Method and kit for identifying bacteria causing sepsis

Abstract

Disclosed is a method for identifying bacteria that cause sepsis, wherein the method includes: a step for performing PCR using a sample collected from a subject and a combination of sets of primers each specific to the full length or a partial region of the gyrB gene of Escherichia coli, the nuc gene of Staphylococcus aureus, the atlE gene of Staphylococcus epidermidis, the gyrB gene of Klebsiella pneumoniae, and the rpoB gene of Enterococcus spp.; and a step for detecting any amplification of the full length or the partial region of each of these genes using a combination of probes each specific to the DNA of the full length or the partial region.

Description

[Technical field]

[0001] The present invention relates to a method and a kit for identifying the causative bacterium of sepsis. [Background technology]

[0002] Sepsis is a secondary symptom caused by an infection and can be caused by infection with various bacteria, etc. If sepsis becomes severe, it can lead to respiratory failure, kidney failure, pulmonary failure, septic shock, etc., and the risk of losing life increases. For this reason, it is important to quickly identify the pathogen causing the infection and start treatment.

[0003] Regarding pathogenic factors of sepsis, for example, Patent Document 1 describes a method for producing a ligand of an adsorption material that adsorbs multiple types of pathogenic factors of sepsis. However, there is no report on a method for identifying the pathogen causing the infection, i.e., the causative bacterium of sepsis, from multiple types of pathogens. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] US Patent Publication No. 2019054227 Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide a method and a kit for efficiently identifying bacteria causing sepsis. [Means for solving the problem]

[0006] The present invention provides, for example, the following inventions. [1] 1. A method for identifying a causative bacterium of sepsis, comprising: carrying out a PCR method using a sample collected from a subject and a combination of primer sets specific to the full length or a partial region of the gyrB gene of Escherichia coli, the nuc gene of Staphylococcus aureus, the atlE gene of Staphylococcus epidermidis, the gyrB gene of Klebsiella pneumoniae, and the rpoB gene of Enterococcus spp. (e.g., Enterococcus faecalis); detecting the presence or absence of amplification of the full length or a portion of the region of each gene using a combination of probes specific to the DNA of the full length or a portion of the region of each gene; A method comprising: [2] the set of primers specific to the full length or a partial region of the gyrB gene of Escherichia coli includes a primer containing a nucleotide sequence of SEQ ID NO: 10 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 10, and a primer containing a nucleotide sequence of SEQ ID NO: 11 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 11, The set of primers specific to the full length or a partial region of the nuc gene of Staphylococcus aureus includes one or more primers selected from a primer including a nucleotide sequence of SEQ ID NO: 32 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 32, a primer including a nucleotide sequence of SEQ ID NO: 34 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 34, and a combination thereof, and a primer including a nucleotide sequence of SEQ ID NO: 33 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 33; the set of primers specific to the full length or a partial region of the atlE gene of Staphylococcus epidermidis comprises a primer containing a nucleotide sequence of SEQ ID NO: 37 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 37, and a primer containing a nucleotide sequence of SEQ ID NO: 38 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 38; the set of primers specific to the full length or a partial region of the gyrB gene of Klebsiella pneumoniae comprises a primer including a nucleotide sequence of SEQ ID NO: 14 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 14, and a primer including a nucleotide sequence of SEQ ID NO: 15 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 15; The method according to [1], wherein the set of primers specific to the full length or a partial region of the rpoB gene of the Enterococcus genus comprises one or more primers selected from a primer having a nucleotide sequence of SEQ ID NO: 26 or a nucleotide sequence that has 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 26, a primer having a nucleotide sequence of SEQ ID NO: 27 or a nucleotide sequence that has 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 27, a primer having a nucleotide sequence of SEQ ID NO: 28 or a nucleotide sequence that has 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 28, and a combination thereof, and a primer having a nucleotide sequence of SEQ ID NO: 29 or a nucleotide sequence that has 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 29. [3] the DNA of the full length or a partial region of the gyrB gene of Escherichia coli comprises the nucleotide sequence of SEQ ID NO: 51 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 51, The DNA of the full length or a partial region of the nuc gene of Staphylococcus aureus comprises the nucleotide sequence of SEQ ID NO: 61 or a nucleotide sequence having a sequence identity of 90% or more to the nucleotide sequence of SEQ ID NO: 61, the DNA of the full length or a partial region of the atlE gene of Staphylococcus epidermidis comprises the nucleotide sequence of SEQ ID NO: 63 or a nucleotide sequence having a sequence identity of 90% or more to the nucleotide sequence of SEQ ID NO: 63, the DNA of the full length or a partial region of the gyrB gene of Klebsiella pneumoniae contains the nucleotide sequence of SEQ ID NO: 53 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 53, The method according to [1] or [2], wherein the DNA of the full length or a partial region of the rpoB gene of the Enterococcus genus comprises the nucleotide sequence of SEQ ID NO: 59 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 59. [4] The method according to [3], wherein the probe hybridizes to the full-length or partial region of DNA under stringent conditions. [5] the probe specific to the DNA of the full length or a partial region of the gyrB gene of Escherichia coli contains the nucleotide sequence of SEQ ID NO: 121 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 121; the probe specific to the DNA of the full length or a partial region of the nuc gene of Staphylococcus aureus comprises the nucleotide sequence of SEQ ID NO: 134 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 134; the probe specific to the DNA of the full length or a partial region of the atlE gene of Staphylococcus epidermidis contains the nucleotide sequence of SEQ ID NO: 136 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 136; the probe specific to the full length or a partial region of the DNA of the gyrB gene of Klebsiella pneumoniae contains the nucleotide sequence of SEQ ID NO: 124 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 124; The method according to any of [1] to [4], wherein the probe specific to DNA of the full length or a partial region of the rpoB gene of the Enterococcus genus is selected from a probe comprising the nucleotide sequence of SEQ ID NO: 129 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 129, a probe comprising the nucleotide sequence of SEQ ID NO: 130 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 130, a probe comprising the nucleotide sequence of SEQ ID NO: 131 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 131, and a combination thereof. [6] The method according to any one of [1] to [5], wherein the probes each contain a different identifier or are each bound to a different identifier on a solid support. [7] The method according to any one of [1] to [6], wherein the DNA amplified by the PCR method contains a label. [8] The method according to any one of [1] to [7], wherein the sample collected from the subject is blood. [9] The method according to [8], further comprising a step of culturing the blood.

[10] A kit for identifying a causative bacterium of sepsis, comprising: a first reagent including a set of PCR primers specific to the full length or a partial region of the gyrB gene of Escherichia coli, the nuc gene of Staphylococcus aureus, the atlE gene of Staphylococcus epidermidis, the gyrB gene of Klebsiella pneumoniae, and the rpoB gene of Enterococcus spp.; and a second reagent comprising a combination of probes specific to the DNA of the full length or partial region of each gene.

[11] the set of primers specific to the full length or a partial region of the gyrB gene of Escherichia coli includes a primer including a nucleotide sequence of SEQ ID NO: 10 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 10, and a primer including a nucleotide sequence of SEQ ID NO: 11 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 11, The set of primers specific to the full length or a partial region of the nuc gene of Staphylococcus aureus includes one or more primers selected from a primer including a nucleotide sequence of SEQ ID NO: 32 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 32, a primer including a nucleotide sequence of SEQ ID NO: 34 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 34, and a combination thereof, and a primer including a nucleotide sequence of SEQ ID NO: 33 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 33; the set of primers specific to the full length or a partial region of the atlE gene of Staphylococcus epidermidis comprises a primer containing a nucleotide sequence of SEQ ID NO: 37 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 37, and a primer containing a nucleotide sequence of SEQ ID NO: 38 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 38; the set of primers specific to the full length or a partial region of the gyrB gene of Klebsiella pneumoniae comprises a primer including a nucleotide sequence of SEQ ID NO: 14 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 14, and a primer including a nucleotide sequence of SEQ ID NO: 15 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 15; The kit described in

[10] , wherein the set of primers specific to the full length or a partial region of the rpoB gene of the Enterococcus genus comprises one or more primers selected from a primer having a nucleotide sequence of SEQ ID NO: 26 or a nucleotide sequence that has 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 26, a primer having a nucleotide sequence of SEQ ID NO: 27 or a nucleotide sequence that has 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 27, a primer having a nucleotide sequence of SEQ ID NO: 28 or a nucleotide sequence that has 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 28, and a combination thereof, and a primer having a nucleotide sequence of SEQ ID NO: 29 or a nucleotide sequence that has 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 29.

[12] the DNA of the full length or a partial region of the gyrB gene of Escherichia coli comprises the nucleotide sequence of SEQ ID NO: 51 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 51, The DNA of the full length or a partial region of the nuc gene of Staphylococcus aureus comprises the nucleotide sequence of SEQ ID NO: 61 or a nucleotide sequence having a sequence identity of 90% or more to the nucleotide sequence of SEQ ID NO: 61, the DNA of the full length or a partial region of the atlE gene of Staphylococcus epidermidis comprises the nucleotide sequence of SEQ ID NO: 63 or a nucleotide sequence having a sequence identity of 90% or more to the nucleotide sequence of SEQ ID NO: 63, the DNA of the full length or a partial region of the gyrB gene of Klebsiella pneumoniae contains the nucleotide sequence of SEQ ID NO: 53 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 53, The kit according to

[10] or

[11] , wherein the DNA of the full length or a partial region of the rpoB gene of the Enterococcus genus comprises the base sequence of SEQ ID NO: 59 or a base sequence having 90% or more sequence identity to the base sequence of SEQ ID NO: 59.

[13] The kit described in

[12] , wherein the probes hybridize to the DNA of the full length or a portion of the region under stringent conditions.

[14] the probe specific to the DNA of the full length or a partial region of the gyrB gene of Escherichia coli contains the nucleotide sequence of SEQ ID NO: 121 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 121; the probe specific to the DNA of the full length or a partial region of the nuc gene of Staphylococcus aureus comprises the nucleotide sequence of SEQ ID NO: 134 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 134; the probe specific to the DNA of the full length or a partial region of the atlE gene of Staphylococcus epidermidis contains the nucleotide sequence of SEQ ID NO: 136 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 136; the probe specific to the full length or a partial region of the DNA of the gyrB gene of Klebsiella pneumoniae contains the nucleotide sequence of SEQ ID NO: 124 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 124; The kit according to any of

[10] to

[13] , wherein the probe specific to DNA of the full length or a partial region of the rpoB gene of the Enterococcus genus is selected from a probe containing the nucleotide sequence of SEQ ID NO: 129 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 129, a probe containing the nucleotide sequence of SEQ ID NO: 130 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 130, a probe containing the nucleotide sequence of SEQ ID NO: 131 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 131, and combinations thereof.

[15] The kit according to any one of

[10] to

[14] , wherein the probes contained in the second reagent each contain a different identifier or are each bound to a different identifier on a solid support.

[16] The kit according to any one of

[10] to

[15] , wherein the primer set is designed so that DNA amplified by PCR using the primer set contains a label, or the kit further contains a third reagent for labeling the amplified DNA. Effect of the Invention

[0007] According to the present invention, it is possible to simultaneously examine a plurality of bacteria causing sepsis, and to efficiently identify the bacteria causing sepsis. Since the bacteria causing sepsis can be efficiently identified, it becomes possible to select a treatment promptly. [Brief description of the drawings]

[0008] [Figure 1] 1 is a graph showing the results (amplification curves) of real-time PCR carried out using primer set 1 and primer set 2 in Example 1. [Diagram 2] 1 is a graph showing the results (detected fluorescence values) of the PCR product obtained using primer set 1 in Example 1, detected with two types of probes (E. coli_531P16 and E. coli_526P15). [Diagram 3] 1 is a graph showing the results (amplification curves) of real-time PCR performed using primer set 1 in Example 2. [Figure 4] 1 is a graph showing the results (amplification curves) of real-time PCR performed using primer set 2 in Example 2. [Diagram 5] 1 is a graph showing the results (amplification curves) of real-time PCR performed using primer set 3 in Example 2. [Figure 6]This is a graph showing the results (detection fluorescence values) of PCR products obtained using primer set 1 and Citrobacter freundii as a template in Example 2, detected using three types of probes (C.spp_24P17, C.spp_55P15Y, and C.spp_65P19). [Figure 7] This is a graph showing the results (detection fluorescence values) of the PCR product obtained using primer set 1 and Citrobacter koseri as a template in Example 2, detected using three types of probes (C.spp_24P17, C.spp_55P15Y, and C.spp_65P19). [Figure 8] 1 is a graph showing the results (detected fluorescence values) of evaluation of the reactivity and specificity of the probes for the PCR products of each bacterial species in Example 3. [Figure 9] 1 is a graph showing the results (detected fluorescence values) of evaluation of the reactivity and specificity of the probes for the PCR products of each bacterial species in Example 3. [Figure 10] 1 is a graph showing the results (detected fluorescence values) of evaluation of the reactivity and specificity of the probes for the PCR products of each bacterial species in Example 3. [Figure 11] 1 is a graph showing the results (detection fluorescence values) of evaluation of the reactivity and specificity of the probes to the PCR products of each drug resistance gene in Example 3. [Figure 12] 1 is a graph showing the results (detection fluorescence values) of evaluation of the reactivity and specificity of the probes to the PCR products of each drug resistance gene in Example 3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] Hereinafter, an embodiment of the present invention will be described in detail. Note that the present invention is not limited to the following embodiment.

[0010] <Identification method> As one embodiment, the present invention provides A sample taken from the subject; and carrying out a PCR method using a combination of primer sets specific to the full length or a partial region of the gyrB gene of Escherichia coli, the nuc gene of Staphylococcus aureus, the atlE gene of Staphylococcus epidermidis, the gyrB gene of Klebsiella pneumoniae, and the rpoB gene of Enterococcus spp.; detecting the presence or absence of amplification of the full length or a portion of the region of each gene using a combination of probes specific to the DNA of the full length or a portion of the region of each gene; The present invention provides a method for identifying the causative bacterium of sepsis, comprising:

[0011] [Step of carrying out PCR method] In one embodiment, the step of performing the PCR method is a step of performing the PCR method using a sample collected from a subject and a combination of primer sets specific to the full-length or partial region of the gyrB gene of Escherichia coli, the nuc gene of Staphylococcus aureus, the atlE gene of Staphylococcus epidermidis, the gyrB gene of Klebsiella pneumoniae, and the rpoB gene of Enterococcus spp.

[0012] The subject from which the sample is taken may be, for example, a subject with sepsis or a subject suspected of having sepsis, or a subject with or suspected of having a bacterial infection. The sample may be, for example, a sample containing or suspected of containing DNA of a bacterium causing sepsis. The subject may be, for example, a mammal, a human, or a non-human mammal.

[0013] The sample taken from the subject may be a bodily fluid, such as, for example, blood, saliva, urine, lymphatic fluid, etc. Preferably, the sample is blood.

[0014] The sample collected from the subject may be cultured as necessary. For example, the method according to the present embodiment may further include a step of culturing blood. The method of culturing blood may be performed by a method known in the art.

[0015] The step of performing the PCR method may further include the step of preparing a template from a sample taken from the subject.

[0016] The template may be, for example, a DNA extract. DNA may be extracted from a sample collected from a subject by a method known in the art.

[0017] In this specification, a set of primers specific to the full length or a part of a region of each gene means a set of primers that specifically bind to the full length or a part of a region of each gene as a target and amplify the DNA of the targeted region by polymerase chain reaction (PCR). The set of primers includes at least one type of forward primer and at least one type of reverse primer. The set of primers may be, for example, a combination of multiple forward primers and one type of reverse primer, a combination of one type of forward primer and multiple reverse primers, or a combination of multiple forward primers and multiple reverse primers. For example, hereinafter, the above-mentioned targeted region may be referred to as a "target region".

[0018] The sequence of each gene that is the target sequence can be obtained from a public database in which sequence information is registered, such as GenBank provided by the National Center for Biotechnology Information (NCBI) in the United States. Information such as GenBank ID of the genes described in this specification is listed in Tables 1 and 2 below. In this specification, in principle, each gene name does not only refer to a gene having a specific sequence, but also refers to, for example, a gene having a naturally occurring single nucleotide polymorphism.

[0019] Exemplary strain names and GenBank IDs for the bacteria described herein are shown below. The gene names are exemplary of the target genes and drug resistance genes described herein.

[0020] [Table 1] TIFF0007673363000002.tif50149

[0021] [Table 2]

[0022] A set of specific primers (forward primer and reverse primer) for amplifying the full length or a partial region of each gene as a target sequence can be designed based on the sequence of the target sequence. The primers can be synthesized by a method known to those skilled in the art. The primers may be, for example, oligonucleotides of 10 to 50 bases long, 15 to 30 bases long, or 17 to 25 bases long. The primers do not need to reflect the exact sequence of the target sequence, but must be sufficiently complementary to hybridize with the template and initiate DNA synthesis.

[0023] In the present specification, the primer may comprise a specific base sequence or a base sequence having sequence identity of 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. The primer may comprise a specific base sequence or a base sequence having sequence identity of 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. The primer may comprise a specific base sequence or a base sequence having 0 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 nucleotide addition or deletion at the 3' end or 5' end.

[0024] In this specification, the one-letter codes for nucleotide bases are used in accordance with the base symbols defined by the International Union of Pure and Applied Chemistry (IUPAC) as shown in the table below.

[0025] [Table 3]

[0026] Primers may consist of the bases A, G, C, T or analogs, or degenerate bases (M, R, W, S, Y, K). By including degenerate bases at specific positions where the base sequence differs depending on the bacterial strain, bacterial genes can be more appropriately amplified.

[0027] Primers may also have a label that is detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Detectable labels include, for example, biotin detected with labeled avidin (e.g., fluorescently labeled streptavidin), haptens, fluorescent dyes (e.g., fluorescein, Texas Red, and rhodamine), electron-dense reagents, enzymes (e.g., horseradish peroxidase and alkaline phosphatase), and radioisotopes (e.g., 32 P, 3 H, 14 C and125 I). For example, the 5'-end, 3'-end or internal of the primer may have a label, or the primer may be labeled via a linker. For example, the 5'-end of the primer may be labeled by being modified with biotin, and in this case, the 5'-end of the primer may be modified with biotin via a linker.

[0028] In the step of carrying out the PCR method, a primer having a label can be used to obtain amplified DNA containing the label. The label can also be used to capture the primer in order to immobilize the primer or the amplified DNA on a solid support.

[0029] In this embodiment, PCR is performed simultaneously using a combination of primer sets specific to the full length or partial regions of multiple genes, respectively, so that the primer sets are designed so that the amplification step can be performed under similar or uniform amplification conditions for each gene. When the sample contains DNA of the full length or partial region of the gene targeted by the primers, the region is amplified.

[0030] A method for carrying out PCR using a combination of primer sets specific to the entire length or a partial region of a plurality of genes can be carried out by a method known to those skilled in the art. PCR usually involves an initial denaturation step, followed by PCR amplification cycles. In each cycle of PCR amplification, the double-stranded target sequence is denatured (denaturation step), primers are annealed to each strand of the denatured target (annealing step), and the primers are extended by the action of DNA polymerase (extension step).

[0031] An example of a method for amplifying various genomic regions with different sequences in a single PCR reaction system is multiplex polymerase chain reaction (multiplex PCR). Multiplex PCR is a method in which multiple primers that amplify different target nucleic acids are combined in a multiplex format and tests are performed simultaneously on one sample.

[0032] In this embodiment, in order to simultaneously amplify different gene regions in one PCR reaction system, a set of primers specific to the full length or a part of each gene is used collectively. For example, under conditions suitable for nucleic acid amplification, a sample collected from a subject is contacted with a mixture of primer sets, so that the primer sets specifically bind to the full length or a part of the bacterial gene or the bacterial gene and the drug resistance gene, respectively, and the region can be amplified by DNA polymerase.

[0033] An amplification reaction mixture can be formed by carrying out a PCR method using a combination of a set of primers targeting a bacterial gene or a bacterial gene and a drug resistance gene contained or suspected to be contained in a sample collected from a subject. If the target gene is contained in the sample, the amplification reaction mixture contains DNA in which the bacterial gene or the bacterial gene and the drug resistance gene have been amplified in full length or in part.

[0034] In the process of carrying out the PCR method, for example, a mixture containing a combination of a template obtained from a sample and the above-mentioned set of primers can be referred to as a reaction mixture.

[0035] The reaction mixture may contain a PCR standard reagent. An example of the PCR standard reagent is Multiplex PCR Assay Kit Ver.2 (Takara Bio Inc.). The reaction mixture may further contain one or more selected from the group consisting of DNA polymerase, deoxynucleoside triphosphate (dNTP), magnesium ion, one or more salts, Tris buffer (Tris-HCL), EDTA, glycerol, and a pH buffer.

[0036] DNA polymerases include, for example, taq (Thermus aquaticus) polymerase and pfu (Pyrococcus furiosus) polymerase.

[0037] The one or more salts include, for example, potassium chloride and magnesium chloride. The salt concentration of the buffer used in PCR may be, for example, 1 mM to 200 mM, and in the case of potassium chloride, it may be 125 mM to 175 mM, and in the case of magnesium chloride, it may be 1 mM to 4 mM.

[0038] An example of the pH buffer is a Tris-pH buffer. The pH of the pH buffer may be, for example, 7.5 to 9.0, or may be 8.0 to 9.0.

[0039] The concentration of each primer used in PCR (final concentration in the PCR reaction solution) may be, for example, 0.01 μM to 3 μM, 0.1 μM to 0.3 μM, 0.05 μM to 2.5 μM, or 0.1 μM to 0.4 μM.

[0040] The denaturation step in PCR may be, for example, at 90° C. to 95° C. for 30 to 60 seconds, or at 94° C. for 30 seconds.

[0041] The annealing step may be, for example, 30 to 65 seconds at 58 to 64° C. and 20 to 40 seconds at 90 to 95° C., 50 to 60 seconds at 60 to 62° C. and 25 to 35 seconds at 92 to 95° C., or 60 seconds at 60° C. and 30 seconds at 94° C. The number of annealing cycles may be, for example, 25 to 50 times, 30 to 40 times, or 30 times.

[0042] The extension step may be, for example, at 70°C to 75°C for 100 to 700 seconds, at 70°C to 75°C for 400 to 700 seconds, or at 72°C for 600 seconds.

[0043] The combination of the primer set used in this embodiment is preferably designed so that the denaturation step, annealing step, and extension step in the PCR method can be performed using constant temperature cycle conditions.

[0044] In the step of carrying out the PCR method, a combination of primer sets specific to the full length or a partial region of the gyrB gene of Escherichia coli, the nuc gene of Staphylococcus aureus, the atlE gene of Staphylococcus epidermidis, the gyrB gene of Klebsiella pneumoniae, and the rpoB gene of Enterococcus spp. (e.g., Enterococcus faecalis) is used.

[0045] In the step of carrying out the PCR method, a set of primers specific to the entire length or a partial region of a gene other than the gyrB gene of Escherichia coli, the nuc gene of Staphylococcus aureus, the atlE gene of Staphylococcus epidermidis, the gyrB gene of Klebsiella pneumoniae, and the rpoB gene of Enterococcus spp. may further be used.

[0046] For example, in the step of carrying out the PCR method, the rpoB gene of Acinetobacter spp. (e.g., Acinetobacter baumannii), the 16S-23S ITS gene of Acinetobacter baumannii, the ompA gene of Citrobacter spp. (e.g., Citrobacter koseri, Citrobacter_freundii), the rpoS gene of Enterobacter spp. (e.g., Enterobacter cloacae), the rpoS gene of Klebsiella aerogenes, the rpoB gene of Acinetobacter spp. (e.g., Acinetobacter baumannii), the 16S-23S ITS gene of Acinetobacter baumannii, the ompA gene of Citrobacter spp. (e.g., Citrobacter koseri, Citrobacter_freundii), the rpoS gene of Enterobacter spp. (e.g., Enterobacter cloacae), the rpoS gene of Klebsiella aerogenes, the rpoS gene of Klebsiella aerogenes gyrB gene, Klebsiella oxytoca pehX gene, Klebsiella variicola gyrB gene, Pseudomonas aeruginosa oprL gene, Proteus spp. (e.g. Proteus mirabilis) rpoB gene, Serratia marcescens hasA gene, Listeria spp. (e.g. Listeria monocytogenes) iap p60 gene, Staphylococcus argenteus nuc gene, Staphylococcus spp. (e.g., Staphylococcus aureus), tuf genes in Streptococcus spp.A set of primers specific to the entire length or a partial region of one or more genes selected from the group consisting of the rnpB gene of Streptococcus pneumoniae, the xisco gene of Streptococcus pneumoniae, and the gyrB gene of Streptococcus pyogenes may also be used.

[0047] In addition, for example, in the step of carrying out the PCR method, drug resistance genes such as the beta-lactamase_KPC gene of Klebsiella pneumoniae, the beta-lactamase_NDM gene of Klebsiella pneumoniae, the beta-lactamase_IMP85 gene of Pseudomonas aeruginosa, the beta-lactamase_VIM gene of Citrobacter freundii, the beta-lactamase_VIM gene of Salmonella enterica, beta-lactamase_CTX-M1 gene of Salmonella enterica, beta-lactamase_CTX-M2 gene of Salmonella enterica, beta-lactamase_CTX-M8 gene of Citrobacter amalonatics, beta-lactamase_CTX-M25 gene of Escherichia coli, beta-lactamase_CTX-M9 gene of Escherichia coli, and Acinetobacter baumannii baumannii beta-lactamase_OXA-23 gene, Acinetobacter baumannii beta-lactamase_OXA-40 gene, Klebsiella pneumoniaeA set of primers specific to the entire length or a partial region of one or more genes selected from the group consisting of the beta-lactamase_OXA-48 gene of P. pneumoniae, the beta-lactamase_OXA-58 gene of Acinetobacter haemolyticus, the mecA gene of Staphylococcus aureus, the vanA gene of Enterococcus faecalis, and the vanB gene of Enterococcus faecium may further be used.

[0048] Furthermore, for example, in the step of carrying out the PCR method, the rpoB gene of Acinetobacter genus, the 16S-23S ITS gene of Acinetobacter baumannii, the ompA gene of Citrobacter genus (e.g., Citrobacter koseri, Citrobacter freundii), the rpoS gene of Enterobacter genus (e.g., Enterobacter cloacae), the gyrB gene of Klebsiella aerogenes, the pehX gene of Klebsiella oxytoca, the gyrB gene of Klebsiella variicola, the oprL gene of Pseudomonas aeruginosa, the rpoB gene of Proteus genus (e.g., Proteus mirabilis), the hasA gene of Serratia marcescens, the iapL gene of Listeria genus, p60 gene, nuc gene of Staphylococcus argenteus, tuf gene of Staphylococcus spp., rnpB gene of Streptococcus spp., xisco gene of Streptococcus pneumoniae, gyrB gene of Streptococcus pyogenes, beta-lactamase_KPC gene of Klebsiella pneumoniae, beta-lactamase_NDM gene of Klebsiella pneumoniae, beta-lactamase_IMP85 gene of Pseudomonas aeruginosa, beta-lactamase_VIM gene of Citrobacter freundii, beta-lactamase_CTX-M1 gene of Salmonella enterica, beta-lactamase_CTX-M2 gene of Salmonella enterica, beta-lactamase_VIM gene of Citrobacter amalonatis A set of primers specific to the entire length or a partial region of one or more genes selected from the group consisting of the CTX-M8 gene, the beta-lactamase_CTX-M25 gene of Escherichia coli, the beta-lactamase_CTX-M9 gene of Escherichia coli, the beta-lactamase_OXA-23 gene of Acinetobacter baumannii, the beta-lactamase_OXA-40 gene of Acinetobacter baumannii, the beta-lactamase_OXA-48 gene of Klebsiella pneumoniae, the beta-lactamase_OXA-58 gene of Acinetobacter haemolyticus, the mecA gene of Staphylococcus aureus, the vanA gene of Enterococcus faecalis, and the vanB gene of Enterococcus faecium may be used.

[0049] The bacteria or genes selected from the group of multiple genes may be, for example, 2 or more, 3 or more, 5 or more, 7 or more, 10 or more, 15 or more, 20 or more, 25 or more, 28 or more, 30 or more, or 32 or more. The bacteria or genes selected from the group of multiple genes may be, for example, 37 or less, 34 or less, 21 or less, 16 or less, or 11 or less. The bacteria or genes selected from the group of multiple genes may be, for example, 2 to 40, 5 to 37, 10 to 37, or 15 to 25.

[0050] By further using the above primer set, it is possible to investigate more bacteria causing sepsis and also to investigate drug resistance genes. By further using a primer set for drug resistance genes, it is possible to simultaneously identify the bacteria causing sepsis and the presence or absence of drug resistance genes in the bacteria, so that treatment can be selected promptly. In addition, it is possible to select an appropriate treatment and determine a treatment policy, taking into account the presence or absence of resistance of the causative bacteria to the therapeutic drug.

[0051] In the step of carrying out the PCR method, the set of primers specific to the full length or a partial region of the gyrB gene of E. coli may include a primer containing a nucleotide sequence of SEQ ID NO: 10 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 10, and a primer containing a nucleotide sequence of SEQ ID NO: 11 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 11, The set of primers specific to the full length or a partial region of the nuc gene of Staphylococcus aureus may include one or more primers selected from a primer including a nucleotide sequence of SEQ ID NO: 32 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 32, a primer including a nucleotide sequence of SEQ ID NO: 34 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 34, and a combination thereof, and a primer including a nucleotide sequence of SEQ ID NO: 33 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 33, The set of primers specific to the full length or a partial region of the atlE gene of Staphylococcus epidermidis may include a primer containing a nucleotide sequence of SEQ ID NO: 37 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 37, and a primer containing a nucleotide sequence of SEQ ID NO: 38 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 38, The set of primers specific to the full length or a partial region of the gyrB gene of Klebsiella pneumoniae may include a primer including a nucleotide sequence of SEQ ID NO: 14 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 14, and a primer including a nucleotide sequence of SEQ ID NO: 15 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 15, The set of primers specific to the entire length or a partial region of the rpoB gene of Enterococcus may include one or more primers selected from a primer containing the nucleotide sequence of SEQ ID NO: 26 or a nucleotide sequence that has 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 26, a primer containing the nucleotide sequence of SEQ ID NO: 27 or a nucleotide sequence that has 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 27, a primer containing the nucleotide sequence of SEQ ID NO: 28 or a nucleotide sequence that has 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 28, and combinations thereof, and a primer containing the nucleotide sequence of SEQ ID NO: 29 or a nucleotide sequence that has 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 29.

[0052] By using the above primer set, the interaction between primers is reduced, and the specificity and reactivity when amplifying each region are improved. In addition, the amplification efficiency is improved under uniform conditions of temperature, reagents, etc. when performing PCR.

[0053] In addition, when a set of primers specific to the full length or a partial region of a gene other than the gyrB gene of Escherichia coli, the nuc gene of Staphylococcus aureus, the atlE gene of Staphylococcus epidermidis, the gyrB gene of Klebsiella pneumoniae, and the rpoB gene of Enterococcus spp. is further used, The set of primers specific to the full length or a partial region of the rpoB gene of Acinetobacter may include a primer including a nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 1, and a primer including a nucleotide sequence of SEQ ID NO: 2 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 2, The set of primers specific to the full length or a partial region of the 16S-23S ITS gene of Acinetobacter baumannii may include a primer including a nucleotide sequence of SEQ ID NO: 3 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 3, and a primer including a nucleotide sequence of SEQ ID NO: 4 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 4, A set of primers specific to the full length or a partial region of the ompA gene of Citrobacter (e.g., Citrobacter koseri, Citrobacter freundii) may include one or more primers selected from a primer containing the nucleotide sequence of SEQ ID NO:5 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO:5, a primer containing the nucleotide sequence of SEQ ID NO:6 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO:6, and a combination thereof, and a primer containing the nucleotide sequence of SEQ ID NO:7 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO:7, The set of primers specific to the full length or a partial region of the rpoS gene of Enterobacter may include a primer containing a nucleotide sequence of SEQ ID NO: 8 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 8, and a primer containing a nucleotide sequence of SEQ ID NO: 9 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 9, The set of primers specific to the full length or a partial region of the gyrB gene of Klebsiella aerogenes may include a primer including a nucleotide sequence of SEQ ID NO: 12 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 12, and a primer including a nucleotide sequence of SEQ ID NO: 13 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 13, The set of primers specific to the full length or a partial region of the pehX gene of Klebsiella oxytoca may include a primer including a nucleotide sequence of SEQ ID NO: 16 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 16, and a primer including a nucleotide sequence of SEQ ID NO: 17 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 17, The set of primers specific to the full length or a partial region of the gyrB gene of Klebsiella variicola may include a primer including a nucleotide sequence of SEQ ID NO: 18 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 18, and a primer including a nucleotide sequence of SEQ ID NO: 19 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 19, The set of primers specific to the full length or a partial region of the oprL gene of Pseudomonas aeruginosa may include a primer containing a nucleotide sequence of SEQ ID NO: 20 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 20, and a primer containing a nucleotide sequence of SEQ ID NO: 21 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 21, The set of primers specific to the full length or a partial region of the rpoB gene of the genus Proteus may include a primer including a nucleotide sequence of SEQ ID NO: 22 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 22, and a primer including a nucleotide sequence of SEQ ID NO: 23 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 23, The set of primers specific to the full length or a partial region of the hasA gene of Serratia marcescens may include a primer including a nucleotide sequence of SEQ ID NO: 24 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 24, and a primer including a nucleotide sequence of SEQ ID NO: 25 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 25, The set of primers specific to the full length or a partial region of the iap p60 gene of the Listeria genus may include a primer including a nucleotide sequence of SEQ ID NO: 30 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 30, and a primer including a nucleotide sequence of SEQ ID NO: 31 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 31, The set of primers specific to the full length or a partial region of the nuc gene of Staphylococcus argenteus may include a primer including a nucleotide sequence of SEQ ID NO: 35 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 35, and a primer including a nucleotide sequence of SEQ ID NO: 36 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 36, The set of primers specific to the full length or a partial region of the tuf gene of the genus Staphylococcus may include a primer containing a nucleotide sequence of SEQ ID NO: 39 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 39, and a primer containing a nucleotide sequence of SEQ ID NO: 40 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 40, The set of primers specific to the full length or a partial region of the rnpB gene of the genus Streptococcus may include a primer containing a nucleotide sequence of SEQ ID NO: 41 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 41, and a primer containing a nucleotide sequence of SEQ ID NO: 42 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 42, The set of primers specific to the full length or a partial region of the xisco gene of Streptococcus pneumoniae may include a primer including a nucleotide sequence of SEQ ID NO: 43 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 43, and a primer including a nucleotide sequence of SEQ ID NO: 44 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 44, The set of primers specific to the full length or a partial region of the gyrB gene of Streptococcus pyogenes may include a primer including a nucleotide sequence of SEQ ID NO: 45 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 45, and a primer including a nucleotide sequence of SEQ ID NO: 46 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 46, The set of primers specific to the full length or a partial region of the beta-lactamase_KPC gene of Klebsiella pneumoniae may include a primer containing a nucleotide sequence of SEQ ID NO: 68 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 68, and a primer containing a nucleotide sequence of SEQ ID NO: 69 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 69, The set of primers specific to the full length or a partial region of the beta-lactamase_NDM gene of Klebsiella pneumoniae may include a primer including a nucleotide sequence of SEQ ID NO: 70 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 70, and a primer including a nucleotide sequence of SEQ ID NO: 71 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 71, The set of primers specific to the full length or a partial region of the beta-lactamase_IMP85 gene of Pseudomonas aeruginosa may include a primer including a nucleotide sequence of SEQ ID NO: 72 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 72, and a primer including a nucleotide sequence of SEQ ID NO: 73 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 73, The set of primers specific to the full length or a partial region of the beta-lactamase_VIM gene of Citrobacter freundii may include a primer including a nucleotide sequence of SEQ ID NO: 74 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 74, and a primer including a nucleotide sequence of SEQ ID NO: 75 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 75, The set of primers specific to the full length or a partial region of the beta-lactamase_CTX-M1 gene of Salmonella enterica may include a primer including a nucleotide sequence of SEQ ID NO: 76 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 76, and a primer including a nucleotide sequence of SEQ ID NO: 77 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 77, The set of primers specific to the full length or a partial region of the beta-lactamase_CTX-M2 gene of Salmonella enterica may include a primer including a nucleotide sequence of SEQ ID NO: 78 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 78, and a primer including a nucleotide sequence of SEQ ID NO: 79 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 79, The set of primers specific to the full length or a partial region of the beta-lactamase_CTX-M8 gene of Citrobacter amalonaticus may include a primer including a nucleotide sequence of SEQ ID NO: 80 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 80, and a primer including a nucleotide sequence of SEQ ID NO: 81 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 81, The set of primers specific to the full length or a partial region of the E. coli beta-lactamase_CTX-M25 gene may include a primer containing a nucleotide sequence of SEQ ID NO: 82 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 82, and a primer containing a nucleotide sequence of SEQ ID NO: 83 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 83, The set of primers specific to the full length or a partial region of the E. coli beta-lactamase_CTX-M9 gene may include a primer containing a nucleotide sequence of SEQ ID NO: 84 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 84, and a primer containing a nucleotide sequence of SEQ ID NO: 85 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 85, The set of primers specific to the full length or a partial region of the beta-lactamase_OXA-23 gene of Acinetobacter baumannii may include a primer including a nucleotide sequence of SEQ ID NO: 86 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 86, and a primer including a nucleotide sequence of SEQ ID NO: 87 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 87, The set of primers specific to the full length or a partial region of the beta-lactamase_OXA-40 gene of Acinetobacter baumannii may include a primer including a nucleotide sequence of SEQ ID NO: 88 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 88, and a primer including a nucleotide sequence of SEQ ID NO: 89 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 89, The set of primers specific to the full length or a partial region of the beta-lactamase_OXA-48 gene of Klebsiella pneumoniae may include a primer including a nucleotide sequence of SEQ ID NO: 90 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 90, and a primer including a nucleotide sequence of SEQ ID NO: 91 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 91, The set of primers specific to the full length or a partial region of the beta-lactamase_OXA-58 gene of Acinetobacter haemolyticus may include a primer including a nucleotide sequence of SEQ ID NO: 92 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 92, and a primer including a nucleotide sequence of SEQ ID NO: 93 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 93, The set of primers specific to the full length or a partial region of the mecA gene of Staphylococcus aureus may include a primer including a nucleotide sequence of SEQ ID NO: 94 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 94, and a primer including a nucleotide sequence of SEQ ID NO: 95 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 95, The set of primers specific to the full length or a partial region of the vanA gene of Enterococcus faecalis may include a primer including a nucleotide sequence of SEQ ID NO: 96 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 96, and a primer including a nucleotide sequence of SEQ ID NO: 97 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 97, A set of primers specific to the entire length or a partial region of the vanB gene of Enterococcus faecium may include a primer containing the nucleotide sequence of SEQ ID NO: 98 or a nucleotide sequence that has 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 98, and a primer containing the nucleotide sequence of SEQ ID NO: 99 or a nucleotide sequence that has 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 99.

[0054] By further using the above primer set, it is possible to investigate more causative bacteria of sepsis or causative bacterial genes and drug resistance genes. By using the above primer set, the interaction between primers is reduced, and the specificity and reactivity when performing the PCR method are improved. In addition, the amplification efficiency is also improved under uniform conditions such as temperature and reagents when performing the PCR method.

[0055] In this embodiment, the length of the entire length or a partial region of each gene (target region, and / or the amplified DNA) may be, for example, 50 to 300 bases long, or 100 to 200 bases long.

[0056] In the present specification, the DNA of the target region of each gene and / or the amplified DNA may include a specific base sequence or a base sequence having sequence identity of 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. The amplified DNA of the gene may include a specific base sequence or a base sequence having sequence identity of 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. The amplified DNA of the gene may include a specific base sequence or a base sequence having 0 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 nucleotide addition or deletion at the 3' end or 5' end.

[0057] The DNA of the target region of each gene and / or the amplified DNA may also have a label detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Detectable labels include, for example, biotin detected with labeled streptavidin, haptens, fluorescent dyes (e.g., fluorescein, Texas Red, and rhodamine), electron-dense reagents, enzymes (e.g., horseradish peroxidase and alkaline phosphatase), and radioisotopes (e.g., 32 P, 3 H, 14 C and 125 I). For example, the 5'-end, 3'-end or internal of the DNA of the amplified gene may be labeled, or the DNA may be labeled via a linker. For example, the 5'-end of the DNA of the amplified gene may be labeled by being modified with biotin, and in this case, the 5'-end of the primer may be modified with biotin via a linker. By amplifying DNA using a primer having a label as described above, amplified DNA containing a label can be obtained.

[0058] The DNA of the full length or a partial region of the gyrB gene of Escherichia coli may contain the nucleotide sequence of SEQ ID NO: 51 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 51, The DNA of the full length or a partial region of the nuc gene of Staphylococcus aureus may contain the nucleotide sequence of SEQ ID NO: 61 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 61, The DNA of the full length or a partial region of the atlE gene of Staphylococcus epidermidis may contain the nucleotide sequence of SEQ ID NO: 63 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 63, The DNA of the full length or a partial region of the gyrB gene of Klebsiella pneumoniae may contain the nucleotide sequence of SEQ ID NO: 53 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 53, The DNA of the full-length or partial region of the rpoB gene of Enterococcus may contain the nucleotide sequence of SEQ ID NO:59 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO:59.

[0059] By using the full-length or partial region of DNA described above, interactions between multiple DNAs are reduced, improving specificity and reactivity when detecting the presence or absence of amplification of each region.

[0060] In addition, when a set of primers specific to the full length or a partial region of a gene other than the gyrB gene of Escherichia coli, the nuc gene of Staphylococcus aureus, the atlE gene of Staphylococcus epidermidis, the gyrB gene of Klebsiella pneumoniae, and the rpoB gene of Enterococcus spp. is further used, The DNA of the full length or a partial region of the rpoB gene of Acinetobacter may contain the nucleotide sequence of SEQ ID NO: 47 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 47, The DNA of the full length or a partial region of the 16S-23S ITS gene of Acinetobacter baumannii may contain the nucleotide sequence of SEQ ID NO: 48 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 48, The DNA of the full length or a partial region of the ompA gene of the genus Citrobacter may contain the nucleotide sequence of SEQ ID NO: 49 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 49, The DNA of the full length or a partial region of the rpoS gene of the Enterobacter genus may contain the nucleotide sequence of SEQ ID NO: 50 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 50, The DNA of the full length or a partial region of the gyrB gene of Klebsiella aerogenes may contain the nucleotide sequence of SEQ ID NO: 52 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 52, The DNA of the full length or a partial region of the pehX gene of Klebsiella oxytoca may contain the nucleotide sequence of SEQ ID NO: 54 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 54, The DNA of the full length or a partial region of the gyrB gene of Klebsiella variicola may contain the nucleotide sequence of SEQ ID NO: 55 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 55, The DNA of the full length or a partial region of the oprL gene of Pseudomonas aeruginosa may contain the nucleotide sequence of SEQ ID NO: 56 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 56, The DNA of the full length or a partial region of the rpoB gene of the genus Proteus may contain the nucleotide sequence of SEQ ID NO: 57 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 57, The DNA of the full length or a partial region of the hasA gene of Serratia marcescens may contain the nucleotide sequence of SEQ ID NO: 58 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 58, The DNA of the full length or a partial region of the iap p60 gene of Listeria may contain the nucleotide sequence of SEQ ID NO: 60 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 60, The DNA of the full length or a partial region of the nuc gene of Staphylococcus argenteus may contain the nucleotide sequence of SEQ ID NO: 62 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 62, The DNA of the full length or a partial region of the tuf gene of the genus Staphylococcus may contain the nucleotide sequence of SEQ ID NO: 64 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 64, The DNA of the full length or a partial region of the rnpB gene of the genus Streptococcus may contain the nucleotide sequence of SEQ ID NO: 65 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 65, The DNA of the full length or a partial region of the xisco gene of Streptococcus pneumoniae may contain the nucleotide sequence of SEQ ID NO: 66 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 66, The DNA of the full length or a partial region of the gyrB gene of Streptococcus pyogenes may contain the nucleotide sequence of SEQ ID NO: 67 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 67, The DNA of the full length or a partial region of the beta-lactamase_KPC gene of Klebsiella pneumoniae may contain the nucleotide sequence of SEQ ID NO: 100 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 100, The DNA of the full length or a partial region of the beta-lactamase_NDM gene of Klebsiella pneumoniae may contain the nucleotide sequence of SEQ ID NO: 101 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 101, The DNA of the full length or a partial region of the beta-lactamase_IMP85 gene of Pseudomonas aeruginosa may contain the nucleotide sequence of SEQ ID NO: 102 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 102, The DNA of the full length or a partial region of the beta-lactamase_VIM gene of Citrobacter freundii may contain the nucleotide sequence of SEQ ID NO: 103 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 103; The DNA of the full length or a partial region of the beta-lactamase_CTX-M1 gene of Salmonella enterica may contain the nucleotide sequence of SEQ ID NO: 104 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 104, The DNA of the full length or a partial region of the beta-lactamase_CTX-M2 gene of Salmonella enterica may contain the nucleotide sequence of SEQ ID NO: 105 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 105, The DNA of the full length or a partial region of the beta-lactamase_CTX-M8 gene of Citrobacter amalonaticus may contain the nucleotide sequence of SEQ ID NO: 106 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 106, The DNA of the full length or a partial region of the E. coli beta-lactamase_CTX-M25 gene may contain the nucleotide sequence of SEQ ID NO: 107 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 107, The DNA of the full length or a partial region of the beta-lactamase_CTX-M9 gene of E. coli may contain the nucleotide sequence of SEQ ID NO: 108 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 108, The DNA of the full length or a partial region of the beta-lactamase_OXA-23 gene of Acinetobacter baumannii may contain the nucleotide sequence of SEQ ID NO: 109 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 109, The DNA of the full length or a partial region of the beta-lactamase_OXA-40 gene of Acinetobacter baumannii may contain the nucleotide sequence of SEQ ID NO: 110 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 110, The DNA of the full length or a partial region of the beta-lactamase_OXA-48 gene of Klebsiella pneumoniae may contain the nucleotide sequence of SEQ ID NO: 111 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 111, The DNA of the full length or a partial region of the beta-lactamase_OXA-58 gene of Acinetobacter haemolyticus may contain the nucleotide sequence of SEQ ID NO: 112 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 112, The DNA of the full length or a partial region of the mecA gene of Staphylococcus aureus may contain the nucleotide sequence of SEQ ID NO: 113 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 113, The full length or partial region of the vanA gene of Enterococcus faecalis may contain the nucleotide sequence of SEQ ID NO: 114 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 114, The DNA of the full length or a partial region of the vanB gene of Enterococcus faecium may contain the nucleotide sequence of SEQ ID NO:115 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO:115.

[0061] By using the full-length or partial region of DNA described above, interactions between multiple DNAs are reduced, improving specificity and reactivity when detecting the presence or absence of amplification of each region.

[0062] [Detection process] In one embodiment, the detection step is a step of detecting the presence or absence of amplification of the full length or a partial region of each gene using a probe specific to the DNA of the full length or a partial region of each gene.

[0063] A probe specific to the DNA of the full length or a part of the region (target region) of each gene can be designed based on the sequence of the DNA of the target region. The probe can be synthesized by a method known to those skilled in the art. The probe may be, for example, an oligonucleotide having a length of 10 to 30 bases, or an oligonucleotide having a length of 15 to 20 bases. The primer does not need to reflect the exact sequence of the target region, but must be sufficiently complementary to hybridize with and detect at least a part of the DNA of the target region (for example, the sequence of an internal region).

[0064] In the present specification, the probe may comprise a specific base sequence or a base sequence having sequence identity of 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. The probe may comprise a specific base sequence or a base sequence having sequence identity of 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. The probe may comprise a specific base sequence or a base sequence having 0 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 nucleotide addition or deletion at the 3' end or 5' end.

[0065] The probes may consist of the bases A, G, C, T or analogs, or degenerate bases (M, R, W, S, Y, K). By including degenerate bases at specific positions where the base sequence differs depending on the bacterial strain, the probes can more appropriately hybridize with the DNA of the target region.

[0066] Probes may also have a label that is detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Detectable labels include, for example, biotin for detection with labeled avidin, haptens, fluorescent dyes (e.g., fluorescein, Texas Red, and rhodamine), electron-dense reagents, enzymes (e.g., horseradish peroxidase and alkaline phosphatase), and radioisotopes (e.g., 32 P, 3 H, 14 C and 125 I). The labeling may be carried out, for example, at the 5'-end, 3'-end or internal of the probe, or via a linker. For example, the 5'-end of the probe may be modified with biotin to be labeled, and in this case, the 5'-end of the probe may be modified with biotin via a linker.

[0067] The label can be used to detect the amplified DNA by hybridizing the amplified DNA with a labeled probe. The label can also be used to capture the probe, for immobilization on a solid support.

[0068] In this embodiment, in order to simultaneously detect the presence or absence of amplification of DNA in the target region of each gene, the probes are designed so that detection can be performed under similar or uniform conditions for the DNA of each gene.

[0069] The probe may be, for example, mixed with the amplified DNA to hybridize with the amplified DNA, or may be immobilized on a solid support to hybridize with the amplified DNA. Examples of the solid support include substrates, arrays, magnetic beads, columns, and colored beads. The method of immobilizing on the solid support may be a multiplex format. The amplified DNA may be captured by the probe immobilized on the solid support.

[0070] When the probes are immobilized on a solid support, for example, the probes may each include a different identifiable identifier (e.g., ID), or each probe may be immobilized on each identifier of a solid support that includes an identifiable identifier. In other words, the probes may each include a different identifier, or each probe may be bound to a different identifier on a solid support. When the probes are immobilized on a solid support, the amplified DNA can be captured by specifically binding to each corresponding probe. The captured amplified DNA is labeled, and the amplified DNA can be detected based on the label. For example, when the label is a fluorescent label, the amplified DNA can be detected by measuring the fluorescence value. An example of an apparatus that can measure the fluorescence value due to the binding of each probe and the amplified DNA by identification by the identifier is the 100 Fluorescent Analyzer (PlexBio), which is a fluorescence measuring apparatus.

[0071] The method of detecting the presence or absence of amplification of DNA in the target region of each gene using a probe can be carried out by a method known to those skilled in the art, and can be carried out, for example, as follows.

[0072] The PCR product obtained in the step of carrying out the PCR method (if the sample contains DNA of the target region, it contains amplified DNA) is thermally denatured (for example, 95°C for 5 minutes and then rapidly cooled to 4°C) and hybridized with the probe. The thermal denaturation may be, for example, 90°C to 95°C for 3 to 10 minutes and then rapidly cooled to 0 to 5°C, or 95°C for 5 minutes and then rapidly cooled to 4°C. The hybridization may be, for example, by incubating at 35°C to 39°C for 10 to 30 minutes, or by incubating at 37°C for 20 minutes. The probe may be a probe that hybridizes with the DNA of the target region under stringent conditions. In this specification, the term "stringent conditions" refers to conditions under which a complementary strand of a nucleotide strand having homology to the sequence of the target region preferentially hybridizes to the sequence of the target region, and a complementary strand of a nucleotide strand having no homology does not substantially hybridize. The stringent conditions are sequence-dependent and differ in various situations. Longer sequences hybridize specifically at higher temperatures. Stringent conditions include, for example, 50% formamide, 5xSSC (150 mM sodium chloride, 15 mM trisodium citrate, 10 mM sodium phosphate, 1 mM ethylenediaminetetraacetic acid, pH 7.2), 5xDenhardt's solution, 0.1% SDS, 10% dextran sulfate, and 100 μg / mL denatured salmon sperm DNA incubated at 42°C, followed by washing the filter in 0.2xSSC at 42°C.

[0073] After hybridization, for example, detection can be performed using the label of the amplified DNA and / or the probe. Detection can be performed appropriately depending on the type of label. Furthermore, after hybridization, biotin-modified amplified DNA can also be detected by labeling with labeled avidin (e.g., Streptoavidin-Phycoerythrin (PlexBio)). Labeling can be performed appropriately depending on the type of label, and may be performed, for example, by incubation at 35°C to 39°C for 10 to 30 minutes, or by incubation at 37°C for 10 minutes.

[0074] In the detection step of this embodiment, the presence or absence of amplification of DNA in the target region is simultaneously detected using a probe specific to each of them. The method of simultaneously detecting the presence or absence of amplification of DNA in the target region using a probe specific to each of them can be performed by a method known to those skilled in the art.

[0075] A method for simultaneously detecting the presence or absence of amplification of DNA of different target regions includes, for example, a multiplex PCR assay. Therefore, the method according to this embodiment can also be carried out by a multiplex PCR assay. The detection step may include hybridizing (for example, using an amplification reaction mixture) probes specific to the DNA of the target regions immobilized on a solid-phase substrate in a multiplex format with the DNA of the target regions amplified in the step of carrying out the PCR method, and simultaneously detecting the hybridized DNA of the amplified target regions.

[0076] In this embodiment, the presence or absence of amplification of DNA of different target regions is detected simultaneously in one reaction system (e.g., hybridization reaction system), so that the probes specific to the DNA of the target region of each gene are used collectively. For example, under conditions suitable for hybridization reaction, a combination (e.g., mixture) of amplified DNA is contacted with a combination (e.g., mixture) of probes, so that the probes specifically bind to at least a part of the amplified DNA of each gene, and the amplified DNA can be detected by real-time PCR detection (e.g., TaqMan probe, molecular beacon), or solid support hybridization (e.g., microarray hybridization of nucleic acid, bead-based capture), etc., by labeling the amplified DNA, labeling the probe, or labeling after the amplified DNA is captured.

[0077] Detection of the presence or absence of amplification of DNA of the bacterial target gene, or the target region of the bacterial target gene and the drug resistance gene can be appropriately determined depending on the detection method, but for example, the presence or absence of amplification of the DNA of the target region may be determined by comparison with a control sample not containing bacterial DNA, or the presence or absence of amplification may be determined by comparison with a reference value. When the presence or absence of amplification is determined by comparison with a predetermined reference value, for example, if the reference value is exceeded, it may be determined that the DNA of the bacterial target gene, or the target region of the bacterial target gene and the drug resistance gene has been detected. The reference value may be set for each gene, or may be set uniformly for all genes. The reference value may be, for example, a value set from a value in a control sample not containing bacterial DNA, a value set from a value in a sample containing bacterial DNA, or a value set by comparing the value in the control sample with the value in a sample containing bacterial DNA. In addition, a cutoff value may be used to appropriately determine the presence or absence of amplification of DNA of the target region of each gene. The cutoff value may also be set for each gene, or may be set uniformly for all genes. The cutoff value can be set by, for example, using an ROC curve to select a cutoff value that can ensure the target sensitivity and specificity. For example, for a probe that shows a slight cross-reaction, the accuracy of the determination can be improved by setting the cutoff value a little higher.

[0078] Based on the DNA of the target region in which amplification has been confirmed, the causative bacterium present in the sample, or the causative bacterium and the drug resistance gene of the bacterium, can be identified. For example, when amplification of the DNA of the target region of a gene of a particular bacterium is detected, the bacterium may be identified as the causative bacterium.

[0079] In the detection step, the probe specific to the full length or a partial region of the DNA of the gyrB gene of Escherichia coli may contain the nucleotide sequence of SEQ ID NO: 121 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 121; The probe specific to the full length or a partial region of the DNA of the nuc gene of Staphylococcus aureus may contain the nucleotide sequence of SEQ ID NO: 134 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 134, The probe specific to the full length or a partial region of the DNA of the atlE gene of Staphylococcus epidermidis may contain the nucleotide sequence of SEQ ID NO: 136 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 136, The probe specific to the full length or a partial region of the DNA of the gyrB gene of Klebsiella pneumoniae may contain the nucleotide sequence of SEQ ID NO: 124 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 124, The probe specific to the DNA of the full length or a partial region of the rpoB gene of Enterococcus may be selected from a probe containing the nucleotide sequence of SEQ ID NO: 129 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 129, a probe containing the nucleotide sequence of SEQ ID NO: 130 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 130, a probe containing the nucleotide sequence of SEQ ID NO: 131 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 131, and combinations thereof.

[0080] By using the above-mentioned probes, the interaction between multiple probes is reduced, and the specificity and reactivity when detecting the presence or absence of amplification of each region are improved. At the same time, the hybridization efficiency under uniform conditions such as temperature and reagents during the hybridization reaction is also improved.

[0081] In addition, when a probe specific to the full length or a partial region of a gene other than the gyrB gene of Escherichia coli, the nuc gene of Staphylococcus aureus, the atlE gene of Staphylococcus epidermidis, the gyrB gene of Klebsiella pneumoniae, and the rpoB gene of Enterococcus spp. is further used, The probe specific to the full length or a partial region of the DNA of the rpoB gene of Acinetobacter sp. may be selected from a probe containing the nucleotide sequence of SEQ ID NO: 116 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 116, a probe containing the nucleotide sequence of SEQ ID NO: 117 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 117, and a combination thereof; The probe specific to the full length or a partial region of the DNA of the 16S-23S ITS gene of Acinetobacter baumannii may contain the nucleotide sequence of SEQ ID NO: 118 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 118, The probe specific to the full length or a partial region of the DNA of the ompA gene of the genus Citrobacter may contain the nucleotide sequence of SEQ ID NO: 119 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 119, The probe specific to the full length or a partial region of the DNA of the rpoS gene of Enterobacter may contain the nucleotide sequence of SEQ ID NO: 120 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 120, The probe specific to the full length or a partial region of the DNA of the gyrB gene of Klebsiella aerogenes may contain the nucleotide sequence of SEQ ID NO: 122 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 122, The probe specific to the full length or a partial region of the DNA of the pehX gene of Klebsiella oxytoca may contain the nucleotide sequence of SEQ ID NO: 123 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 123, The probe specific to the full length or a partial region of the DNA of the gyrB gene of Klebsiella variicola may contain the nucleotide sequence of SEQ ID NO: 125 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 125, The probe specific to the full length or a partial region of the DNA of the oprL gene of Pseudomonas aeruginosa may contain the nucleotide sequence of SEQ ID NO: 126 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 126, The probe specific to the full length or a partial region of the DNA of the rpoB gene of the genus Proteus may contain the nucleotide sequence of SEQ ID NO: 127 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 127, The probe specific to the full length or a partial region of the DNA of the hasA gene of Serratia marcescens may contain the nucleotide sequence of SEQ ID NO: 128 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 128, The probe specific to the full length or a partial region of the DNA of the iap p60 gene of the Listeria genus may be selected from a probe containing the nucleotide sequence of SEQ ID NO: 132 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 132, a probe containing the nucleotide sequence of SEQ ID NO: 133 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 133, and a combination thereof; The probe specific to the DNA of the full length or a part of the nuc gene of Staphylococcus argenteus may contain the nucleotide sequence of SEQ ID NO: 135 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 135, The probe specific to the DNA of the full length or a part of the region of the tuf gene of the genus Staphylococcus may contain the nucleotide sequence of SEQ ID NO: 137 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 137; The probe specific to the full length or a partial region of the DNA of the rnpB gene of the genus Streptococcus may contain the nucleotide sequence of SEQ ID NO: 140 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 140, The probe specific to the full length or a partial region of the DNA of the xisco gene of Streptococcus pneumoniae may contain the nucleotide sequence of SEQ ID NO: 138 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 138, The probe specific to the DNA of the full length or a part of the region of the gyrB gene of Streptococcus pyogenes may contain the nucleotide sequence of SEQ ID NO: 139 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 139; The probe specific to the full length or a partial region of the DNA of the beta-lactamase_KPC gene of Klebsiella pneumoniae may contain the nucleotide sequence of SEQ ID NO: 142 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 142, The probe specific to the full length or a partial region of the DNA of the beta-lactamase_NDM gene of Klebsiella pneumoniae may contain the nucleotide sequence of SEQ ID NO: 141 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 141, The probe specific to the full length or a partial region of the DNA of the beta-lactamase_IMP85 gene of Pseudomonas aeruginosa may contain the nucleotide sequence of SEQ ID NO: 144 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 144, The probe specific to the full length or a partial region of the DNA of the beta-lactamase_VIM gene of Citrobacter freundii may contain the nucleotide sequence of SEQ ID NO: 143 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 143; The probe specific to the full length or a partial region of the DNA of the beta-lactamase_CTX-M1 gene of Salmonella enterica may contain the nucleotide sequence of SEQ ID NO: 149 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 149, The probe specific to the full length or a partial region of the DNA of the beta-lactamase_CTX-M2 gene of Salmonella enterica may contain the nucleotide sequence of SEQ ID NO: 150 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 150, The probe specific to the full length or a partial region of the DNA of the beta-lactamase_CTX-M8 gene of Citrobacter amalonaticus may contain the nucleotide sequence of SEQ ID NO: 151 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 151, The probe specific to the full length or a partial region of the E. coli beta-lactamase_CTX-M25 gene may contain the nucleotide sequence of SEQ ID NO: 153 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 153, The probe specific to the full length or a partial region of the DNA of the E. coli beta-lactamase_CTX-M9 gene may contain the nucleotide sequence of SEQ ID NO: 152 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 152, The probe specific to the full length or a partial region of the DNA of the beta-lactamase_OXA-23 gene of Acinetobacter baumannii may contain the nucleotide sequence of SEQ ID NO: 145 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 145, The probe specific to the full length or a partial region of the DNA of the beta-lactamase_OXA-40 gene of Acinetobacter baumannii may contain the nucleotide sequence of SEQ ID NO: 146 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 146, The probe specific to the full length or a partial region of the DNA of the beta-lactamase_OXA-48 gene of Klebsiella pneumoniae may contain the nucleotide sequence of SEQ ID NO: 147 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 147, The probe specific to the full length or a partial region of the DNA of the beta-lactamase_OXA-58 gene of Acinetobacter haemolyticus may contain the nucleotide sequence of SEQ ID NO: 148 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 148, The probe specific to the full length or a partial region of the DNA of the mecA gene of Staphylococcus aureus may contain the nucleotide sequence of SEQ ID NO: 154 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 154, The probe specific to the full length or a partial region of the vanA gene of Enterococcus faecalis may contain the nucleotide sequence of SEQ ID NO: 155 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 155; A probe specific to the DNA of the entire length or a partial region of the vanB gene of Enterococcus faecium may contain the nucleotide sequence of SEQ ID NO:156 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO:156.

[0082] By further using the above-mentioned probes, it is possible to examine a larger number of bacteria causing sepsis and also to examine drug resistance genes.

[0083] By using the above-mentioned probes, the interaction between multiple probes is reduced, and the specificity and reactivity when detecting the presence or absence of amplification of each region are improved. In addition, the hybridization efficiency is improved under uniform conditions such as temperature and reagents during the hybridization reaction.

[0084] In one embodiment, the present invention relates to a method for detecting a gyrB gene of Escherichia coli, a nuc gene of Staphylococcus aureus, an atlE gene of Staphylococcus epidermidis, a gyrB gene of Klebsiella pneumoniae, an rpoB gene of Enterococcus spp. (e.g., Enterococcus faecalis), an rpoB gene of Acinetobacter spp. (e.g., Acinetobacter baumannii), an 16S-23S gene of Acinetobacter baumannii, or an 18S-23S gene of Acinetobacter baumannii. ITS gene, ompA gene of Citrobacter spp. (e.g., Citrobacter koseri, Citrobacter freundii), rpoS gene of Enterobacter cloacae, gyrB gene of Klebsiella aerogenes, pehX gene of Klebsiella oxytoca, gyrB gene of Klebsiella variicola, oprL gene of Pseudomonas aeruginosa, rpoB gene of Proteus spp. (e.g., Proteus mirabilis), hasA gene of Serratia marcescens, and Listeria spp. spp. (e.g., Listeria monocytogenes), the iap p60 gene, Staphylococcus argenteus, the nuc gene, Staphylococcus spp. (e.g., Staphylococcus aureus),aureus), tuf gene of Streptococcus spp. (e.g., Streptococcus pneumoniae), rnpB gene of Streptococcus pneumoniae, xisco gene of Streptococcus pneumoniae, gyrB gene of Streptococcus pyogenes, beta-lactamase_KPC gene of Klebsiella pneumoniae, beta-lactamase_NDM gene of Klebsiella pneumoniae, Pseudomonas aeruginosa, beta-lactamase_IMP85 gene of A. aeruginosa, beta-lactamase_VIM gene of Citrobacter freundii, beta-lactamase_CTX-M1 gene of Salmonella enterica, beta-lactamase_CTX-M2 gene of Salmonella enterica, beta-lactamase_CTX-M8 gene of Citrobacter amalonatics, and Escherichia coli beta-lactamase_CTX-M25 gene of Escherichia coli, beta-lactamase_CTX-M9 gene of Escherichia coli, beta-lactamase_OXA-23 gene of Acinetobacter baumannii,performing a PCR method using a combination of primer sets specific to the full length or a partial region of a gene and / or drug resistance gene of two or more types of bacteria selected from the group consisting of the beta-lactamase_OXA-40 gene of Klebsiella pneumoniae, the beta-lactamase_OXA-48 gene of Klebsiella pneumoniae, the beta-lactamase_OXA-58 gene of Acinetobacter haemolyticus, the mecA gene of Staphylococcus aureus, the vanA gene of Enterococcus faecalis, and the vanB gene of Enterococcus faecium; detecting the presence or absence of amplification of DNA of the full length or a part of the region of each gene using a combination of probes specific to the DNA of the full length or a part of the region of each gene; Also provided is a method for identifying a causative bacterium of sepsis and / or a drug resistance gene, comprising:

[0085] The bacteria or genes selected from the group of multiple genes may be, for example, 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 28 or more, 30 or more, 32 or more, 35 or more, or 37 or more. The bacterial genes selected from the group of multiple genes may be, for example, 3 or more, 5 or more, 7 or more, 10 or more, 12 or more, 15 or more, 17 or more, 19 or more, or 21 or more. The drug resistance genes selected from the group of multiple genes may be, for example, 3 or more, 5 or more, 7 or more, 10 or more, 12 or more, 14 or more, or 16 or more. The bacteria or genes selected from the group of multiple genes may be, for example, 37 or less, 34 or less, 21 or less, 16 or less, or 11 or less. Furthermore, the bacteria or genes selected from the group of multiple genes may be, for example, 2 to 40 types, 5 to 37 types, 10 to 37 types, or 15 to 25 types.

[0086] In the step of carrying out the PCR method, the set of primers specific to the full length or a partial region of the gyrB gene of E. coli may include a primer containing a nucleotide sequence of SEQ ID NO: 10 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 10, and a primer containing a nucleotide sequence of SEQ ID NO: 11 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 11, The set of primers specific to the full length or a partial region of the nuc gene of Staphylococcus aureus may include one or more primers selected from a primer including a nucleotide sequence of SEQ ID NO: 32 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 32, a primer including a nucleotide sequence of SEQ ID NO: 34 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 34, and a combination thereof, and a primer including a nucleotide sequence of SEQ ID NO: 33 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 33, The set of primers specific to the full length or a partial region of the atlE gene of Staphylococcus epidermidis may include a primer containing a nucleotide sequence of SEQ ID NO: 37 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 37, and a primer containing a nucleotide sequence of SEQ ID NO: 38 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 38, The set of primers specific to the full length or a partial region of the gyrB gene of Klebsiella pneumoniae may include a primer including a nucleotide sequence of SEQ ID NO: 14 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 14, and a primer including a nucleotide sequence of SEQ ID NO: 15 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 15, The set of primers specific to the full length or a partial region of the rpoB gene of the genus Enterococcus may include one or more primers selected from a primer including a nucleotide sequence of SEQ ID NO: 26 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 26, a primer including a nucleotide sequence of SEQ ID NO: 27 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 27, a primer including a nucleotide sequence of SEQ ID NO: 28 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 28, and a combination thereof, and a primer including a nucleotide sequence of SEQ ID NO: 29 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 29, The set of primers specific to the full length or a partial region of the rpoB gene of Acinetobacter may include a primer including a nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 1, and a primer including a nucleotide sequence of SEQ ID NO: 2 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 2, The set of primers specific to the full length or a partial region of the 16S-23S ITS gene of Acinetobacter baumannii may include a primer including a nucleotide sequence of SEQ ID NO: 3 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 3, and a primer including a nucleotide sequence of SEQ ID NO: 4 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 4, The set of primers specific to the full length or a partial region of the ompA gene of the genus Citrobacter may include one or more primers selected from a primer containing the nucleotide sequence of SEQ ID NO: 5 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 5, a primer containing the nucleotide sequence of SEQ ID NO: 6 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 6, and a combination thereof, and a primer containing the nucleotide sequence of SEQ ID NO: 7 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 7, The set of primers specific to the full length or a partial region of the rpoS gene of Enterobacter may include a primer containing a nucleotide sequence of SEQ ID NO: 8 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 8, and a primer containing a nucleotide sequence of SEQ ID NO: 9 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 9, The set of primers specific to the full length or a partial region of the gyrB gene of Klebsiella aerogenes may include a primer including a nucleotide sequence of SEQ ID NO: 12 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 12, and a primer including a nucleotide sequence of SEQ ID NO: 13 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 13, The set of primers specific to the full length or a partial region of the pehX gene of Klebsiella oxytoca may include a primer including a nucleotide sequence of SEQ ID NO: 16 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 16, and a primer including a nucleotide sequence of SEQ ID NO: 17 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 17, The set of primers specific to the full length or a partial region of the gyrB gene of Klebsiella variicola may include a primer including a nucleotide sequence of SEQ ID NO: 18 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 18, and a primer including a nucleotide sequence of SEQ ID NO: 19 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 19, The set of primers specific to the full length or a partial region of the oprL gene of Pseudomonas aeruginosa may include a primer containing a nucleotide sequence of SEQ ID NO: 20 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 20, and a primer containing a nucleotide sequence of SEQ ID NO: 21 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 21, The set of primers specific to the full length or a partial region of the rpoB gene of the genus Proteus may include a primer including a nucleotide sequence of SEQ ID NO: 22 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 22, and a primer including a nucleotide sequence of SEQ ID NO: 23 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 23, The set of primers specific to the full length or a partial region of the hasA gene of Serratia marcescens may include a primer including a nucleotide sequence of SEQ ID NO: 24 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 24, and a primer including a nucleotide sequence of SEQ ID NO: 25 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 25, The set of primers specific to the full length or a partial region of the iap p60 gene of the Listeria genus may include a primer including a nucleotide sequence of SEQ ID NO: 30 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 30, and a primer including a nucleotide sequence of SEQ ID NO: 31 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 31, The set of primers specific to the full length or a partial region of the nuc gene of Staphylococcus argenteus may include a primer including a nucleotide sequence of SEQ ID NO: 35 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 35, and a primer including a nucleotide sequence of SEQ ID NO: 36 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 36, The set of primers specific to the full length or a partial region of the tuf gene of the genus Staphylococcus may include a primer containing a nucleotide sequence of SEQ ID NO: 39 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 39, and a primer containing a nucleotide sequence of SEQ ID NO: 40 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 40, The set of primers specific to the full length or a partial region of the rnpB gene of the genus Streptococcus may include a primer containing a nucleotide sequence of SEQ ID NO: 41 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 41, and a primer containing a nucleotide sequence of SEQ ID NO: 42 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 42, The set of primers specific to the full length or a partial region of the xisco gene of Streptococcus pneumoniae may include a primer including a nucleotide sequence of SEQ ID NO: 43 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 43, and a primer including a nucleotide sequence of SEQ ID NO: 44 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 44, The set of primers specific to the full length or a partial region of the gyrB gene of Streptococcus pyogenes may include a primer including a nucleotide sequence of SEQ ID NO: 45 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 45, and a primer including a nucleotide sequence of SEQ ID NO: 46 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 46, The set of primers specific to the full length or a partial region of the beta-lactamase_KPC gene of Klebsiella pneumoniae may include a primer containing a nucleotide sequence of SEQ ID NO: 68 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 68, and a primer containing a nucleotide sequence of SEQ ID NO: 69 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 69, The set of primers specific to the full length or a partial region of the beta-lactamase_NDM gene of Klebsiella pneumoniae may include a primer including a nucleotide sequence of SEQ ID NO: 70 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 70, and a primer including a nucleotide sequence of SEQ ID NO: 71 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 71, The set of primers specific to the full length or a partial region of the beta-lactamase_IMP85 gene of Pseudomonas aeruginosa may include a primer including a nucleotide sequence of SEQ ID NO: 72 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 72, and a primer including a nucleotide sequence of SEQ ID NO: 73 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 73, The set of primers specific to the full length or a partial region of the beta-lactamase_VIM gene of Citrobacter freundii may include a primer including a nucleotide sequence of SEQ ID NO: 74 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 74, and a primer including a nucleotide sequence of SEQ ID NO: 75 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 75, The set of primers specific to the full length or a partial region of the beta-lactamase_CTX-M1 gene of Salmonella enterica may include a primer including a nucleotide sequence of SEQ ID NO: 76 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 76, and a primer including a nucleotide sequence of SEQ ID NO: 77 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 77, The set of primers specific to the full length or a partial region of the beta-lactamase_CTX-M2 gene of Salmonella enterica may include a primer including a nucleotide sequence of SEQ ID NO: 78 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 78, and a primer including a nucleotide sequence of SEQ ID NO: 79 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 79, The set of primers specific to the full length or a partial region of the beta-lactamase_CTX-M8 gene of Citrobacter amalonaticus may include a primer including a nucleotide sequence of SEQ ID NO: 80 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 80, and a primer including a nucleotide sequence of SEQ ID NO: 81 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 81, The set of primers specific to the full length or a partial region of the E. coli beta-lactamase_CTX-M25 gene may include a primer containing a nucleotide sequence of SEQ ID NO: 82 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 82, and a primer containing a nucleotide sequence of SEQ ID NO: 83 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 83, The set of primers specific to the full length or a partial region of the E. coli beta-lactamase_CTX-M9 gene may include a primer containing a nucleotide sequence of SEQ ID NO: 84 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 84, and a primer containing a nucleotide sequence of SEQ ID NO: 85 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 85, The set of primers specific to the full length or a partial region of the beta-lactamase_OXA-23 gene of Acinetobacter baumannii may include a primer including a nucleotide sequence of SEQ ID NO: 86 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 86, and a primer including a nucleotide sequence of SEQ ID NO: 87 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 87, The set of primers specific to the full length or a partial region of the beta-lactamase_OXA-40 gene of Acinetobacter baumannii may include a primer including a nucleotide sequence of SEQ ID NO: 88 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 88, and a primer including a nucleotide sequence of SEQ ID NO: 89 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 89, The set of primers specific to the full length or a partial region of the beta-lactamase_OXA-48 gene of Klebsiella pneumoniae may include a primer including a nucleotide sequence of SEQ ID NO: 90 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 90, and a primer including a nucleotide sequence of SEQ ID NO: 91 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 91, The set of primers specific to the full length or a partial region of the beta-lactamase_OXA-58 gene of Acinetobacter haemolyticus may include a primer including a nucleotide sequence of SEQ ID NO: 92 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 92, and a primer including a nucleotide sequence of SEQ ID NO: 93 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 93, The set of primers specific to the full length or a partial region of the mecA gene of Staphylococcus aureus may include a primer including a nucleotide sequence of SEQ ID NO: 94 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 94, and a primer including a nucleotide sequence of SEQ ID NO: 95 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 95, The set of primers specific to the full length or a partial region of the vanA gene of Enterococcus faecalis may include a primer including a nucleotide sequence of SEQ ID NO: 96 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 96, and a primer including a nucleotide sequence of SEQ ID NO: 97 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 97, A set of primers specific to the entire length or a partial region of the vanB gene of Enterococcus faecium may include a primer containing the nucleotide sequence of SEQ ID NO: 98 or a nucleotide sequence that has 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 98, and a primer containing the nucleotide sequence of SEQ ID NO: 99 or a nucleotide sequence that has 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 99.

[0087] The use of the above combination of primer sets improves the specificity and reactivity when performing PCR, and also improves the amplification efficiency under uniform conditions of temperature, reagents, etc., when performing PCR. In the step of carrying out the PCR method, the DNA of the full length or a partial region of the gyrB gene of Escherichia coli may contain the nucleotide sequence of SEQ ID NO: 51 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 51, The DNA of the full length or a partial region of the nuc gene of Staphylococcus aureus may contain the nucleotide sequence of SEQ ID NO: 61 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 61, The DNA of the full length or a partial region of the atlE gene of Staphylococcus epidermidis may contain the nucleotide sequence of SEQ ID NO: 63 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 63, The DNA of the full length or a partial region of the gyrB gene of Klebsiella pneumoniae may contain the nucleotide sequence of SEQ ID NO: 53 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 53, The DNA of the full length or a partial region of the rpoB gene of the genus Enterococcus may contain the nucleotide sequence of SEQ ID NO: 59 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 59, The DNA of the full length or a partial region of the rpoB gene of Acinetobacter may contain the nucleotide sequence of SEQ ID NO: 47 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 47, The DNA of the full length or a partial region of the 16S-23S ITS gene of Acinetobacter baumannii may contain the nucleotide sequence of SEQ ID NO: 48 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 48, The DNA of the full length or a partial region of the ompA gene of the genus Citrobacter may contain the nucleotide sequence of SEQ ID NO: 49 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 49, The DNA of the full length or a partial region of the rpoS gene of the Enterobacter genus may contain the nucleotide sequence of SEQ ID NO: 50 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 50, The DNA of the full length or a partial region of the gyrB gene of Klebsiella aerogenes may contain the nucleotide sequence of SEQ ID NO: 52 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 52, The DNA of the full length or a partial region of the pehX gene of Klebsiella oxytoca may contain the nucleotide sequence of SEQ ID NO: 54 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 54, The DNA of the full length or a partial region of the gyrB gene of Klebsiella variicola may contain the nucleotide sequence of SEQ ID NO: 55 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 55, The DNA of the full length or a partial region of the oprL gene of Pseudomonas aeruginosa may contain the nucleotide sequence of SEQ ID NO: 56 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 56, The DNA of the full length or a partial region of the rpoB gene of the genus Proteus may contain the nucleotide sequence of SEQ ID NO: 57 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 57, The DNA of the full length or a partial region of the hasA gene of Serratia marcescens may contain the nucleotide sequence of SEQ ID NO: 58 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 58, The DNA of the full length or a partial region of the iap p60 gene of Listeria may contain the nucleotide sequence of SEQ ID NO: 60 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 60, The DNA of the full length or a partial region of the nuc gene of Staphylococcus argenteus may contain the nucleotide sequence of SEQ ID NO: 62 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 62, The DNA of the full length or a partial region of the tuf gene of the genus Staphylococcus may contain the nucleotide sequence of SEQ ID NO: 64 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 64, The DNA of the full length or a partial region of the rnpB gene of the genus Streptococcus may contain the nucleotide sequence of SEQ ID NO: 65 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 65, The DNA of the full length or a partial region of the xisco gene of Streptococcus pneumoniae may contain the nucleotide sequence of SEQ ID NO: 66 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 66, The DNA of the full length or a partial region of the gyrB gene of Streptococcus pyogenes may contain the nucleotide sequence of SEQ ID NO: 67 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 67, The DNA of the full length or a partial region of the beta-lactamase_KPC gene of Klebsiella pneumoniae may contain the nucleotide sequence of SEQ ID NO: 100 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 100, The DNA of the full length or a partial region of the beta-lactamase_NDM gene of Klebsiella pneumoniae may contain the nucleotide sequence of SEQ ID NO: 101 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 101, The DNA of the full length or a partial region of the beta-lactamase_IMP85 gene of Pseudomonas aeruginosa may contain the nucleotide sequence of SEQ ID NO: 102 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 102, The DNA of the full length or a partial region of the beta-lactamase_VIM gene of Citrobacter freundii may contain the nucleotide sequence of SEQ ID NO: 103 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 103; The DNA of the full length or a partial region of the beta-lactamase_CTX-M1 gene of Salmonella enterica may contain the nucleotide sequence of SEQ ID NO: 104 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 104, The DNA of the full length or a partial region of the beta-lactamase_CTX-M2 gene of Salmonella enterica may contain the nucleotide sequence of SEQ ID NO: 105 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 105, The DNA of the full length or a partial region of the beta-lactamase_CTX-M8 gene of Citrobacter amalonaticus may contain the nucleotide sequence of SEQ ID NO: 106 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 106, The DNA of the full length or a partial region of the E. coli beta-lactamase_CTX-M25 gene may contain the nucleotide sequence of SEQ ID NO: 107 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 107, The DNA of the full length or a partial region of the beta-lactamase_CTX-M9 gene of E. coli may contain the nucleotide sequence of SEQ ID NO: 108 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 108, The DNA of the full length or a partial region of the beta-lactamase_OXA-23 gene of Acinetobacter baumannii may contain the nucleotide sequence of SEQ ID NO: 109 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 109, The amplified DNA of the full length or a partial region of the beta-lactamase_OXA-40 gene of Acinetobacter baumannii may contain the nucleotide sequence of SEQ ID NO: 110 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 110, The amplified DNA of the full length or a partial region of the beta-lactamase_OXA-48 gene of Klebsiella pneumoniae may contain the nucleotide sequence of SEQ ID NO: 111 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 111, The DNA of the full length or a partial region of the beta-lactamase_OXA-58 gene of Acinetobacter haemolyticus may contain the nucleotide sequence of SEQ ID NO: 112 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 112, The DNA of the full length or a partial region of the mecA gene of Staphylococcus aureus may contain the nucleotide sequence of SEQ ID NO: 113 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 113, The full length or partial region of the vanA gene of Enterococcus faecalis may contain the nucleotide sequence of SEQ ID NO: 114 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 114, The DNA of the full length or a partial region of the vanB gene of Enterococcus faecium may contain the nucleotide sequence of SEQ ID NO:115 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO:115.

[0088] By using the full-length or partial region of DNA described above, interactions between multiple DNAs are reduced, improving specificity and reactivity when detecting the presence or absence of amplification of each region. In the identification step, the probe specific to the full length or a partial region of the amplified DNA of the gyrB gene of Escherichia coli may contain the nucleotide sequence of SEQ ID NO: 121 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 121; The probe specific to the full length or a partial region of the DNA of the nuc gene of Staphylococcus aureus may contain a nucleotide sequence of SEQ ID NO: 134 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 134, The probe specific to the full length or a partial region of the DNA of the atlE gene of Staphylococcus epidermidis may contain the nucleotide sequence of SEQ ID NO: 136 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 136, The probe specific to the full length or a partial region of the DNA of the gyrB gene of Klebsiella pneumoniae may contain the nucleotide sequence of SEQ ID NO: 124 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 124, The probe specific to the DNA of the full length or a partial region of the rpoB gene of the genus Enterococcus may be selected from a probe containing the nucleotide sequence of SEQ ID NO: 129 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 129, a probe containing the nucleotide sequence of SEQ ID NO: 130 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 130, a probe containing the nucleotide sequence of SEQ ID NO: 131 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 131, and combinations thereof. The probe specific to the full length or a partial region of the DNA of the rpoB gene of Acinetobacter sp. may be selected from a probe containing the nucleotide sequence of SEQ ID NO: 116 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 116, a probe containing the nucleotide sequence of SEQ ID NO: 117 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 117, and a combination thereof; The probe specific to the full length or a partial region of the DNA of the 16S-23S ITS gene of Acinetobacter baumannii may contain the nucleotide sequence of SEQ ID NO: 118 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 118, The probe specific to the full length or a partial region of the DNA of the ompA gene of the genus Citrobacter may contain the nucleotide sequence of SEQ ID NO: 119 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 119, The probe specific to the full length or a partial region of the DNA of the rpoS gene of Enterobacter may contain the nucleotide sequence of SEQ ID NO: 120 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 120, The probe specific to the full length or a partial region of the DNA of the gyrB gene of Klebsiella aerogenes may contain the nucleotide sequence of SEQ ID NO: 122 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 122, The probe specific to the full length or a partial region of the DNA of the pehX gene of Klebsiella oxytoca may contain the nucleotide sequence of SEQ ID NO: 123 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 123, The probe specific to the full length or a partial region of the DNA of the gyrB gene of Klebsiella variicola may contain the nucleotide sequence of SEQ ID NO: 125 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 125, The probe specific to the full length or a partial region of the DNA of the oprL gene of Pseudomonas aeruginosa may contain the nucleotide sequence of SEQ ID NO: 126 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 126, The probe specific to the full length or a partial region of the DNA of the rpoB gene of the genus Proteus may contain the nucleotide sequence of SEQ ID NO: 127 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 127, The probe specific to the full length or a partial region of the DNA of the hasA gene of Serratia marcescens may contain the nucleotide sequence of SEQ ID NO: 128 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 128, The probe specific to the full length or a partial region of the DNA of the iap p60 gene of the Listeria genus may be selected from a probe containing the nucleotide sequence of SEQ ID NO: 132 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 132, a probe containing the nucleotide sequence of SEQ ID NO: 133 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 133, and a combination thereof; The probe specific to the DNA of the full length or a part of the nuc gene of Staphylococcus argenteus may contain the nucleotide sequence of SEQ ID NO: 135 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 135, The probe specific to the DNA of the full length or a part of the region of the tuf gene of the genus Staphylococcus may contain the nucleotide sequence of SEQ ID NO: 137 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 137; The probe specific to the full length or a partial region of the DNA of the rnpB gene of the genus Streptococcus may contain the nucleotide sequence of SEQ ID NO: 140 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 140, The probe specific to the full length or a partial region of the DNA of the xisco gene of Streptococcus pneumoniae may contain the nucleotide sequence of SEQ ID NO: 138 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 138, The probe specific to the DNA of the full length or a part of the region of the gyrB gene of Streptococcus pyogenes may contain the nucleotide sequence of SEQ ID NO: 139 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 139; The probe specific to the full length or a partial region of the DNA of the beta-lactamase_KPC gene of Klebsiella pneumoniae may contain the nucleotide sequence of SEQ ID NO: 142 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 142, The probe specific to the full length or a partial region of the DNA of the beta-lactamase_NDM gene of Klebsiella pneumoniae may contain the nucleotide sequence of SEQ ID NO: 141 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 141, The probe specific to the full length or a partial region of the DNA of the beta-lactamase_IMP85 gene of Pseudomonas aeruginosa may contain the nucleotide sequence of SEQ ID NO: 144 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 144, The probe specific to the full length or a partial region of the DNA of the beta-lactamase_VIM gene of Citrobacter freundii may contain the nucleotide sequence of SEQ ID NO: 143 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 143; The probe specific to the full length or a partial region of the DNA of the beta-lactamase_CTX-M1 gene of Salmonella enterica may contain the nucleotide sequence of SEQ ID NO: 149 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 149, The probe specific to the full length or a partial region of the DNA of the beta-lactamase_CTX-M2 gene of Salmonella enterica may contain the nucleotide sequence of SEQ ID NO: 150 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 150, The probe specific to the full length or a partial region of the DNA of the beta-lactamase_CTX-M8 gene of Citrobacter amalonaticus may contain the nucleotide sequence of SEQ ID NO: 151 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 151, The probe specific to the full length or a partial region of the E. coli beta-lactamase_CTX-M25 gene may contain the nucleotide sequence of SEQ ID NO: 153 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 153, The probe specific to the full length or a partial region of the DNA of the E. coli beta-lactamase_CTX-M9 gene may contain the nucleotide sequence of SEQ ID NO: 152 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 152, The probe specific to the full length or a partial region of the DNA of the beta-lactamase_OXA-23 gene of Acinetobacter baumannii may contain the nucleotide sequence of SEQ ID NO: 145 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 145, The probe specific to the full length or a partial region of the DNA of the beta-lactamase_OXA-40 gene of Acinetobacter baumannii may contain the nucleotide sequence of SEQ ID NO: 146 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 146, The probe specific to the full length or a partial region of the DNA of the beta-lactamase_OXA-48 gene of Klebsiella pneumoniae may contain the nucleotide sequence of SEQ ID NO: 147 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 147, The probe specific to the full length or a partial region of the DNA of the beta-lactamase_OXA-58 gene of Acinetobacter haemolyticus may contain the nucleotide sequence of SEQ ID NO: 148 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 148, The probe specific to the full length or a partial region of the DNA of the mecA gene of Staphylococcus aureus may contain the nucleotide sequence of SEQ ID NO: 154 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 154, The probe specific to the full length or a partial region of the vanA gene of Enterococcus faecalis may contain the nucleotide sequence of SEQ ID NO: 155 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 155; A probe specific to the DNA of the entire length or a partial region of the vanB gene of Enterococcus faecium may contain the nucleotide sequence of SEQ ID NO:156 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO:156.

[0089] By using the above-mentioned probes, the interaction between multiple probes is reduced, and the specificity and reactivity when detecting the presence or absence of amplification of each region are improved. At the same time, the hybridization efficiency under uniform conditions such as temperature and reagents during the hybridization reaction is also improved.

[0090] The specific aspects of the method according to this embodiment can be the same as those described above without any limitations.

[0091] <Kit> As one embodiment, the present invention provides a first reagent including a set of PCR primers specific to the full length or a partial region of the gyrB gene of Escherichia coli, the nuc gene of Staphylococcus aureus, the atlE gene of Staphylococcus epidermidis, the gyrB gene of Klebsiella pneumoniae, and the rpoB gene of Enterococcus spp.; and a second reagent comprising a combination of probes specific to the DNA of the full length or partial region of each gene.

[0092] The step of carrying out the PCR method in the above-mentioned <Identification method> can be carried out using the first reagent. The first reagent may further contain a set of primers specific to the full length or a partial region of a gene other than the gyrB gene of Escherichia coli, the nuc gene of Staphylococcus aureus, the atlE gene of Staphylococcus epidermidis, the gyrB gene of Klebsiella pneumoniae, and the rpoB gene of Enterococcus.

[0093] For example, the first reagent may further include a set of primers specific to the entire length or a partial region of one or more genes selected from the group consisting of the rpoB gene of Acinetobacter spp., the 16S-23S ITS gene of Acinetobacter baumannii, the ompA gene of Citrobacter spp., the rpoS gene of Enterobacter spp., the gyrB gene of Klebsiella aerogenes, the pehX gene of Klebsiella oxytoca, the gyrB gene of Klebsiella variicola, the oprL gene of Pseudomonas aeruginosa, the rpoB gene of Proteus spp., the hasA gene of Serratia marcescens, the iap p60 gene of Listeria spp., the nuc gene of Staphylococcus argenteus, the tuf gene of Staphylococcus spp., the rnpB gene of Streptococcus spp., the xisco gene of Streptococcus pneumoniae, and the gyrB gene of Streptococcus pyogenes.

[0094] For example, the first reagent is a drug resistance gene, such as a beta-lactamase_KPC gene of Klebsiella pneumoniae, a beta-lactamase_NDM gene of Klebsiella pneumoniae, a beta-lactamase_IMP85 gene of Pseudomonas aeruginosa, a beta-lactamase_VIM gene of Citrobacter freundii, a beta-lactamase_CTX-M1 gene of Salmonella enterica, a beta-lactamase_CTX-M2 gene of Salmonella enterica, a beta-lactamase_CTX-M8 gene of Citrobacter amalonaticus, a beta-lactamase_CTX-M25 gene of Escherichia coli, or a beta-lactamase_CTX-M3 gene of Escherichia coli. The nucleic acid sequence may further comprise a set of primers specific to the entire length or a partial region of one or more genes selected from the group consisting of the beta-lactamase_CTX-M9 gene of Acinetobacter baumannii, the beta-lactamase_OXA-23 gene of Acinetobacter baumannii, the beta-lactamase_OXA-40 gene of Acinetobacter baumannii, the beta-lactamase_OXA-48 gene of Klebsiella pneumoniae, the beta-lactamase_OXA-58 gene of Acinetobacter haemolyticus, the mecA gene of Staphylococcus aureus, the vanA gene of Enterococcus faecalis, and the vanB gene of Enterococcus faecium.

[0095] For example, the first reagent may contain the rpoB gene of Acinetobacter spp., the 16S-23S ITS gene of Acinetobacter baumannii, the ompA gene of Citrobacter spp., the rpoS gene of Enterobacter spp., the gyrB gene of Klebsiella aerogenes, the pehX gene of Klebsiella oxytoca, the gyrB gene of Klebsiella variicola, the oprL gene of Pseudomonas aeruginosa, the rpoB gene of Proteus spp., the hasA gene of Serratia marcescens, the iapA gene of Listeria spp. p60 gene, nuc gene of Staphylococcus argenteus, tuf gene of Staphylococcus spp., rnpB gene of Streptococcus spp., xisco gene of Streptococcus pneumoniae, gyrB gene of Streptococcus pyogenes, beta-lactamase_KPC gene of Klebsiella pneumoniae, beta-lactamase_NDM gene of Klebsiella pneumoniae, beta-lactamase_IMP85 gene of Pseudomonas aeruginosa, beta-lactamase_VIM gene of Citrobacter freundii, beta-lactamase_CTX-M1 gene of Salmonella enterica, beta-lactamase_CTX-M2 gene of Salmonella enterica, beta-lactamase_VIM gene of Citrobacter amalonatis The set of primers may include a set of primers specific to the entire length or a partial region of one or more genes selected from the group consisting of the CTX-M8 gene, the beta-lactamase_CTX-M25 gene of Escherichia coli, the beta-lactamase_CTX-M9 gene of Escherichia coli, the beta-lactamase_OXA-23 gene of Acinetobacter baumannii, the beta-lactamase_OXA-40 gene of Acinetobacter baumannii, the beta-lactamase_OXA-48 gene of Klebsiella pneumoniae, the beta-lactamase_OXA-58 gene of Acinetobacter haemolyticus, the mecA gene of Staphylococcus aureus, the vanA gene of Enterococcus faecalis, and the vanB gene of Enterococcus faecium.

[0096] The bacteria or genes selected from the group of multiple genes may be, for example, 2 or more, 3 or more, 5 or more, 7 or more, 10 or more, 15 or more, 20 or more, 25 or more, 28 or more, 30 or more, or 32 or more. The bacteria or genes selected from the group of multiple genes may be, for example, 37 or less, 34 or less, 21 or less, 16 or less, or 11 or less. The bacteria or genes selected from the group of multiple genes may be, for example, 2 to 40, 5 to 37, 10 to 37, or 15 to 25.

[0097] For example, in the first reagent, the set of primers specific to the full length or a partial region of the gyrB gene of Escherichia coli may include a primer including a nucleotide sequence of SEQ ID NO: 10 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 10, and a primer including a nucleotide sequence of SEQ ID NO: 11 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 11, The set of primers specific to the full length or a partial region of the nuc gene of Staphylococcus aureus may include one or more primers selected from a primer including a nucleotide sequence of SEQ ID NO: 32 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 32, a primer including a nucleotide sequence of SEQ ID NO: 34 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 34, and a combination thereof, and a primer including a nucleotide sequence of SEQ ID NO: 33 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 33, The set of primers specific to the full length or a partial region of the atlE gene of Staphylococcus epidermidis may include a primer containing a nucleotide sequence of SEQ ID NO: 37 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 37, and a primer containing a nucleotide sequence of SEQ ID NO: 38 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 38, The set of primers specific to the full length or a partial region of the gyrB gene of Klebsiella pneumoniae may include a primer including a nucleotide sequence of SEQ ID NO: 14 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 14, and a primer including a nucleotide sequence of SEQ ID NO: 15 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 15, The set of primers specific to the entire length or a partial region of the rpoB gene of Enterococcus may include one or more primers selected from a primer containing the nucleotide sequence of SEQ ID NO: 26 or a nucleotide sequence that has 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 26, a primer containing the nucleotide sequence of SEQ ID NO: 27 or a nucleotide sequence that has 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 27, a primer containing the nucleotide sequence of SEQ ID NO: 28 or a nucleotide sequence that has 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 28, and combinations thereof, and a primer containing the nucleotide sequence of SEQ ID NO: 29 or a nucleotide sequence that has 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 29.

[0098] By using the above primer set, the interaction between the primers is reduced, and the specificity and reactivity when amplifying each region are improved. In addition, the amplification efficiency under uniform conditions of temperature, reagents, etc. when amplifying simultaneously is also improved.

[0099] The first reagent may further include a set of primers specific to the full length or a partial region of a gene other than the gyrB gene of Escherichia coli, the nuc gene of Staphylococcus aureus, the atlE gene of Staphylococcus epidermidis, the gyrB gene of Klebsiella pneumoniae, and the rpoB gene of Enterococcus. Each primer is as described above in <Identification method>.

[0100] The DNA of the full length or a partial region of the gyrB gene of Escherichia coli may contain the nucleotide sequence of SEQ ID NO: 51 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 51, The DNA of the full length or a partial region of the nuc gene of Staphylococcus aureus may contain the nucleotide sequence of SEQ ID NO: 61 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 61, The DNA of the full length or a partial region of the atlE gene of Staphylococcus epidermidis may contain the nucleotide sequence of SEQ ID NO: 63 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 63, The DNA of the full length or a partial region of the gyrB gene of Klebsiella pneumoniae may contain the nucleotide sequence of SEQ ID NO: 53 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 53, The DNA of the full-length or partial region of the rpoB gene of Enterococcus may contain the nucleotide sequence of SEQ ID NO:59 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO:59.

[0101] By using the full-length or partial region of DNA described above, interactions between multiple DNAs are reduced, improving specificity and reactivity when detecting the presence or absence of amplification of each region.

[0102] In addition, when PCR is performed using the first reagent for the full length or a part of a gene other than the gyrB gene of Escherichia coli, the nuc gene of Staphylococcus aureus, the atlE gene of Staphylococcus epidermidis, the gyrB gene of Klebsiella pneumoniae, and the rpoB gene of Enterococcus, the DNA of the full length or a part of each gene is as described above in <Identification method>.

[0103] The second reagent contains a probe specific to the DNA of the full length or a partial region of each gene. The detection step in the above-mentioned <Identification method> can be carried out using the second reagent. In order to detect the presence or absence of amplification of the full length or a partial region of a plurality of genes, the probe is designed so that detection can be carried out under similar or uniform conditions for the DNA of each gene. The probe may be a probe that hybridizes with the amplified DNA under stringent conditions. The stringent conditions are as described above in the <Identification method>. The second reagent may contain a combination of probes, for example, as a mixture, or in a form immobilized on a solid support. Examples of solid supports include substrates, arrays, magnetic beads, columns, and colored beads. The combination of probes may be immobilized on a solid support in a multiplex format.

[0104] When the probes contained in the second reagent are immobilized on a solid support, the solid support may be, for example, a solid support on which probes each containing a different identifiable identifier (e.g., ID) are immobilized, or a solid support containing a distinguishable identifier on which each probe is immobilized. In other words, the probes may each contain a different identifier, or each be bound to a different identifier on the solid support.

[0105] In the second reagent, the probe specific to the full length or a partial region of DNA of the gyrB gene of Escherichia coli may contain the nucleotide sequence of SEQ ID NO: 121 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 121; The probe specific to the full length or a partial region of the DNA of the nuc gene of Staphylococcus aureus may contain the nucleotide sequence of SEQ ID NO: 134 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 134, The probe specific to the full length or a partial region of the DNA of the atlE gene of Staphylococcus epidermidis may contain the nucleotide sequence of SEQ ID NO: 136 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 136, The probe specific to the full length or a partial region of the DNA of the gyrB gene of Klebsiella pneumoniae may contain the nucleotide sequence of SEQ ID NO: 124 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 124, The probe specific to the DNA of the full length or a partial region of the rpoB gene of Enterococcus may be selected from a probe containing the nucleotide sequence of SEQ ID NO: 129 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 129, a probe containing the nucleotide sequence of SEQ ID NO: 130 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 130, a probe containing the nucleotide sequence of SEQ ID NO: 131 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 131, and combinations thereof.

[0106] By using the above-mentioned probes, the interaction between multiple probes is reduced, and the specificity and reactivity when detecting each region are improved. In addition, the hybridization efficiency under uniform conditions such as temperature and reagents during the hybridization reaction is also improved.

[0107] The second reagent may further contain a probe specific to the full length or a partial region of DNA of a gene other than the gyrB gene of Escherichia coli, the nuc gene of Staphylococcus aureus, the atlE gene of Staphylococcus epidermidis, the gyrB gene of Klebsiella pneumoniae, and the rpoB gene of Enterococcus. Each probe is as described above in <Identification method>.

[0108] The kit according to the present embodiment may be designed so that the DNA amplified by carrying out the PCR method using the above primer set contains a label, and may further include a third reagent for labeling the amplified DNA. The primers designed so that the amplified DNA contains a label are as specifically explained in <Identification method>. For example, the primers may be designed so that they have a label at the 5' end, 3' end, or internally, since amplified DNA containing a label can be obtained by carrying out the PCR method using a primer having a label. For example, the 5' end of the primer may be modified with biotin. The labeling of the amplified DNA is also as specifically explained in <Identification method>. For example, labeled avidin may be included as a third reagent to label the biotin-modified amplified DNA.

[0109] The kit according to the present embodiment may further include a means for collecting a sample from a subject. The means for collecting the sample varies depending on the type of sample, etc., but examples of the means include blood collection means (e.g., a blood collection kit) when the sample is blood, and a urine collection container when the sample is urine.

[0110] The kit according to this embodiment may further include preparation materials for preparing a sample collected from a control, such as a cell lysis agent and a DNA extraction material.

[0111] The kit according to this embodiment may further include a PCR standard reagent. The kit may further include one or more selected from the group consisting of DNA polymerase, deoxynucleoside triphosphate (dNTP), magnesium ion, one or more salts, and a pH buffer. These reagents may be included in the first reagent together with the primer.

[0112] The kit according to this embodiment may further comprise a targeting reagent for labeling the amplified DNA and / or the probe, for example, labeled avidin.

[0113] The kit according to this embodiment may further include a control (eg, a control sample) for comparing the detection results.

[0114] The kit of this embodiment is used to identify the causative bacterium of sepsis and may further include instructions describing a method for identifying the causative bacterium of sepsis or instructions for using the reagents included in the kit.

[0115] As for specific aspects of the kit according to this embodiment, such as the primers, the target regions of each gene, the probes, and the like, the specific aspects described above in the <Identification method> and the like can be applied without any restrictions.

[0116] As one embodiment, the present invention provides gyrB gene of Escherichia coli, nuc gene of Staphylococcus aureus, atlE gene of Staphylococcus epidermidis, gyrB gene of Klebsiella pneumoniae, rpoB gene of Enterococcus spp., rpoB gene of Acinetobacter spp., 16S-23S ITS gene of Acinetobacter baumannii, ompA gene of Citrobacter spp., rpoS gene of Enterobacter spp., gyrB gene of Klebsiella aerogenes, pehX gene of Klebsiella oxytoca, gyrB gene of Klebsiella variicola, oprL gene of Pseudomonas aeruginosa, rpoB gene of Proteus spp., hasA gene of Serratia marcescens, iap gene of Listeria spp. p60 gene, nuc gene of Staphylococcus argenteus, tuf gene of Staphylococcus spp., rnpB gene of Streptococcus spp., xisco gene of Streptococcus pneumoniae, gyrB gene of Streptococcus pyogenes, beta-lactamase_KPC gene of Klebsiella pneumoniae, beta-lactamase_NDM gene of Klebsiella pneumoniae, beta-lactamase_IMP85 gene of Pseudomonas aeruginosa, beta-lactamase_VIM gene of Citrobacter freundii, beta-lactamase_CTX-M1 gene of Salmonella enterica, beta-lactamase_CTX-M2 gene of Salmonella enterica, beta-lactamase_CTX-M8 gene of Citrobacter amalonatis, beta-lactamase_CTX-M1 gene of Salmonella enterica, beta-lactamase_CTX-M2 gene of Salmonella enterica, beta-lactamase_CTX-M3 gene of Citrobacter amalonatis, a first reagent comprising a set of primers specific to the full length or a partial region of a gene and / or a drug resistance gene of two or more kinds of bacteria selected from the group consisting of the beta-lactamase_CTX-M25 gene of Enterocoli, the beta-lactamase_CTX-M9 gene of Escherichia coli, the beta-lactamase_OXA-23 gene of Acinetobacter baumannii, the beta-lactamase_OXA-40 gene of Acinetobacter baumannii, the beta-lactamase_OXA-48 gene of Klebsiella pneumoniae, the beta-lactamase_OXA-58 gene of Acinetobacter haemolyticus, the mecA gene of Staphylococcus aureus, the vanA gene of Enterococcus faecalis, and the vanB gene of Enterococcus faecium; and a second reagent comprising a combination of probes specific to the full-length or partial region of DNA, respectively, for identifying a bacterium causing sepsis and / or a drug resistance gene.

[0117] The bacteria or genes selected from the group of multiple genes may be, for example, 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 28 or more, 30 or more, 32 or more, 35 or more, or 37 or more. The bacterial genes selected from the group of multiple genes may be, for example, 3 or more, 5 or more, 7 or more, 10 or more, 12 or more, 15 or more, 17 or more, 19 or more, or 21 or more. The drug resistance genes selected from the group of multiple genes may be, for example, 3 or more, 5 or more, 7 or more, 10 or more, 12 or more, 14 or more, or 16 or more. The bacteria or genes selected from the group of multiple genes may be, for example, 37 or less, 34 or less, 21 or less, 16 or less, or 11 or less. Furthermore, the bacteria or genes selected from the group of multiple genes may be, for example, 2 to 40 types, 5 to 37 types, 10 to 37 types, or 15 to 25 types.

[0118] The specific aspects of the kit according to this embodiment can be the same as those described above without any limitations.

[0119] <Methods for diagnosing sepsis, methods for assisting diagnosis, and methods for treating sepsis> As one embodiment, the present invention also provides a method for diagnosing or assisting in the diagnosis of sepsis, which comprises identifying a causative bacterium of sepsis. The method for identifying a causative bacterium of sepsis is as described above in <Identification method>.

[0120] As an example, the method for diagnosing sepsis of the present embodiment includes: an amplification step of carrying out a PCR method using a combination of primer sets specific to the full length or a partial region of the gyrB gene of Escherichia coli, the nuc gene of Staphylococcus aureus, the atlE gene of Staphylococcus epidermidis, the gyrB gene of Klebsiella pneumoniae, and the rpoB gene of Enterococcus spp.; detecting the presence or absence of amplification of the full-length or partial region by using a combination of probes specific to the full-length or partial region of DNA, respectively; If the causative bacterium is identified, the method may include a step of diagnosing sepsis or suspected sepsis.

[0121] In addition, as an example, the method for assisting diagnosis of sepsis of the present embodiment includes: an amplification step of carrying out a PCR method using a combination of primer sets specific to the full length or a partial region of the gyrB gene of Escherichia coli, the nuc gene of Staphylococcus aureus, the atlE gene of Staphylococcus epidermidis, the gyrB gene of Klebsiella pneumoniae, and the rpoB gene of Enterococcus spp.; detecting the presence or absence of amplification of the full-length or partial region by using a combination of probes specific to the full-length or partial region of DNA, respectively; If a causative bacterium is identified, the method may include providing an indication of sepsis or suspected sepsis.

[0122] When the causative bacterium of sepsis is identified, it is possible to diagnose that sepsis is caused by the identified bacterium or that sepsis is suspected, or to assist in the diagnosis by providing an indicator that sepsis is caused by the identified bacterium or that sepsis is suspected.

[0123] The diagnostic or diagnostic support method according to this embodiment can be carried out in vitro.

[0124] As one embodiment, the present invention also provides a method for treating sepsis, which comprises identifying a bacterium causing sepsis. The method for identifying a bacterium causing sepsis is as described above in <Identification method>.

[0125] The treatment method of this embodiment may include a step of treating sepsis in a subject who has been diagnosed with sepsis or who has been provided with an indicator of sepsis by the above-mentioned diagnostic or diagnostic support method. The treatment method may also include a step of selecting an appropriate treatment based on the identified causative bacterium. Furthermore, when a drug resistance gene is also identified, the treatment method may include a step of selecting an appropriate treatment based on the identified causative bacterium and drug resistance gene.

[0126] The specific aspects of the diagnostic method, diagnostic support method, and treatment method according to this embodiment can be the same as those described above without any restrictions. EXAMPLES

[0127] [Example 1] Development of primers and probes for detecting E. coli Primers and probes for detecting the bacterial species included in the sepsis panel were developed for each bacterial species item. As a representative example, a specific method for developing primers and probes for detecting the genomic DNA of Escherichia coli will be described. The detection method in this example includes a step of PCR amplifying a target gene in the bacterial genomic DNA and a step of detecting the amplified PCR product using a probe on a substrate.

[0128] 1. Primer development and selection Two types of primer sets were designed as candidates to amplify a specific region of the gene (gyrB) of E. coli genomic DNA. PCR reaction solutions were prepared using each of the primer sets 1 and 2 shown in the table below and E. coli-derived genomic DNA, and amplification by real-time PCR was performed using a Light Cycler (registered trademark) 480 II (Roche). The reagents used for amplification were Takara multiplex assay kit Ver2 (Takara Bio Inc.), and 20xEvaGreen (biotium) was used as the intercalator dye. Each primer was added to a final concentration of 0.2uM, and 1x 2xMultiplex PCR Buffer and 1U Multiplex PCR Enzyme Mix in the Takara multiplex assay kit Ver2 were used, and E. coli genomic DNA was mixed as a template to prepare a PCR reaction solution.

[0129] [Table 4]

[0130] The amplification reaction was carried out under the following conditions. Denaturation step: 94℃ 30 seconds Annealing step: 60℃ 60 seconds → 94℃ 30 seconds (40 cycles) Extension process: 72℃ 600 seconds

[0131] The composition of the PCR reaction solution is as shown in the table below. [Table 5]

[0132] 2.Results The results of the amplification curves of primer sets 1 and 2 are shown in Figure 1. As a result of the study using real-time PCR, primer set 1 showed a rapid rise in the amplification curve for the E. coli template. It was shown that primer set 1 can amplify the target gene of E. coli. For primer set 2, the rise in the amplification curve for the E. coli template was slow and did not reach a plateau, so sufficient amplification of the PCR product was not observed. This showed that the primer function of primer set 2 for selectively amplifying the target gene of E. coli was insufficient. Based on the above results, primer set 1 (E. coli_468F19 / E. coli_567R18) was adopted as the primer for identifying E. coli.

[0133] 3. Probe Development and Selection Regarding the probe to be used in the step of detecting the amplified PCR product by the probe on the substrate, multiple candidate probes were evaluated as described below, and the probe to be used for identifying the causative bacterium of sepsis was selected. In a real-time PCR study, it was shown that primer set 1 can generate PCR products specific to the target gene of E. coli by performing PCR using E. coli as a template. E. coli_531P16 and E. coli_526P15 were designed as probes to detect this PCR product. For primer set 1, PCR amplification, hybridization reaction with the probe on the substrate, labeling, and fluorescence measurement were performed on the Escherichia coli template. At this time, a primer modified with biotin via a linker on the 5' end was used as the reverse primer. The sequence numbers and sequences of the candidate probes are shown in the table below. The measurement procedure is shown in the outline of the detection process below.

[0134] [Table 6]

[0135] Overview of the detection process (1) PCR amplification reaction Using E. coli genomic DNA as a template, PCR amplification reaction was carried out under the following conditions. Denaturation step: 94℃ 30 seconds Annealing step: 60℃ 60 seconds → 94℃ 30 seconds (30 cycles) Extension process: 72℃ 600 seconds

[0136] The composition of the PCR reaction solution is as shown in the table below. [Table 7]

[0137] (2) Thermal denaturation of PCR products Before applying the PCR product to the probe immobilized on the substrate, a thermal denaturation reaction was carried out under the following conditions. Thermal denaturation conditions: 95℃ for 5 minutes, then rapidly cooled to 4℃

[0138] (3) Hybridization reaction between the probe on the substrate and the PCR product and labeling A hybridization reaction was carried out between each probe (see Table 6) immobilized on the substrate and the PCR product. The reaction solution contained saline-sodium-phosphate-EDTA buffer (SSPE-buffer). The PCR product captured by the probe on the substrate was modified with biotin at the 5' end, which was labeled with Streptoavidin-Phycoerythrin (PlexBio). The hybridization reaction and labeling on the substrate were carried out under the following conditions using an IntelliPlex 1000 πcode Processor, a device manufactured by PlexBio. Hybridization conditions: Incubate at 37℃ for 20 minutes Labeling conditions: Incubate at 37℃ for 10 minutes

[0139] (4) Detection of labeled PCR products on the substrate Each substrate is given an identifiable ID, and a specific probe is immobilized on each ID. The PCR product corresponding to each probe is captured on the substrate, and the PCR product is labeled. The PCR product labeled on the substrate emits fluorescence, so the fluorescence value can be measured by a detector. The fluorescence value was measured using a fluorescence measuring device (100 Fluorescent Analyzer PlexBio). PlexBio's 100 Fluorescent Analyzer can identify the ID of the substrate and measure the fluorescence value on a substrate-by-substrate basis, so the reactivity of the probe with the PCR product can be evaluated by measuring the fluorescence value of the substrate on which a specific probe is immobilized.

[0140] 4.Results The PCR products obtained using primer set 1 (E.coli_468F19 / E.coli_567R18) and E. coli genomic DNA as a template were evaluated with two probes (E.coli_531P16 and E.coli_526P15). The fluorescence value of E.coli_526P15 was below 9000, while E.coli_531P16 showed a fluorescence value of above 40000. This shows that the PCR products of E. coli can be significantly detected when E.coli_531P16 is used.

[0141] These results indicate that E. coli can be detected by using primer set 1 and E. coli_531P16 as a probe.

[0142] [Example 2] Development of primers and probes for detecting Citrobacter sp. As another representative example, a specific method for developing primers and probes for detecting genomic DNA of Citrobacter spp. is described below. The detection method of this example also includes a step of PCR amplifying a target gene in bacterial genomic DNA and a step of detecting the amplified PCR product by a probe on a substrate, similar to Example 1.

[0143] 1. Primer development and selection Three types of primer sets were designed as candidates to amplify a specific region of the gene (ompA) of Citrobacter genus DNA. PCR reaction solutions were prepared using primer sets 1, 2, and 3 shown in the table below and genomic DNA derived from Citrobacter freundii and genomic DNA derived from Citrobacter koseri as templates, and real-time PCR amplification was performed using a Light Cycler (registered trademark) 480 II (Roche). The reagents used for amplification were Takara multiplex assay kit Ver2 (Takara Bio Inc.) and 20xEvaGreen (biotium) was used as an intercalator dye. Each primer was added to a final concentration of 0.2uM, and template genomic DNA was added to 1ug / L. 1x 2xMultiplex PCR Buffer and 1U Multiplex PCR Enzyme Mix in the Takara multiplex assay kit Ver2 were used, and the template DNA was mixed to prepare a PCR reaction solution.

[0144] [Table 8]

[0145] The amplification reaction was carried out under the following conditions. Denaturation step: 94℃ 30 seconds Annealing step: 60℃ 60 seconds → 94℃ 30 seconds (40 cycles) Extension process: 72℃ 600 seconds

[0146] The composition of the PCR reaction solution is as shown in the table below. [Table 9]

[0147] 2.Results The results of the amplification curves for each template of primer sets 1, 2, and 3 are shown in Figures 3 to 5. As a result of the examination using real-time PCR, primer set 1 showed a rapid rise in the amplification curve for the templates of Citrobacter freundii and Citrobacter koseri. Primer set 1 also matched the target genes of Citrobacter braakii, Citrobacter youngae, and Citrobacter werkmanii in terms of sequence design, so it was shown that it can amplify the target genes of Citrobacter genus bacteria. With primer set 2, the amplification curves rose for Citrobacter koseri and Citrobacter freundii, but the rise was relatively slow and did not reach a plateau. With primer set 3, the amplification curve rose selectively for the template of Citrobacter koseri, but sufficient PCR product amplification was not observed for the template of Citrobacter freundii. This indicates that the primer function of primer set 3 for selectively amplifying the target gene of Citrobacter is insufficient.

[0148] Primer set 2 is designed to have low sequence coverage for the target genes of Citrobacter freundii and Citrobacter koseri, but high sequence coverage for the target genes of Citrobacter other than Citrobacter freundii and Citrobacter koseri. In other words, by using both primer sets 1 and 2 in the reaction system, the target genes of Citrobacter can be amplified comprehensively and selectively. Three types of primers (C. spp_449F17, C. spp_443F18, and C. spp_551R19) were used to detect the genomic DNA of Citrobacter.

[0149] 3. Probe Development and Selection Regarding the probe to be used in the step of detecting the amplified PCR product by the probe on the substrate, multiple candidate probes were evaluated as described below, and the probe to be used for identifying the causative bacterium of sepsis was selected.

[0150] In real-time PCR, we demonstrated that PCR products specific to Citrobacter freundii and Citrobacter koseri templates could be obtained. Probes C.spp_24P17R, C.spp_55P15Y, and C.spp_65P19R were designed to detect the PCR products.

[0151] For primer set 1, PCR amplification, hybridization reaction with the probe on the substrate, labeling, and fluorescence measurement were performed on the templates of Citrobacter freundii and Citrobacter koseri. A primer modified with biotin via a linker on the 5' end was used as the reverse primer. The sequence numbers and sequences of the candidate probes are shown in the table below. The measurement procedure is shown in the overview of the detection process below.

[0152] [Table 10]

[0153] Overview of the detection process (1) PCR amplification reaction PCR amplification reactions were performed using the genomic DNA of Citrobacter freundii and Citrobacter koseri as templates under the following conditions. Denaturation step: 94℃ 30 seconds Annealing step: 60℃ 60 seconds → 94℃ 30 seconds (30 cycles) Extension process: 72℃ 600 seconds

[0154] [Table 11]

[0155] (2) Thermal denaturation of PCR products Before applying the PCR product to the probe immobilized on the substrate, a thermal denaturation reaction was carried out under the following conditions. Thermal denaturation conditions: 95℃ for 5 minutes, then rapidly cooled to 4℃

[0156] (3) Hybridization reaction between the probe on the substrate and the PCR product and labeling A hybridization reaction was carried out between each probe (see Table 10) immobilized on the substrate and the PCR product. The reaction solution contained saline-sodium-phosphate-EDTA buffer (SSPE-buffer). The PCR product captured by the probe on the substrate was modified with biotin at the 5' end, which was labeled with Streptoavidin-Phycoerythrin (PlexBio). The hybridization reaction and labeling on the substrate were carried out under the following conditions using an IntelliPlex 1000 πcode Processor, a device manufactured by PlexBio. Hybridization conditions: Incubate at 37℃ for 20 minutes Labeling conditions: Incubate at 37℃ for 10 minutes

[0157] (4) Detection of labeled PCR products on the substrate Each substrate is provided with an identifiable ID, and a specific probe is immobilized on each ID. The PCR product corresponding to each probe is captured on the substrate, and the PCR product is labeled. The PCR product labeled on the substrate emits fluorescence, so that the fluorescence value can be measured by a detector. The fluorescence value was measured using a fluorescence measuring device (100 Fluorescent Analyzer PlexBio) in the same manner as in Example 1.

[0158] 4.Results The PCR products obtained using primer set 1 (C.spp_449F17R / C.spp_551R19R) and the genomic DNA of Citrobacter freundii and Citrobacter koseri as templates were evaluated with three probes (C.spp_24P17R, C.spp_55P15Y, and C.spp_65P19R) as shown in Figure 6. For Citrobacter freundii and Citrobacter koseri, C.spp_55P15Y and C.spp_65P19R showed fluorescence values ​​of less than 2000, while C.spp_24P17R showed a fluorescence value of more than 6000. When water was used as a template, no fluorescence value was observed with any of the probes. This indicates that PCR products of Citrobacter spp. can be significantly detected when C.spp_24P17R is used.

[0159] These results indicate that Citrobacter species can be detected by using primer set 1 and C.spp_24P17R as a probe.

[0160] [Example 3] Performance evaluation of primers and probes in a multiplex system In the investigations so far, E.coli_468F19 / E.coli_567R18 were selected as the primers used to detect the genomic DNA of E. coli. For the probe, E.coli_531P16 was selected based on the evaluation of reactivity. For the probe, C.spp_449F17, C.spp_443F18, and C.spp_551R19 were selected as the primers used to detect the genomic DNA of Citrobacter. For the probe, C.spp_24P17R was selected based on the evaluation of reactivity. Primers and probes were developed using the same procedure for bacterial species other than E. coli and Citrobacter. Primers and probes were also developed using the same procedure for simultaneous detection of bacterial drug resistance genes (AMR).

[0161] The primer sets selected for detecting bacterial target genes and the DNA sequences amplified by the primer sets are shown in Table 12.

[0162] [Table 12] TIFF0007673363000014.tif185149TIFF0007673363000015.tif211149TIFF0007673363000016.tif221149 TIFF0007673363000017.tif198149TIFF0007673363000018.tif154149TIFF0007673363000019.tif223149

[0163] The primer sets selected for detecting bacterial drug resistance genes and the DNA amplified by the primer sets are shown in Table 13.

[0164] [Table 13] TIFF0007673363000021.tif223149TIFF0007673363000022.tif217149TIFF0007673363000023.tif211149TIFF0007673363000024.tif141149

[0165] The probes selected to detect bacterial target genes are shown in Table 14. [Table 14]

[0166] The probes selected to detect bacterial drug resistance genes are shown in Table 15. [Table 15]

[0167] Since the present invention is a technology that enables simultaneous multi-item measurements of multiple bacterial species and drug resistance genes, it is necessary to evaluate the reactivity and specificity for each template in evaluation using multiplex PCR and a substrate on which multiple probes are immobilized.

[0168] The PCR products amplified by multiplex PCR using templates of representative bacterial species were reacted in a system containing all probes, and simultaneous multi-item measurements were performed. The PCR products amplified by multiplex PCR using templates of drug resistance genes were reacted in a system containing all probes, and simultaneous multi-item measurements were performed. The measurement procedure is shown in the overview of the detection process below.

[0169] Overview of the detection process (1) PCR amplification reaction PCR amplification was performed using the genomic DNA or drug resistance genes of the target bacterial species as templates. Denaturation step: 94℃ 30 seconds Annealing step: 60℃ 60 seconds → 94℃ 30 seconds (30 cycles) Extension process: 72℃ 600 seconds

[0170] The composition of the PCR reaction solution is as shown in the table below.

[0171] [Table 16]

[0172] [Table 17] TIFF0007673363000029.tif119149

[0173] [Table 18]

[0174] (2) Thermal denaturation of PCR products Before applying the PCR product to the probe immobilized on the substrate, a thermal denaturation reaction was carried out under the following conditions. Thermal denaturation conditions: 95℃ for 5 minutes, then rapidly cooled to 4℃

[0175] (3) Hybridization reaction between the probe on the substrate and the PCR product and labeling A hybridization reaction between each probe immobilized on the substrate and the PCR product was carried out. The reaction solution contained saline-sodium-phosphate-EDTA buffer (SSPE-buffer). The PCR products captured by the probes on the substrate were modified with biotin at the 5' end, which was then labeled with Streptoavidin-Phycoerythrin (PlexBio). The hybridization reaction and labeling on the substrate were carried out under the following conditions using an IntelliPlex 1000 πcode Processor, a PlexBio device. Hybridization conditions: Incubate at 37℃ for 20 minutes Labeling conditions: Incubate at 37℃ for 10 minutes

[0176] (4) Detection of labeled PCR products on the substrate Each substrate is provided with an identifiable ID, and a specific probe is immobilized on each ID. The PCR product corresponding to each probe is captured on the substrate, and the PCR product is labeled. The PCR product labeled on the substrate emits fluorescence, so that the fluorescence value can be measured by a detector. The fluorescence value was measured using a fluorescence measuring device (100 Fluorescent Analyzer PlexBio) in the same manner as in Example 1.

[0177] 4.Results The results of evaluating the reactivity and specificity of all probes for the PCR products of each bacterial species are shown in Table 19 and Figures 8 to 10. The graphs in Figures 8 to 10 were created using the values ​​obtained by subtracting the cutoff value (uniformly 3000) from the fluorescence value of each probe. The probe E.coli_531P16 gave a fluorescent signal in E. coli, but no fluorescent signal was observed in other bacterial species. This indicated a specific reaction to E. coli. Therefore, even in the multiplex system (all probes simultaneous multiplex measurement system of Multiplex PCR), the primers (two types, E.coli_468F19 and E.coli_567R18) and probe (E.coli_531P16) for detecting E. coli were shown to be capable of specifically detecting the genomic DNA of E. coli. For each bacterial species other than E. coli, each probe emitted a fluorescent signal for the PCR product of the corresponding bacterial genome. Each probe did not emit a fluorescent signal for the PCR product of the non-corresponding bacterial genome. Therefore, it was demonstrated that the primers and probes corresponding to each bacterial species functioned in the multiplex system as well, enabling specific detection of each bacterial species.

[0178] [Table 19] TIFF0007673363000032.tif226149

[0179] The results of evaluating the reactivity and specificity of all probes for the PCR products of each drug resistance gene are shown in Table 20, Figures 11 and 12. The graphs in Figures 11 and 12 were created using values ​​obtained by subtracting the cutoff value (see Table 20) from the fluorescence value of each probe. The probes for detecting each drug resistance gene emitted fluorescent signals for the PCR products of the corresponding drug resistance gene. No fluorescent signals were observed for the PCR products of drug resistance genes that did not correspond to each probe. This demonstrated that the primers and probes corresponding to each drug resistance gene functioned even in the multiplex system, and each drug resistance gene could be specifically detected.

[0180]

Table 20

Claims

1. 1. A method for identifying a causative bacterium of sepsis, comprising: A step of carrying out a PCR method using a sample collected from a subject and a combination of primer sets specific to the full length or a partial region of the gyrB gene of Escherichia coli, the nuc gene of Staphylococcus aureus, the atlE gene of Staphylococcus epidermidis, the gyrB gene of Klebsiella pneumoniae, and the rpoB gene of Enterococcus spp.; detecting the presence or absence of amplification of the full-length or partial region of each gene using a combination of probes specific to the DNA of the full-length or partial region of each gene; Including, the set of primers specific to the full length or a partial region of the gyrB gene of Escherichia coli comprises a primer including a nucleotide sequence of SEQ ID NO: 10 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 10, and a primer including a nucleotide sequence of SEQ ID NO: 11 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 11; The set of primers specific to the full length or a partial region of the nuc gene of Staphylococcus aureus includes one or more primers selected from a primer including a base sequence of SEQ ID NO: 32 or a base sequence having a sequence identity of 90% or more with the base sequence of SEQ ID NO: 32, a primer including a base sequence of SEQ ID NO: 34 or a base sequence having a sequence identity of 90% or more with the base sequence of SEQ ID NO: 34, and a combination thereof, and a primer including a base sequence of SEQ ID NO: 33 or a base sequence having a sequence identity of 90% or more with the base sequence of SEQ ID NO: 33; the set of primers specific to the full length or a partial region of the atlE gene of Staphylococcus epidermidis comprises a primer including a nucleotide sequence of SEQ ID NO: 37 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 37, and a primer including a nucleotide sequence of SEQ ID NO: 38 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 38; the set of primers specific to the full length or a partial region of the gyrB gene of Klebsiella pneumoniae comprises a primer comprising a nucleotide sequence of SEQ ID NO: 14 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 14, and a primer comprising a nucleotide sequence of SEQ ID NO: 15 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 15; the set of primers specific to the full length or a partial region of the rpoB gene of the genus Enterococcus comprises one or more primers selected from a primer comprising the nucleotide sequence of SEQ ID NO: 26 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 26, a primer comprising the nucleotide sequence of SEQ ID NO: 27 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 27, a primer comprising the nucleotide sequence of SEQ ID NO: 28 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 28, and a combination thereof; and a primer comprising the nucleotide sequence of SEQ ID NO: 29 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 29, the probe specific to the full length or a partial region of DNA of the gyrB gene of Escherichia coli comprises the nucleotide sequence of SEQ ID NO: 121 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 121; the probe specific to the DNA of the full length or a partial region of the nuc gene of Staphylococcus aureus comprises the base sequence of SEQ ID NO: 134 or a base sequence having a sequence identity of 90% or more to the base sequence of SEQ ID NO: 134; the probe specific to the DNA of the full length or a partial region of the atlE gene of Staphylococcus epidermidis comprises the nucleotide sequence of SEQ ID NO: 136 or a nucleotide sequence having a sequence identity of 90% or more to the nucleotide sequence of SEQ ID NO: 136; the probe specific to the DNA of the full length or a partial region of the gyrB gene of Klebsiella pneumoniae comprises the nucleotide sequence of SEQ ID NO: 124 or a nucleotide sequence having a sequence identity of 90% or more to the nucleotide sequence of SEQ ID NO: 124; the probe specific to DNA of the full length or a partial region of the rpoB gene of the Enterococcus genus is selected from a probe containing the nucleotide sequence of SEQ ID NO: 129 or a nucleotide sequence having 90% or more sequence identity to the nucleotide sequence of SEQ ID NO: 129, a probe containing the nucleotide sequence of SEQ ID NO: 130 or a nucleotide sequence having 90% or more sequence identity to the nucleotide sequence of SEQ ID NO: 130, a probe containing the nucleotide sequence of SEQ ID NO: 131 or a nucleotide sequence having 90% or more sequence identity to the nucleotide sequence of SEQ ID NO: 131, and combinations thereof.

2. the DNA of the full length or a partial region of the gyrB gene of Escherichia coli comprises the nucleotide sequence of SEQ ID NO:51 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO:51, The DNA of the full length or a partial region of the nuc gene of Staphylococcus aureus comprises the base sequence of SEQ ID NO: 61 or a base sequence having a sequence identity of 90% or more to the base sequence of SEQ ID NO: 61, the DNA of the full length or a partial region of the atlE gene of Staphylococcus epidermidis comprises the nucleotide sequence of SEQ ID NO: 63 or a nucleotide sequence having a sequence identity of 90% or more to the nucleotide sequence of SEQ ID NO: 63, the DNA of the full length or a partial region of the gyrB gene of Klebsiella pneumoniae comprises the nucleotide sequence of SEQ ID NO: 53 or a nucleotide sequence having a sequence identity of 90% or more to the nucleotide sequence of SEQ ID NO: 53; The method according to claim 1, wherein the DNA of the full length or a partial region of the rpoB gene of the Enterococcus genus comprises the base sequence of SEQ ID NO:59 or a base sequence having a sequence identity of 90% or more to the base sequence of SEQ ID NO:

59.

3. The method according to claim 1 or 2, wherein the probe hybridizes to the entire length or a portion of the DNA under stringent conditions.

4. The method of any one of claims 1 to 3, wherein the probes each comprise a different identifier or are each bound to a different identifier on a solid support.

5. The method according to any one of claims 1 to 4, wherein the DNA amplified by PCR contains a label.

6. A kit for identifying a causative bacterium of sepsis, comprising: a first reagent including a set of PCR primers specific to the full length or a partial region of the gyrB gene of Escherichia coli, the nuc gene of Staphylococcus aureus, the atlE gene of Staphylococcus epidermidis, the gyrB gene of Klebsiella pneumoniae, and the rpoB gene of Enterococcus spp.; a second reagent including a combination of probes specific to the DNA of the full length or a partial region of each gene, a set of primers specific to the full length or a partial region of the gyrB gene of Escherichia coli, comprising a primer including a nucleotide sequence of SEQ ID NO: 10 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 10, and a primer including a nucleotide sequence of SEQ ID NO: 11 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 11; A set of primers specific to the full length or a partial region of the nuc gene of Staphylococcus aureus includes one or more primers selected from a primer including a base sequence of SEQ ID NO: 32 or a base sequence having 90% or more sequence identity with the base sequence of SEQ ID NO: 32, a primer including a base sequence of SEQ ID NO: 34 or a base sequence having 90% or more sequence identity with the base sequence of SEQ ID NO: 34, and a combination thereof, and a primer including a base sequence of SEQ ID NO: 33 or a base sequence having 90% or more sequence identity with the base sequence of SEQ ID NO: 33; a set of primers specific to the full length or a partial region of the atlE gene of Staphylococcus epidermidis, comprising a primer including a nucleotide sequence of SEQ ID NO: 37 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 37, and a primer including a nucleotide sequence of SEQ ID NO: 38 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 38; A set of primers specific to the full length or a partial region of the gyrB gene of Klebsiella pneumoniae includes a primer including a nucleotide sequence of SEQ ID NO: 14 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 14, and a primer including a nucleotide sequence of SEQ ID NO: 15 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 15, a set of primers specific to the full length or a partial region of the rpoB gene of Enterococcus, comprising one or more primers selected from a primer comprising the nucleotide sequence of SEQ ID NO:26 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO:26, a primer comprising the nucleotide sequence of SEQ ID NO:27 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO:27, a primer comprising the nucleotide sequence of SEQ ID NO:28 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO:28, and a combination thereof; and a primer comprising the nucleotide sequence of SEQ ID NO:29 or a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO:29, the probe specific to the full length or a partial region of DNA of the gyrB gene of Escherichia coli comprises the nucleotide sequence of SEQ ID NO: 121 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO: 121; the probe specific to the DNA of the full length or a partial region of the nuc gene of Staphylococcus aureus comprises the base sequence of SEQ ID NO: 134 or a base sequence having a sequence identity of 90% or more to the base sequence of SEQ ID NO: 134; the probe specific to the DNA of the full length or a partial region of the atlE gene of Staphylococcus epidermidis comprises the nucleotide sequence of SEQ ID NO: 136 or a nucleotide sequence having a sequence identity of 90% or more to the nucleotide sequence of SEQ ID NO: 136; the probe specific to the DNA of the full length or a partial region of the gyrB gene of Klebsiella pneumoniae comprises the nucleotide sequence of SEQ ID NO: 124 or a nucleotide sequence having a sequence identity of 90% or more to the nucleotide sequence of SEQ ID NO: 124; the probe specific to DNA of the full length or a partial region of the rpoB gene of the Enterococcus genus is selected from a probe containing the base sequence of SEQ ID NO: 129 or a base sequence that has 90% or more sequence identity to the base sequence of SEQ ID NO: 129, a probe containing the base sequence of SEQ ID NO: 130 or a base sequence that has 90% or more sequence identity to the base sequence of SEQ ID NO: 130, a probe containing the base sequence of SEQ ID NO: 131 or a base sequence that has 90% or more sequence identity to the base sequence of SEQ ID NO: 131, and combinations thereof.

7. the DNA of the full length or a partial region of the gyrB gene of Escherichia coli comprises the nucleotide sequence of SEQ ID NO:51 or a nucleotide sequence having a sequence identity of 90% or more with the nucleotide sequence of SEQ ID NO:51, The DNA of the full length or a partial region of the nuc gene of Staphylococcus aureus comprises the base sequence of SEQ ID NO: 61 or a base sequence having a sequence identity of 90% or more to the base sequence of SEQ ID NO: 61, the DNA of the full length or a partial region of the atlE gene of Staphylococcus epidermidis comprises the nucleotide sequence of SEQ ID NO: 63 or a nucleotide sequence having a sequence identity of 90% or more to the nucleotide sequence of SEQ ID NO: 63, the DNA of the full length or a partial region of the gyrB gene of Klebsiella pneumoniae comprises the nucleotide sequence of SEQ ID NO: 53 or a nucleotide sequence having a sequence identity of 90% or more to the nucleotide sequence of SEQ ID NO: 53; The kit according to claim 6, wherein the DNA of the full length or partial region of the rpoB gene of the Enterococcus genus comprises the base sequence of SEQ ID NO:59 or a base sequence having a sequence identity of 90% or more to the base sequence of SEQ ID NO:

59.

8. The kit according to claim 6 or 7, wherein the probe hybridizes to the full length or partial region of DNA under stringent conditions.

9. The kit according to any one of claims 6 to 8, wherein the probes contained in the second reagent each contain a different identifier or are each bound to a different identifier on a solid support.

10. The kit according to any one of claims 6 to 9, wherein the set of primers is designed so that DNA amplified by PCR using the set of primers contains a label, or the kit further comprises a third reagent for labeling the amplified DNA.

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