               
                   
                 
                   M. smegunatis 
                 
                 19420 
                 24.4 
                 UD 
                 UD 
               
               
                   
                 
                   M. triviale 
                 
                 23292 
                 22.66 
                 UD 
                 UD 
               
               
                   
                 
                   M. aurum 
                 
                 23366 
                 24.83 
                 UD 
                 UD 
               
               
                   
                 
                   M. farcinogen 
                 
                 35753 
                 20 
                 UD 
                 UD 
               
               
                   
                 
                   M. gilvum 
                 
                 43909 
                 19.4 
                 UD 
                 UD 
               
               
                   
                 
                   M. ncoaurum 
                 
                 25795 
                 17.83 
                 UD 
                 UD 
               
               
                   
                 
                   M. parafortninum 
                 
                 19686 
                 19.06 
                 UD 
                 UD 
               
               
                   
                 
                   M. peregrinum 
                 
                 14467 
                 18.57 
                 UD 
                 UD 
               
               
                   
                 
                   M. septicum 
                 
                 700731 
                 23.47 
                 UD 
                 UD 
               
               
                   
                 
                   M. abscessus 
                 
                 19977 
                 21.73 
                 UD 
                 UD 
               
               
                   
               
            
           
         
       
     
     Table 3 shows detection of specificity of Real GP-GN/Real Can PCR by using 39 reference strain specimens. 
     
       
         
           
               
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                   
                 Real-time PCR TaqMan 
               
               
                   
                 assay (C T  value) 
               
            
           
           
               
               
               
               
               
               
            
               
                 Genus 
                 Species 
                 *ATCC no. 
                 Real-GP ™ 
                 Real-GN ™ 
                 Real-CAN ™ 
               
               
                   
               
            
           
           
               
            
               
                 Gram-negative bacteria (24) 
               
            
           
           
               
               
               
               
               
               
            
               
                   Escherichia  (2) 
                 
                   E. coli 
                 
                 25922 
                 UD 
                 20.46 
                 UD 
               
               
                   
                 
                   E. coli 
                 
                 35218 
                 UD 
                 17.90 
                 UD 
               
               
                   Enterobacter  (1) 
                 
                   E. aerogenes 
                 
                 1304 
                 UD 
                 19.09 
                 UD 
               
               
                   Citrobacter  (1) 
                 
                   C. freundii 
                 
                 6750 
                 UD 
                 14.19 
                 UD 
               
               
                   Shigella  (3) 
                 
                   S. boydii 
                 
                   # DMl.399 
                 UD 
                 25.59 
                 UD 
               
               
                   
                 
                   S. dysenteriae 
                 
                 DMl.400 
                 UD 
                 18.38 
                 UD 
               
               
                   
                 
                   S. flexneri 
                 
                 9199 
                 UD 
                 21.82 
                 UD 
               
               
                   Serratia  (1) 
                 
                   S. liquifaciens 
                 
                 27952 
                 UD 
                 24.96 
                 UD 
               
               
                   Salmonella  (5) 
                 
                   S. typhi 
                 
                 19430 
                 UD 
                 16.54 
                 UD 
               
               
                   
                 
                   S. enteriridis 
                 
                 13076 
                 UD 
                 20.15 
                 UD 
               
               
                   
                 
                   S. paratyphi 
                 
                 11511 
                 UD 
                 19.61 
                 UD 
               
               
                   
                 
                   S. typhimuriun 
                 
                 13311 
                 UD 
                 16.19 
                 UD 
               
               
                   
                 
                   S. newport 
                 
                 6962 
                 UD 
                 17.22 
                 UD 
               
               
                   Klebsiella  (2) 
                 
                   K. pneumaniae 
                 
                 13883 
                 UD 
                 20.60 
                 UD 
               
               
                   
                 
                   K. oxytoca 
                 
                 700324 
                 UD 
                 21.87 
                 UD 
               
               
                   Proteus  (3) 
                 
                   P. alcalifaciens 
                 
                 51902 
                 UD 
                 18.38 
                 UD 
               
               
                   
                 
                   P. vulgaris 
                 
                 49132 
                 UD 
                 17.13 
                 UD 
               
               
                   
                 
                   P. unirabilis 
                 
                 49132 
                 UD 
                 16.34 
                 UD 
               
               
                   Pseudomonas  (2) 
                 
                   P. cepacia 
                 
                 25608 
                 UD 
                 19.66 
                 UD 
               
               
                   
                 
                   P. aeruginosa 
                 
                 27853 
                 UD 
                 16.57 
                 UD 
               
               
                   Acinetobacter  (1) 
                 
                   A. baumannii 
                 
                 17978 
                 UD 
                 19.69 
                 UD 
               
               
                   Haemophilus  (1) 
                 
                   H. influenzae 
                 
                 49247 
                 UD 
                 18.61 
                 UD 
               
               
                   Leclercia  (1) 
                 
                   L. adecarboxylata 
                 
                 23216 
                 UD 
                 15.70 
                 UD 
               
               
                   Bordetella  (1) 
                 
                   H. branchiseptica 
                 
                 10580 
                 UD 
                 19.44 
                 UD 
               
            
           
           
               
            
               
                 Fungi 
               
            
           
           
               
               
               
               
               
               
            
               
                   Candida  (5) 
                 
                   C. albicans 
                 
                 36802 
                 UD 
                 UD 
                 26.42 
               
               
                   
                 
                   C. tropicalis 
                 
                 14506 
                 UD 
                 UD 
                 25.98 
               
               
                   
                 
                   C. glabrata 
                 
                 38326 
                 UD 
                 UD 
                 17.09 
               
               
                   
                 
                   C. parapsilosis 
                 
                 7330 
                 UD 
                 UD 
                 24.27 
               
               
                   
                 
                   C. krusei 
                 
                 20298 
                 UD 
                 UD 
                 19.67 
               
               
                   
               
               
                 *ATCC: American type culture collection. 
               
               
                   # DML: Diagnostic Microbiology Laboratory. Biomedical laboratory science. Yonsci University 
               
            
           
         
       
     
     Table 4 shows detection of specificity of Real GP-GN/Real Can PCR by using 29 reference strain specimens. 
     
       
         
           
               
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                   
                 Real-time PCR TaqMan 
               
               
                   
                 assay (C T  value) 
               
            
           
           
               
               
               
               
               
            
               
                 Culture identification 
                 No. of samples 
                 GP/GN or Fungi 
                 Ranged C T  Value 
                 Mean C T  Value 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 
                   Staphylococcus aureus 
                 
                 12 
                 GP 
                 22.44-26.65 
                 24.47 
               
               
                   Staphylococcus  spp. (CoNS) 
                 8 
                 GP 
                 19.46-28.71 
                 21.5 
               
               
                   Streptococcus  spp. 
                 5 
                 GP 
                 17.35-30.46 
                 24.25 
               
               
                 
                   Enterococcus faecalis 
                 
                 4 
                 GP 
                 25.2-27.3 
                 26.37 
               
               
                 
                   E. faecium 
                 
                 10 
                 GP 
                 21.3-31.6 
                 26.58 
               
               
                 
                   E. mundtii 
                 
                 1 
                 GP 
                 27.85 
                 27.85 
               
               
                   Corynebacterium  spp. 
                 1 
                 GP 
                 24.51 
                 24.51 
               
               
                 
                   Escherichia coli 
                 
                 16 
                 GN 
                 12.68-30.65 
                 23.26 
               
               
                 
                   Klebsiella pneumoniae 
                 
                 13 
                 GN 
                 15.48-26.08 
                 20.73 
               
               
                 
                   Pseudomonas aeruginosa 
                 
                 13 
                 GN 
                 15.23-19.96 
                 18.11 
               
               
                 
                   Acinetobacter baumannii 
                 
                 11 
                 GN 
                 18.09-24.65 
                 21.04 
               
               
                 
                   Enterobacter asburiae 
                 
                 1 
                 GN 
                 15.79 
                 15.79 
               
               
                 
                   E. coloacae 
                 
                 1 
                 GN 
                 15.41 
                 15.41 
               
               
                 
                   E. asburiae 
                 
                 1 
                 GN 
                 15.79 
                 15.79 
               
               
                 
                   Moraxella catarrhalis 
                 
                 1 
                 GN 
                 33.54 
                 33.54 
               
               
                 
                   Serratia marcescens 
                 
                 1 
                 GN 
                 21.64 
                 21.64 
               
               
                 
                   Providencia rettgeri 
                 
                 1 
                 GN 
                 24.3 
                 24.3 
               
               
                 
                   Morganella morganii 
                 
                 1 
                 GN 
                 20.6 
                 20.6 
               
               
                 
                   Proteus mirabilis 
                 
                 1 
                 GN 
                 24.88 
                 24.88 
               
               
                   Aeromonas  spp. 
                 1 
                 GN 
                 25.97 
                 25.97 
               
               
                 
                   Citrobacter fruendii 
                 
                 2 
                 GN 
                 17.11-18.01 
                 17.56 
               
               
                 
                   Candida albicans 
                 
                 5 
                 Can 
                 17.61-29.56 
                 23.9 
               
               
                 
                   C. parapsilosis 
                 
                 3 
                 Can 
                 24.73-30.95 
                 27.58 
               
               
                 
                   C. tropicalis 
                 
                 1 
                 Can 
                 26.62 
                 26.62 
               
               
                 
                   C. glabrata 
                 
                 1 
                 Can 
                 17.68 
                 17.68 
               
               
                 total 
                 115 
               
               
                   
               
            
           
         
       
     
     Table 5 shows detection of specificity of Real GP-GN/Rea. Can PCR by using 115 solid culture bacteria specimens. 
     
       
         
           
               
               
               
               
               
               
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                   
                 Real-time PCR 
                   
                   
                   
                   
                   
               
               
                   
                 (No. of samples) 
                 Sensitivity 
                 Specificity 
                 GP 
                 GN 
                 CAN 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 Blood culture result 
                 Positive 
                 Negative 
                 (%) 
                 (%) 
                 (Ranged C τ ) 
                 (Ranged C τ ) 
                 (Ranged C τ ) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 Blood Culture Positive (276) 
                 275 
                 1 
                 99.6% 
                 — 
                   
                   
                   
               
               
                 Gram positive bacteria (176) 
                 175 
                 1 
                 99.4% 
                 — 
                   
                   
                   
               
               
                   Staphylococccus epidermidis  (47) 
                 47 
                 0 
                   
                   
                 13.08-33.99 
                 UD 
                 UD 
               
               
                   S. aureus  (24) 
                 24 
                 0 
                   
                   
                 13.24-34.19 
                 UD 
                 UD 
               
               
                   S. hominis  (17) 
                 16 
                 1 
                   
                   
                 13.69-30.00 
                 UD 
                 UD 
               
               
                   S. capitis  (14) 
                 14 
                 0 
                   
                   
                 13.60-25.10 
                 UD 
                 UD 
               
               
                   S. haemolyticus  (8) 
                 8 
                 0 
                   
                   
                 14.68-33.3  
                 UD 
                 UD 
               
               
                   S. warneri  (1) 
                 1 
                 0 
                   
                   
                 18.50 
                 UD 
                 UD 
               
               
                   S. saprophyticus  (1) 
                 1 
                 0 
                   
                   
                 16.57 
                 UD 
                 UD 
               
               
                   S. xylosus  (1) 
                 1 
                 0 
                   
                   
                 21.23 
                 UD 
                 UD 
               
               
                   S. chleiferi  (1) 
                 1 
                 0 
                   
                   
                 20.44 
                 UD 
                 UD 
               
               
                   Streptococcus salivarius  (5) 
                 5 
                 0 
                   
                   
                 13.55-23.58 
                 UD 
                 UD 
               
               
                   S. mitis  (4) 
                 4 
                 0 
                   
                   
                 11.52-23.12 
                 UD 
                 UD 
               
               
                   S. pneumoniae  (4) 
                 4 
                 0 
                   
                   
                 16.37-17.77 
                 UD 
                 UD 
               
               
                   S. agalactiae  (2) 
                 2 
                 0 
                   
                   
                   
                 UD 
                 UD 
               
               
                   S. pyogenes  (1) 
                 1 
                 0 
                   
                   
                   
                 UD 
                 UD 
               
               
                   S. dysgalactiae  (1) 
                 1 
                 0 
                   
                   
                 15.81 
                 UD 
                 UD 
               
               
                   S. parasangus  (1) 
                 1 
                 0 
                   
                   
                 12.96 
                 UD 
                 UD 
               
               
                   Streptococcus  spp. (2) 
                 2 
                 0 
                   
                   
                   
                 UD 
                 UD 
               
               
                   Enterococcus faecium  (8) 
                 8 
                 0 
                   
                   
                 26.43-27.54 
                 UD 
                 UD 
               
               
                   K. faecalis  (1) 
                 1 
                 0 
                   
                   
                 14.50 
                 UD 
                 UD 
               
               
                   Micrococcus  spp. (5) 
                 5 
                 0 
                   
                   
                 20.96-31.33 
                 UD 
                 UD 
               
               
                   Propionibacterium acnes  (3) 
                 3 
                 0 
                   
                   
                 23.77-26.86 
                 UD 
                 UD 
               
               
                 
                   Peptostreptococcus 
                 
                 1 
                 0 
                   
                   
                 26.72 
                 UD 
                 UD 
               
               
                   asaccharolyticus  (1) 
                   
                   
                   
                   
                   
                   
                   
               
               
                   Peptostreptococcus micros  (1) 
                 1 
                 0 
                   
                   
                 26   
                 UD 
                 UD 
               
               
                   Corynebacterium  spp. (6) 
                 6 
                 0 
                   
                   
                 26.47 
                 UD 
                 UD 
               
               
                 Gram positive rods (16) 
                 16 
                 0 
                   
                   
                 12.43-34.39 
                 UD 
                 UD 
               
               
                   
               
            
           
         
       
     
     Table 6 shows a comparison between a result of detection by Real GP-GN PCR from a blood culture and a result of a culture in a BACTEC 9240. 
     
       
         
           
               
               
               
               
               
               
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                   
                 Real-time PCR 
                   
                   
                   
                   
                   
               
               
                   
                 (No. of samples) 
                 Sensitivity 
                 Specificity 
                 GP 
                 GN 
                 CAN 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 Blood culture result 
                 Positive 
                 Negative 
                 (%) 
                 (%) 
                 (Ranged C τ ) 
                 (Ranged C τ ) 
                 (Ranged C τ ) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 Gram negative bacteria (70) 
                 70 
                 0 
                 100% 
                   
                   
                   
                   
               
               
                   Escherichia coli  (35) 
                 35 
                 0 
                   
                   
                 UD 
                  9.86-21.89 
                 UD 
               
               
                   Klebsiella pneumoniae  (13) 
                 13 
                 0 
                   
                   
                 UD 
                 12.84-13.2  
                 UD 
               
               
                   Acintobacter baumannii  (5) 
                 5 
                 0 
                   
                   
                 UD 
                 10.53-11.89 
                 UD 
               
               
                   A. lwoffii  (1) 
                 1 
                 0 
                   
                   
                 UD 
                 12.22-15.11 
                 UD 
               
               
                   Enterobacter  spp. (2) 
                 2 
                 0 
                   
                   
                 UD 
                  6.56 
                 UD 
               
               
                   Pseudomonas aeruginosa  (3) 
                 3 
                 0 
                   
                   
                 UD 
                 12.08 
                 UD 
               
               
                   Salmonella  group D (1) 
                 1 
                 0 
                   
                   
                 UD 
                 13.62-21.48 
                 UD 
               
               
                   Proteus mirabilis  (1) 
                 1 
                 0 
                   
                   
                 UD 
                 14.38 
                 UD 
               
               
                   Aeromonas  spp. (2) 
                 2 
                 0 
                   
                   
                 UD 
                 20.1  
                 UD 
               
               
                   Morganella morganii  (1) 
                 1 
                 0 
                   
                   
                 UD 
                 10.39 
                 UD 
               
               
                   Haemophillus influenzae  (1) 
                 1 
                 0 
                   
                   
                 UD 
                 20.68 
                 UD 
               
               
                   Chryseobacterium indologenes  (1) 
                 1 
                 0 
                   
                   
                 UD 
                 14.4  
                 UD 
               
               
                   Sphingomonas paucimobilis  (1) 
                 1 
                 0 
                   
                   
                 UD 
                 24.08 
                 UD 
               
               
                   Serratia marcescens  (1) 
                 1 
                 0 
                   
                   
                 UD 
                   
                 UD 
               
               
                   Citrobacter freundii  (1) 
                 1 
                 0 
                   
                   
                 UD 
                   
                 UD 
               
               
                   
               
            
           
         
       
     
     Table 7 shows a comparison between a result of detection by Real GP-GN PCR from a blood culture and a result of a culture in a BACTEC 9240. 
     
       
         
           
               
               
               
               
               
               
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                   
                 Real-time PCR 
                   
                   
                   
                   
                   
               
               
                   
                 (No. of samples) 
                 Sensitivity 
                 Specificity 
                 GP 
                 GN 
                 CAN 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 Blood culture result 
                 Positive 
                 Negative 
                 (%) 
                 (%) 
                 (Ranged C τ ) 
                 (Ranged C τ ) 
                 (Ranged C τ ) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 Fungus** (24) 
                 24 
                 0 
                 100% 
                   
                   
                   
                   
               
               
                   Candida albicans  (2) 
                 2 
                 0 
                   
                   
                 UD 
                 UD 
                 26.98-31.52 
               
               
                   C. parapsilosis  (1) 
                 1 
                 0 
                   
                   
                 UD 
                 UD 
                 31.98 
               
               
                   C. tropicalis  (1) 
                 1 
                 0 
                   
                   
                 UD 
                 UD 
                 22.81 
               
               
                 *Multiple Infection (6) 
                 6 
                 0 
                 100% 
                   
                   
                   
                   
               
               
                   Streptococcus agalactiae , 
                 1 
                 — 
                   
                   
                 14.61 
                 10.61 
                 UD 
               
               
                   Citrobacter koseri  (1) 
                   
                   
                   
                   
                   
                   
                   
               
               
                   Enterococcus faecium , 
                 1 
                 — 
                   
                   
                 17.18 
                 UD 
                 29.51 
               
               
                   Candida albicans  (1) 
                   
                   
                   
                   
                   
                   
                   
               
               
                   Enterococcus faeclis , 
                 1 
                 — 
                   
                   
                 UD 
                 12.14 
                 UD 
               
               
                   Proteus mirabilis  (1) 
                   
                   
                   
                   
                   
                   
                   
               
               
                   Escherichia coli , 
                 1 
                 — 
                   
                   
                 UD 
                 13.25 
                 UD 
               
               
                   Enterococcus gallinarym  (1) 
                   
                   
                   
                   
                   
                   
                   
               
               
                   Klebsiella pneumonia , 
                 1 
                 — 
                   
                   
                 UD 
                 13.31 
                 UD 
               
               
                   Enterococcus casseliflavus  (1) 
                   
                   
                   
                   
                   
                   
                   
               
               
                   Klebsiella pneumonia , 
                 1 
                 — 
                   
                   
                 UD 
                 14.42 
                 UD 
               
               
                   Enterobacter cloacae  (1) 
                   
                   
                   
                   
                   
                   
                   
               
               
                 Blood Culture Negative (200) 
                 21 
                 179  
                   
                 89.5 
                   
                   
                   
               
               
                 Gram positive bacteria 
                 7 
                 0 
                   
                   
                 17.54-27.43 
                 — 
                 — 
               
               
                 Gram negative bacteria 
                 14 
                 0 
                   
                   
                 — 
                 20.48-31.26 
                 — 
               
               
                 Fungi 
                 0 
                 0 
                   
                   
                 — 
                 — 
                 — 
               
               
                   
               
               
                 *GP-GN mixed bacteria 
               
               
                 **The specificity test for real GP-GN PCR 
               
            
           
         
       
     
     Table 8 shows a comparison between a result of detection by Real GP-GN PCR from a blood culture and a result of a culture in a BACTEC 9240. 
     
       
         
           
               
               
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                   
                 Real GP-GN PCR 
                   
               
            
           
           
               
               
               
               
               
            
               
                 Blood culture 
                 Positive 
                 Negative 
                 Sensitivity 
                 Specificity 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Gram positive bacteria 
                 126 
                   
                 100% 
                   
               
               
                 (126) 
                   
                   
                   
                   
               
               
                 Gram negative bacteria (37) 
                 37 
                   
                 100% 
                   
               
               
                 Total (184) 
                 184 
                   
                 100% 
                   
               
               
                 Culture Negative (200) 
                 21 
                 179 
                   
                 89.50% 
               
               
                 Gram positive bacteria 
                 12 
                   
                   
                   
               
               
                 Gram negative bacteria* 
                 9 
               
               
                   
               
            
           
         
       
     
     Table 9 shows sensitivity and specificity of Real GP-GN PCR in a blood culture bottle. 
     
       
         
           
               
               
               
               
               
             
               
                 TABLE 10 
               
               
                   
               
               
                 Case 
                 Real GP-GN 
                   
                   
                 REBA 
               
               
                 no. 
                 PCR 
                 C T  Value 
                 Sequencing results 
                 results 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 1 
                 GN 
                 28.25 
                   Janthinobacterium  sp. 
                 GN 
               
               
                 2 
                 GP 
                 21.23 
                 Unculturued bacterium 
                 GP 
               
               
                 3 
                 GN 
                 30.31 
                   Polynucleobacter  sp. 
                 ND 
               
               
                 4 
                 GP 
                 27.43 
                 Unculturued bacterium 
                 GP 
               
               
                 5 
                 GP 
                 23.28 
                 Unculturued bacterium 
                 GP 
               
               
                 6 
                 GP 
                 19.92 
                 Unculturued bacterium 
                 GP 
               
               
                 7 
                 GP 
                 17.54 
                 Unculturued bacterium 
                 GP 
               
               
                 8 
                 GP 
                 17.85 
                 Unculturued bacterium 
                 GP 
               
               
                 9 
                 GN 
                 17.79 
                 Unculturued bacterium 
                 Pan-bac 
               
               
                 10 
                 GN 
                 29.05 
                 
                   proteobacterium 
                 
                 GN 
               
               
                 11 
                 GN 
                 27.01 
                 
                   proteobacterium 
                 
                 GN 
               
               
                 12 
                 GN 
                 31.26 
                   Hyalangnum  sp. 
                 GN 
               
               
                 13 
                 GN 
                 20.48 
                   Duganella  sp. 
                 GN 
               
               
                 14 
                 GN 
                 30.24 
                   Ochrobacterium  sp. 
                 GN 
               
               
                 15 
                 GN 
                 22.79 
                   Pseudomonas  sp. 
                 GN 
               
               
                 16 
                 GP 
                 27.15 
                 Unculturued bacterium 
                 GP 
               
               
                 17 
                 GN 
                 25.33 
                 
                   Burkholderiales bacterium 
                 
                 GN 
               
               
                 18 
                 GN 
                 28.31 
                   Methylophilus  sp. 
                 GN 
               
               
                 19 
                 GN 
                 22.59 
                 Nitrosomonadaccae 
                 GN 
               
               
                 20 
                 GN 
                 27.15 
                   Pseudomonas  sp. 
                 GN 
               
               
                 21 
                 GN 
                 17.79 
                   Enterobacter  sp. 
                 GN 
               
               
                   
               
            
           
         
       
     
     Table 10 shows a comparison between PCR-REBA and sequencing with respect to 21 Real GP-GN PCR positive and culture negative bacteria specimens. 
     
       
         
           
               
               
               
               
               
             
               
                 TABLE 11 
               
               
                   
               
               
                 No. 
                 Gene 
                 Sequence 
                 Size 
                 Modification 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                  1 
                 16s 
                 16S-F* 
                 T AAY ACA TGC AAG TCG ARC G 
                   
                 Biotin 
               
               
                   
                 rRNA 
                   
                   
                   
                   
               
               
                   
               
               
                  2 
                   
                 16S-R5H-A5 
                 TGG CAC GDA GTT RGC CGK KGC TT 
                 470 bp 
                 Biotin 
               
               
                   
               
               
                  3 
                   
                 407F 
                 T AAY ACA TGC AAG TCG ARC G 
                   
                   
               
               
                   
               
               
                  4 
                   
                 280R* 
                 TGT GGC YGR TCR YCC TCT CAG 
                 170 bp 
                 Biotin 
               
               
                   
               
               
                  5 
                 ITS 
                 MF3 
                 AACGCANMTTGCRCYCHHTG 
                   
                   
               
               
                   
               
               
                  6 
                   
                 CR3* 
                 CAGCGGGTADYCCYACCTGA 
                 230 bp 
                 Biotin 
               
               
                   
               
               
                  7 
                 Nuc 
                 nuc-F 
                 AGCGATTGATGGTGATACGGT 
                   
                   
               
               
                   
               
               
                  8 
                   
                 nuc-R* 
                 ATGCACTTGCTTCAGGACCA 
                 135 bp 
                 Biotin 
               
               
                   
               
               
                  9 
                 MecA 
                 MecA-F 
                 GGTGTTGGTGAAGATATACCAAGTG 
                   
                   
               
               
                   
               
               
                 10 
                   
                 MecA-R* 
                 GAAAGGATCTGTACTGGGTTAATCAT 
                 145 bp 
                 Biotin 
               
               
                   
               
               
                 11 
                 vanA 
                 vanA325-F 
                 TCAATAGCGCGGACGAATTG 
                   
                   
               
               
                   
               
               
                 12 
                   
                 R* 
                 GCGGGAACGGTTATAACTGCGTTT 
                 150 bp 
                 Biotin 
               
               
                   
               
               
                 13 
                 vanB 
                 vanBF-3 
                 TACCTACCCTGTCTTTGTGAAGCC 
                   
                   
               
               
                   
               
               
                 14 
                   
                 R* 
                 GCTGCTTCTATCGCAGCGTTTAGT 
                 100 bp 
                 Biotin 
               
               
                   
               
               
                 15 
                 invA 
                 sal-F 
                 TCTGGCAGTACCTTCCTCAGCC 
                   
                   
               
               
                   
               
               
                 16 
                   
                 sal-R* 
                 TCGACAGACGTAAGGAGGACAAGA 
                 120 bp 
                 Biotin 
               
               
                   
               
               
                 17 
                 ipaH 
                 Shi-F 
                 AGTTGCAGTCTCCTAGGTAAAGGG 
                   
                   
               
               
                   
               
               
                 18 
                   
                 Shi-R* 
                 ACTGCAAACTCTTCCATCTCTGCC 
                 100 bp 
                 Biotin 
               
               
                   
               
               
                 19 
                 spn- 
                 297F 
                 GACCAATCGTTTAAATGCGACTTCT 
                   
                   
               
               
                   
                 cpsA 
                   
                   
                   
                   
               
               
                   
               
               
                 20 
                   
                 416R* 
                 GTCCCAGTCGGTGCTGTCACACT 
                 130 bp 
                 Biotin 
               
               
                   
               
            
           
         
       
     
     Table 11 shows a primer for REBA Sepsis-1a 
     
       
         
           
               
               
               
               
             
               
                 TABLE 12 
               
               
                   
               
               
                 No. 
                 Name 
                 Sequence 
                 Species 
               
               
                   
               
             
            
               
                 21 
                 Pan-bac 
                 AGYGGCGG ACGGGTGAGTAA 
                   
               
               
                   
               
               
                 22 
                 GN350-5 
                 AACKGCGATCCCTAGCTGGTC 
                 Gram negative 
               
               
                   
               
               
                 23 
                 Eco-5 
                 GGAAGGGAGTAAAGTTAATACCTTTGCTCA 
                   E . coli / Shigella   
               
               
                   
               
               
                 24 
                 shi 
                 AGTTCAGTAAGATGGTTGTGCGCA 
                 
                   Shigella 
                 
               
               
                   
               
               
                 25 
                 sal 
                 CGGAAGCCTCCGCTAATTTGAT 
                 
                   Salmonella 
                 
               
               
                   
               
               
                 26 
                 kpn-11 
                 AAAAAAA GGTTAATAACCTCATCGATTGAC 
                   K . pneumoniae   
               
               
                   
               
               
                 27 
                 Pae 
                 ATACGTCCTGAGGGAGAAAGTG 
                   P . aeruginosa   
               
               
                   
               
               
                 28 
                 Cfre 
                 CGCAGAGGAGCTTGCTCCTTG 
                   C . freundii   
               
               
                   
               
               
                 29 
                 Aba-3 
                 AGCTTGCTACCGGACCTAGCG 
                   A . baumannii   
               
               
                   
               
               
                 30 
                 Hinf 
                 CGTATTATCGGAAGATGAAAGTGC 
                   H . influenzae   
               
               
                   
               
               
                 31 
                 GP9 
                 CVACGATRCRTAGCCGAC 
                 Gram positive 
               
               
                   
               
               
                 32 
                 GP11 
                 AAAAAACGATRCRTAGCCGAC 
                 Gram positive 
               
               
                   
               
               
                 33 
                 Baci 
                 AAACCGTTCRAATAGGGCG 
                   Bacillus  spp. 
               
               
                   
               
               
                 34 
                 Ent-3 
                 GGATAACACTTGGAAACAGGTGC 
                   Enterococcus  spp. 
               
               
                   
               
               
                 35 
                 Strep-4 
                 GCGTAGGTAACCTGCCTBRTAGCG 
                   Streptococcus  spp. 
               
               
                   
               
               
                 36 
                 spn 
                 TAGCAGATAGTGAGATCGAAAATGTTAC 
                   S .  pneumoniae   
               
               
                   
               
               
                 37 
                 staphyl-9 
                 CAWAYGTGTAAGTAACTRTGCACRTCT 
                   Staphylococcus  spp. 
               
               
                   
               
               
                 38 
                 nuc 
                 TTGGTTGATACACCTGAAACAAAG 
                   S .  aureus   
               
               
                   
               
               
                 39 
                 MecA 
                 AGCTGATTCAGGTTACGGACAAGGT 
                 MecA 
               
               
                   
               
               
                 40 
                 VanA 
                 TCGTATTCATCAGGAAGTCGAGCC 
                 VanA 
               
               
                   
               
               
                 41 
                 VanB 
                 TCGTCCTTTGGCGTAACCAA 
                 VanB 
               
               
                   
               
               
                 42 
                 alb 
                 AA TAGTGGTAAGGCGGGATC 
                   C .  albicans   
               
               
                   
               
               
                 43 
                   
                 GTAGTGGTAAGGCGGGATCG 
                   
               
               
                   
               
               
                 44 
                 tro 
                 ACG TGGAAACTTATTTT AAGCGA 
                   C .  tropicalis   
               
               
                   
               
               
                 45 
                 gla 
                 AGCGCAAGCTTCTCTATTAATCTG 
                   C .  glabrata   
               
               
                   
               
               
                 46 
                 para 
                 AGGCG GA GTATAAACTAATGGATAGGT 
                   C .  parapsilosis   
               
               
                   
               
               
                 47 
                 kru 
                 AGCGGAGCGGACGACGTGTA 
                   C .  krusei   
               
               
                   
               
            
           
         
       
     
     Table 12 shows a probe for REBA Sepsis-ID. 
     
       
         
           
               
               
               
               
               
               
             
               
                 TABLE 13 
               
               
                   
               
               
                 No. 
                 Gene 
                   
                 Sequence 
                 Size 
                 Modification 
               
               
                   
               
             
            
               
                 48 
                 16S rRNA 
                 300F 
                 ATTAGCTAGTWGGTRRGGTAANGGC 
                 120 bp 
                   
               
               
                   
               
               
                 49 
                   
                 420R 
                 ACTGCTGCCTCCCGTAGGAGT 
                   
                   
               
               
                   
               
               
                 50 
                   
                 GP350S1 
                 C AAG GCA ACG ATR CRTAGCCGAC 
                   
                 HEX-BHQ1 
               
               
                   
               
               
                 51 
                   
                 GP350M1 
                 AAGGCKWCGACGGGTAGCCGGC 
                   
                 HEX-BHQ1 
               
               
                   
               
               
                 52 
                   
                 GN325-3 
                 TCACCTAGGCGACGATCYSTAGCKGGT 
                   
                 FAM-BHQ1 
               
               
                   
               
               
                 53 
                   
                 Pan-bac 
                 GCCACAYTGGRACTGAGACACGG 
                   
                 Cy5-BHQ2 
               
               
                   
               
            
           
         
       
     
     Table 13 shows a primer and a probe for Real GP-GN. 
     
       
         
           
               
               
               
               
               
               
             
               
                 TABLE 14 
               
               
                   
               
               
                 No. 
                 Gene 
                   
                 Sequence 
                 Size 
                 Modification 
               
               
                   
               
             
            
               
                 54 
                 can18-1 
                 F 
                 AGCTCGTAGTTGAACYTTGGGCYTG 
                 120 bp 
                   
               
               
                   
               
               
                 55 
                   
                 R 
                 TCAAAGTAAWMGTCCTGGTTCGCC 
                   
                   
               
               
                   
               
               
                 56 
                   
                 P 
                 CCGRGYCTTTCCTTCTGGSTARCC 
                   
                 FAM-BHQ1 
               
               
                   
               
            
           
         
       
     
     Table 14 shows a primer and a probe for Real Can.