Primer sets and probes for the detection of Klebsiella spp.

Abstract

The present invention provides a primer set for detecting the presence of Klebsiella variicola in a specimen, the primer set comprising a first primer and a second primer that target the gyrB gene of Klebsiella variicola.

Description

[Technical field]

[0001] The present invention relates to an oligonucleotide for detecting bacteria of the genus Klebsiella (Klebsiella spp.), and more particularly to an oligonucleotide probe, a kit, or a method for detecting or differentiating between Klebsiella variicola and Klebsiella pneumoniae. [Background technology]

[0002] Klebsiella bacteria are gram-negative rod-shaped bacteria that are widely distributed in the environment. Among them, Klebsiella pneumoniae, known as Klebsiella pneumoniae in Japanese, is a normal inhabitant of the human intestinal tract and pharynx, and is one of the representative causative agents of opportunistic infections, such as respiratory infections, urinary tract infections, and bloodstream infections, that are pathogenic to humans. It is the species of Klebsiella most frequently observed in clinical specimens.

[0003] There are resistant strains of Klebsiella pneumoniae that possess an enzyme called extended-spectrum β-lactamase (ESBL) that breaks down many types of antibiotics. The bacteria are classified as one of the carbapenem-resistant Enterobacteriacae (CRE), and the increasing difficulty of treatment and its spread have become an international problem.

[0004] As a method for detecting infectious disease-causing bacteria such as Klebsiella pneumoniae, a culture identification method is known in which the causative bacteria are isolated and cultured, and the bacteria are identified based on their biochemical properties. It has been found that some of the bacteria isolated from clinical specimens and identified as Klebsiella pneumoniae, probably at a rate of about 10% to 15%, are actually a different species called Klebsiella variicola (Non-Patent Document 1). Klebsiella variicola, a bacterium of the genus Klebsiella, is a related species of Klebsiella pneumoniae, which was registered as a new species in 2004, and has been reported to be isolated from plants such as bananas and sugar cane, and from mycelium in the nests of leaf-cutting ants, and is known to be not a normal bacterium in humans (Non-Patent Document 1). It has been reported that some properties (e.g., adonitol decomposition ability) of this bacterium tend to differ from Klebsiella pneumoniae in existing phenotypic tests, but there is currently no definite indicator, and differentiation is considered difficult.

[0005] In addition, the genus Klebsiella is currently classified into 14 species and 5 subspecies, and other species that are known to be pathogenic to humans include Klebsiella aerogenes, Klebsiella oxytoca, and Klebsiella planticola.

[0006] As mentioned above, when identifying Klebsiella bacteria in clinical specimens, the many species of Klebsiella must be considered. Differentiation of these species is difficult using existing phenotypic tests, and genetic methods such as sequence analysis, multilocus sequence typing (MLST), and average nucleotide identity (ANI) have been adopted to accurately identify Klebsiella bacteria in clinical specimens.

[0007] Among them, methods for distinguishing between Klebsiella pneumoniae and Klebsiella variicolae have been conventionally performed based on gene sequence information, such as MLST (Non-Patent Document 1) that targets six genes, rpoB, gyrA, mdh, infB, phoE, and nifH. However, these methods lack convenience because they require sequence analysis, and are difficult to adopt as routine work in actual medical settings.

[0008] A simpler method, that is, a discrimination method using multiplex PCR and gel electrophoresis of three genes, blaSHV, blaOKP, and blaLEN, that requires only a small amount of effort and can be adopted as routine work in the medical field, has been reported (Non-Patent Document 2). However, this method focuses on analysis by gel electrophoresis, with the PCR product length being at least 348 bp and at most 995 bp, and is unsuitable for the currently widely used real-time PCR because the product length is too long.

[0009] In addition, a method has been reported in which real-time PCR and Sanger sequencing are combined to identify strains based on sequence information of 16S rDNA and rpoB genes (Non-Patent Document 3). This method also requires sequence analysis for identification, and is therefore not simple. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Klebsiella variicola, a novel species with clinical and plant associated isolates. Syst Appl Microbiol 27, 27-35 [Non-Patent Document 2] :A one-step multiplex PCR to identify Klebsiella pneumoniae, Klebsiella variicola, and Klebsiella quasipneumoniae in the clinical routine, Diagnostic Microbiology and Infectious Disease Volume 87, Issue 4, April 2017, Pages 315-317 [Non-Patent Document 3] Journal of Microbiological Methods Volume 159, April 2019, Pages 148-156 Summary of the Invention [Problem to be solved by the invention]

[0011] In view of the above circumstances, an object of the present invention is to provide an oligonucleotide or the like for detecting or differentiating bacteria of the genus Klebsiella (Klebsiella spp.). [Means for solving the problem]

[0012] As a result of intensive research, the present inventors have focused on the sequences of the gyrB genes of Klebsiella pneumoniae, Klebsiella variicola, and other Klebsiella bacteria, and have discovered a method for distinguishing between Klebsiella pneumoniae and Klebsiella variicola more simply and easily than previously reported methods by focusing on a specific position in the gyrB genes.

[0013] That is, the present application includes the following inventions. [1] A primer set for detecting the presence of one or more Klebsiella bacteria selected from the group consisting of Klebsiella variicola and Klebsiella pneumoniae in a sample, comprising: A primer set consisting of a first primer and a second primer targeting the gyrB gene of Klebsiella bacteria. [2] A primer set described in [1], in which the gyrB gene has a base sequence shown in any one of SEQ ID NOs: 1 to 6, or has a base sequence in which one or more bases have been deleted, substituted, inserted or added in the base sequence shown in any one of SEQ ID NOs: 1 to 6, and encodes a GyrB protein. [3] A primer set described in [1] or [2], wherein the targeted gyrB gene has a base sequence including at least 10 consecutive bases from positions 1887 to 2063 of the base sequence shown in any one of SEQ ID NOs: 1 to 6. [4] A primer set for detecting the presence of Klebsiella variecol, comprising a first primer having a base sequence as set forth in SEQ ID NO: 7, or a base sequence including at least 10 consecutive bases in a base sequence in which one or more bases have been deleted, substituted, inserted or added in the base sequence as set forth in SEQ ID NO: 7, and having the same function as the base sequence as set forth in SEQ ID NO: 7. [5] A primer set for detecting the presence of Klebsiella variecol, wherein the second primer has a base sequence as set forth in SEQ ID NO: 8, or has a base sequence containing at least 10 consecutive bases in the base sequence as set forth in SEQ ID NO: 8 in which one or more bases have been deleted, substituted, inserted or added, and has the same function as the base sequence as set forth in SEQ ID NO: 8. [6] A primer set for detecting the presence of Klebsiella varicol, further comprising a third primer and a fourth primer targeting the gyrB gene of Klebsiella pneumoniae, the primer set being described in any of [1] to [5]. [7] A primer set according to any one of [1] to [6], wherein the third primer has a base sequence as set forth in SEQ ID NO: 10, or has a base sequence containing at least 10 consecutive bases in the base sequence as set forth in SEQ ID NO: 10 in which one or several bases have been deleted, substituted, inserted or added, and has the same function as the base sequence as set forth in SEQ ID NO: 10. [8] A primer set according to any one of [1] to [7], wherein the fourth primer has a base sequence as set forth in SEQ ID NO: 11, or has a base sequence containing at least 10 consecutive bases in the base sequence as set forth in SEQ ID NO: 11 in which one or several bases have been deleted, substituted, inserted or added, and has the same function as the base sequence as set forth in SEQ ID NO: 11. [9] A primer set for detecting the presence of Klebsiella pneumoniae, wherein the first primer has a base sequence set forth in SEQ ID NO: 10, or has a base sequence containing at least 10 consecutive bases in the base sequence set forth in SEQ ID NO: 10 in which one or more bases have been deleted, substituted, inserted or added, and has the same function as the base sequence set forth in SEQ ID NO: 10.

[10] A primer set described in any one of [1], [2] or [9], wherein the second primer has a base sequence as set forth in SEQ ID NO: 11, or a base sequence containing at least 10 consecutive bases in the base sequence as set forth in SEQ ID NO: 11 in which one or more bases have been deleted, substituted, inserted or added, and has the same function as the base sequence as set forth in SEQ ID NO: 11.

[11] A primer set described in any of [1], [2], [9], or

[10] , further comprising a third primer and a fourth primer targeting the gyrB gene of Klebsiella variicola.

[12] A primer set according to any one of [1], [2], and [9] to

[11] , wherein the third primer has a base sequence as set forth in SEQ ID NO: 7, or has a base sequence containing at least 10 consecutive bases in the base sequence as set forth in SEQ ID NO: 7 in which one or several bases have been deleted, substituted, inserted or added, and has the same function as the base sequence as set forth in SEQ ID NO: 7.

[13] A primer set according to any one of [1], [2], and [9] to

[12] , wherein the fourth primer has a base sequence as set forth in SEQ ID NO: 8, or has a base sequence containing at least 10 consecutive bases in the base sequence as set forth in SEQ ID NO: 8 in which one or several bases have been deleted, substituted, inserted or added, and has the same function as the base sequence as set forth in SEQ ID NO: 8.

[14] The primer set according to any one of [1] to

[13] , wherein the primers are labeled with biotin.

[15] A probe for detecting the presence of one or more Klebsiella bacteria selected from the group consisting of Klebsiella varicol and Klebsiella pneumoniae in a sample, comprising: A probe targeting the gyrB gene of Klebsiella.

[16] The probe described in

[15] , wherein the gyrB gene has a base sequence shown in any one of SEQ ID NOs: 1 to 6, or has a base sequence in which one or more bases have been deleted, substituted, inserted or added in the base sequence shown in any one of SEQ ID NOs: 1 to 6, and encodes a GyrB protein.

[17] A probe described in

[15] or

[16] , wherein the targeted gyrB gene has a base sequence including at least 10 consecutive bases from positions 1887 to 2063 of the base sequence shown in any one of SEQ ID NOs: 1 to 6.

[18] The probe according to any one of

[15] to

[17] , which has the base sequence set forth in SEQ ID NO: 9, or has a base sequence containing at least 8 consecutive bases in the base sequence set forth in SEQ ID NO: 9 in which one or several bases have been deleted, substituted, inserted or added, and has the same function as the base sequence set forth in SEQ ID NO: 9.

[19] A kit comprising the primer set according to any one of [1] to

[14] and the probe according to any one of

[15] to

[18] .

[20] A method for detecting the presence of Klebsiella varicol in a sample, the method using a primer set described in any one of [1] to [9].

[21] The method described in

[20] , comprising a step of distinguishing Klebsiella variikola from other Klebsiella bacteria using the first and second primers.

[22] The method according to

[20] or

[21] , further comprising using a probe according to any one of

[15] to

[18] .

[23] The method according to any of

[20] to

[22] , wherein the Klebsiella bacterium to be identified as Klebsiella pneumoniae is one or more species selected from the group consisting of Klebsiella pneumoniae, Klebsiella michiganensis, Klebsiella quasipneumoniae, Klebsiella aerogenes, Klebsiella planticola, Klebsiella granulomatis, Klebsiella grimontii, and Klebsiella oxytoca.

[24] The method according to any of

[20] to

[23] , further comprising using a fourth primer and a fifth primer and / or a probe targeting the gyrB gene of Klebsiella pneumoniae, wherein the Klebsiella bacterium to be differentiated from Klebsiella pneumoniae is Klebsiella pneumoniae.

[25] A method for detecting the presence of Klebsiella pneumoniae in a sample, the method using a primer set described in any one of [1], [2], and [9] to

[14] .

[26] The method according to

[25] , comprising a step of distinguishing Klebsiella pneumoniae from other Klebsiella bacteria using the first and second primers.

[27] The method according to

[26] , further comprising using a probe according to any one of

[15] to

[18] .

[28] The method according to any of

[25] to

[27] , wherein the Klebsiella bacterium to be identified as Klebsiella pneumoniae is one or more species selected from the group consisting of Klebsiella variicola; Klebsiella michiganensis; Klebsiella cassipneumoniae; Klebsiella aerogenes; Klebsiella planticola; Klebsiella granulomatis; Klebsiella grimonti; and Klebsiella oxytoca.

[29] The method according to any of

[25] to

[28] , further comprising using a fourth primer and a fifth primer and / or a probe targeting the gyrB gene of Klebsiella variicola, wherein the Klebsiella bacterium to be differentiated from Klebsiella pneumoniae is Klebsiella variicola. Effect of the Invention

[0014] The oligonucleotide of the present invention makes it possible to more easily distinguish Klebsiella variicola and / or Klebsiella pneumoniae from other bacteria of the genus Klebsiella. Furthermore, the oligonucleotide of the present invention can exhibit superior discrimination ability in comparison with other primers and probes targeting the same gyrB gene. [Brief description of the drawings]

[0015] [Figure 1] Multiple alignment results of a portion of the gyrB gene of K. variicola strains DSM15968, MGH20, and X39 (SEQ ID NOs: 1, 2, and 3) and the gyrB genes of K. pneumoniae strains NU-CRE047, CHS105, and KPNIH33 (SEQ ID NOs: 4, 5, and 6). [Diagram 2]The results of melting curve analysis of the amplification products obtained using the primer sets of SEQ ID NOs: 7 and 8, 10 and 11. The solid line shows the results for the K. variicola JCM12419 strain, the dashed line shows the results for the K. pneumoniae JCM1662T strain, and the dashed dotted line shows the results for the control (water). [Diagram 3] Detection results on a substrate using DNA probes targeting the genomic DNA of K. variicola and K. pneumoniae. The vertical axis shows the fluorescence intensity measured in pCode. [Figure 4] The results for the negative control (water) have been removed from the results in Figure 3. The vertical axis shows the fluorescence intensity measured in pCode. [Diagram 5] Detection results on a substrate using a DNA probe for clinical samples. The vertical axis shows the fluorescence intensity measured in pCode. [Figure 6] Multiple alignment results (1 to 301 bp) of parts of the gyrB genes (SEQ ID NOs: 1, 2, and 3) of K. variikola strains DSM15968, MGH20, and X39. [Figure 7] Multiple alignment results (300 to 600 bp) of parts of the gyrB genes (sequence numbers 1, 2, and 3) of K. variikola strains DSM15968, MGH20, and X39. [Figure 8] Multiple alignment results (701 to 1,000 bp) of parts of the gyrB genes (sequence numbers 1, 2, and 3) of K. variikola strains DSM15968, MGH20, and X39. [Figure 9] Multiple alignment results (1601 to 1900 bp) of parts of the gyrB genes (sequence numbers 1, 2, and 3) of K. variicola strains DSM15968, MGH20, and X39. [Figure 10] Multiple alignment results (2051 to 2350 bp) of parts of the gyrB genes (sequence numbers 1, 2, and 3) of K. variikola strains DSM15968, MGH20, and X39. [Figure 11] Fluorescence measurement results using assay sets 1 to 6 are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Hereinafter, an embodiment of the present invention (hereinafter, referred to as "the present embodiment") will be described, but the scope of the present invention should not be interpreted as being limited to the following embodiment.

[0017] (Primer set) In a first aspect, there is provided a primer set for detecting the presence of one or more Klebsiella bacteria selected from the group consisting of Klebsiella variicola and Klebsiella pneumoniae in a sample, the primer set comprising a first primer and a second primer targeting the gyrB gene of the Klebsiella bacteria.

[0018] The gyrB gene encodes the GyrB protein, a subunit of DNA gyrase.

[0019] The specimen is not particularly limited as long as it is suspected of containing Klebsiella spp. including Klebsiella variicola or Klebsiella pneumoniae, and examples thereof include human or non-human derived body fluids such as blood, serum, plasma, urine, stool, saliva, sputum, tissue fluid, cerebrospinal fluid, swabs, and dilutions thereof, with blood, serum, plasma, urine, stool, cerebrospinal fluid, and dilutions thereof being preferred. The specimen may also be food, soil, plants, and the like.

[0020] Examples of the gyrB gene of Klebsiella variicola include those having a base sequence shown in any one of SEQ ID NOs: 1 to 3, or those having a base sequence in which one or several bases have been deleted, substituted, inserted or added in the base sequence shown in any one of SEQ ID NOs: 1 to 3, and which encode the GyrB protein.

[0021] Examples of the gyrB gene of Klebsiella pneumoniae include those having a base sequence shown in any one of SEQ ID NOs: 4 to 6, or those having a base sequence in which one or several bases have been deleted, substituted, inserted or added in the base sequence shown in any one of SEQ ID NOs: 4 to 6, and which encode the GyrB protein.

[0022] For example, the first primer as a forward primer may have any sequence as long as it can hybridize under stringent conditions to a base sequence shown in any one of SEQ ID NOs: 1 to 6, or a base sequence in which one or several bases have been deleted, substituted, inserted or added.

[0023] Similarly, the second primer as a reverse primer may have any sequence as long as it can hybridize under stringent conditions to a complementary strand of the base sequence shown in any one of SEQ ID NOs: 1 to 6, or to a base sequence in which one or several bases have been deleted, substituted, inserted or added in the complementary strand of the base sequence shown in any one of SEQ ID NOs: 1 to 6.

[0024] As used herein, "stringent conditions" refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed, and can be appropriately determined based on information such as the length of the base sequence. For example, stringent conditions can be set with reference to Molecular Cloning (Fourth Edition, Cold Spring Harbor Laboratory Press, New York). Stringent conditions can also be said to be conditions under which polynucleotides having high homology (preferably identity), for example, polynucleotides having a homology of 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, particularly preferably 99% or more, hybridize with each other, and polynucleotides showing a lower homology do not hybridize with each other.

[0025] Specific stringent conditions include incubating a base sequence complementary to the desired base sequence and a sample suspected of containing Klebsiella variicol in a hybridization solution (50% formamide, 10x SSC (0.15M NaCl, 15mM sodium citrate, pH 7.0), 5x Denhardt's solution, 1% SDS, 10% dextran sulfate, 10μg / ml denatured salmon sperm DNA, and 50mM phosphate buffer (pH 7.5)) at about 42°C to about 50°C, and then washing with 0.1x SSC, 0.1% SDS at about 65°C to about 70°C.

[0026] Primers and probes are designed so as not to cross-react with sequences of other related species, but can be appropriately changed depending on the system used to detect the amplification product and other conditions. When generating an amplification product having a length of 100 to 200 bases, for example, it is preferable to target the base sequence from position 1887 to position 2063 of the base sequence shown in any of SEQ ID NOs: 1 to 6. The primer is designed to have a base sequence containing at least 10 consecutive bases from the base sequence constituting such a target. It is preferable to design the primer so that there is a mismatch with a Klebsiella bacterium other than Klebsiella variicola at the end, particularly the 3' end.

[0027] Examples of a first primer targeting the gyrB gene of Klebsiella variicola include a primer having the nucleotide sequence set forth in SEQ ID NO: 7 below, or a primer having a nucleotide sequence containing at least 10 consecutive nucleotides in the nucleotide sequence set forth in SEQ ID NO: 7 in which one or several nucleotides have been deleted, substituted, inserted or added, and having the same function as the nucleotide sequence set forth in SEQ ID NO: 7. 5'-GGTGAACACCCTGGTTTC-3' (SEQ ID NO: 7)

[0028] Examples of a second primer targeting the gyrB gene of Klebsiella variicola include a primer having the nucleotide sequence set forth in SEQ ID NO: 8 below, or a primer having a nucleotide sequence containing at least 10 consecutive nucleotides in the nucleotide sequence set forth in SEQ ID NO: 8 in which one or several nucleotides have been deleted, substituted, inserted or added, and having the same function as the nucleotide sequence set forth in SEQ ID NO: 8. 5'-CGATATTCCGGCCCCATG-3' (SEQ ID NO: 8)

[0029] Examples of a first primer targeting the gyrB gene of Klebsiella pneumoniae include a primer having the nucleotide sequence set forth in SEQ ID NO: 7 below, or a primer having a nucleotide sequence containing at least 10 consecutive nucleotides in the nucleotide sequence set forth in SEQ ID NO: 7 in which one or several nucleotides have been deleted, substituted, inserted or added, and having the same function as the nucleotide sequence set forth in SEQ ID NO: 7. 5'-GGTGAACACCCTGGTCTC-3' (SEQ ID NO: 10)

[0030] Examples of the second primer targeting the gyrB gene of Klebsiella pneumoniae include a primer having the nucleotide sequence set forth in SEQ ID NO: 8 below, or a primer having a nucleotide sequence containing at least 10 consecutive nucleotides in the nucleotide sequence set forth in SEQ ID NO: 8 in which one or several nucleotides have been deleted, substituted, inserted or added, and having the same function as the nucleotide sequence set forth in SEQ ID NO: 8. 5'-GATATTCCGGCCCCATAATG-3' (SEQ ID NO: 11)

[0031] The length of the first and second primers is arbitrary and can be appropriately set within the range of, for example, about 10 to about 35 bases, but is preferably at least 10 bases or more.

[0032] The ratio of the first primer to the second primer is not particularly limited, and may be the same or different from each other. When the primer set is used in asymmetric PCR, the concentration of one primer may be adjusted to be higher than the concentration of the other primer.

[0033] The first and second primers can specifically amplify the whole or part of the gyrB gene of Klebsiella variicola or Klebsiella pneumoniae, which allows differentiation from other Klebsiella species. Alternatively, Klebsiella variicola or Klebsiella pneumoniae can be differentiated from other Klebsiella species by subjecting the amplified product of the first and second primers to melting curve analysis and comparing the melting temperatures with those of the amplified products of other primer sets.

[0034] Other Klebsiella species include, but are not limited to, Klebsiella michiganensis, Klebsiella quasipneumoniae, Klebsiella aerogenes, Klebsiella planticola, Klebsiella granulomatis, Klebsiella grimontii, Klebsiella oxytoca, etc. The Klebsiella species may be one or more species.

[0035] The primer set may be a combination of the first and second primers for Klebsiella variicola and the first and second primers for Klebsiella pneumoniae, or may contain, individually or in combination, additional primers other than these, such as third, fourth, fifth, sixth, etc. The detection sensitivity can be improved by using appropriate additional primers. Such primers may be, for example, primers for detecting other Klebsiella species expected to be contained in the sample.

[0036] Examples of a third primer targeting the gyrB gene of Klebsiella pneumoniae that can be used for detecting or differentiating Klebsiella variicola include a primer having the nucleotide sequence set forth in SEQ ID NO: 10, or a primer having a nucleotide sequence containing at least 10 consecutive nucleotides in the nucleotide sequence set forth in SEQ ID NO: 10 in which one or several nucleotides have been deleted, substituted, inserted or added, and having the same function as the nucleotide sequence set forth in SEQ ID NO: 10.

[0037] Examples of the fourth primer targeting the gyrB gene of Klebsiella pneumoniae that can be used for detecting or differentiating Klebsiella variicola include a primer having the nucleotide sequence set forth in SEQ ID NO: 11, or a primer having a nucleotide sequence containing at least 10 consecutive nucleotides in the nucleotide sequence set forth in SEQ ID NO: 11 in which one or several nucleotides have been deleted, substituted, inserted or added, and having the same function as the nucleotide sequence set forth in SEQ ID NO: 11.

[0038] Examples of a third primer targeting the gyrB gene of Klebsiella variicola that can be used for detecting or differentiating Klebsiella pneumoniae include a primer having the nucleotide sequence set forth in SEQ ID NO: 7, or a primer having a nucleotide sequence containing at least 10 consecutive nucleotides in the nucleotide sequence set forth in SEQ ID NO: 7 in which one or several nucleotides have been deleted, substituted, inserted or added, and having the same function as the nucleotide sequence set forth in SEQ ID NO: 7.

[0039] Examples of the fourth primer targeting the gyrB gene of Klebsiella variicola that can be used for detecting or differentiating Klebsiella pneumoniae include a fourth primer having the nucleotide sequence set forth in SEQ ID NO: 8, or a nucleotide sequence containing at least 10 consecutive nucleotides in the nucleotide sequence set forth in SEQ ID NO: 8 in which one or several nucleotides have been deleted, substituted, inserted or added, and having the same function as the nucleotide sequence set forth in SEQ ID NO: 8.

[0040] The presence of Klebsiella variicola and / or Klebsiella pneumoniae in a sample can be detected by a nucleic acid amplification reaction using the above primers.

[0041] Examples of nucleic acid amplification methods include PCR, including multiplex PCR, LAMP (Loop-mediated isothermal AMPlification), ICAN (Isothermal and Chimeric primer-initiated Amplification of Nucleic Acids), RCA (Rolling Circle Amplification), LCR (Ligase Chain Reaction), SDA (Strand Displacement Amplification), etc. When a sample is expected to contain a large number of Klebsiella bacteria, multiplex PCR, which can comprehensively detect multiple bacteria simultaneously, is preferred.

[0042] Detection of the amplification product can be performed by known methods such as electrophoresis using polyacrylamide or agarose gels, for example, in which the presence of the amplification product can be identified based on the mobility of the amplification product relative to the mobility of a marker of known molecular weight.

[0043] To detect the amplified product after the nucleic acid amplification reaction, a label capable of specifically recognizing the amplified product may be used. Examples of such a label include fluorescent dyes, biotin, digoxigenin, etc. When a fluorescent substance is used as the label, the fluorescence can be detected using a fluorescent microscope, a fluorescent plate reader, etc.

[0044] (probe) In a second aspect, a probe is provided for detecting the presence of one or more Klebsiella bacteria selected from the group consisting of Klebsiella variicola and Klebsiella pneumoniae in a sample, the probe being capable of targeting the gyrB gene of Klebsiella bacteria.

[0045] The probe is preferably designed to contain a mismatch with other Klebsiella bacteria within the probe, which shortens the base length of the full match portion upon hybridization, improving specificity and reducing false positives.

[0046] Examples of probes that can be used to detect or differentiate Klebsiella variecol include those having the nucleotide sequence set forth in SEQ ID NO:9 below, or those having a nucleotide sequence containing at least 8 consecutive nucleotides in the nucleotide sequence set forth in SEQ ID NO:9 in which one or several nucleotides have been deleted, substituted, inserted or added, and having the same function as the nucleotide sequence set forth in SEQ ID NO:9. 5'-GTTGCAGCAGTTTGAG-3' (SEQ ID NO: 9)

[0047] Examples of probes that can be used to detect or differentiate Klebsiella pneumoniae include those having the nucleotide sequence set forth in SEQ ID NO:12 below, or those having a nucleotide sequence containing at least 8 consecutive nucleotides in the nucleotide sequence set forth in SEQ ID NO:12 in which one or several nucleotides have been deleted, substituted, inserted or added, and having the same function as the nucleotide sequence set forth in SEQ ID NO:12. 5'-GAACTGCAGCATTTTG-3' (SEQ ID NO: 12)

[0048] The probes may be attached to solid phase bodies such as beads. Examples of such solid phase bodies include beads suitable for multiplex assays, such as beads having unique analog code identifiers for storing information about the multiplex assay, as disclosed in WO2018052464A1.

[0049] (kit) In a third aspect, a kit is provided comprising a primer set and a probe for detecting the presence of one or more Klebsiella bacteria selected from the group consisting of Klebsiella variicola and Klebsiella pneumoniae in a sample.

[0050] The kit may contain a reagent used for detecting one or more Klebsiella bacteria selected from the group consisting of Klebsiella variicola and Klebsiella pneumoniae, for example, a reagent for performing an amplification reaction. The reagent for performing the amplification reaction may include, for example, a buffer solution, salts, primers, deoxyribonucleotides, a thermostable DNA polymerase, etc. The contents and reagents of the kit can be appropriately determined by those skilled in the art depending on the amplification method, and the kit may further contain a solid phase body for hybridization with an amplification product obtained using a primer or a probe.

[0051] The kit may further include primers and probes for detecting other Klebsiella species expected to be present in the sample.

[0052] (Detection method) In a fourth aspect, there is provided a method for detecting the presence of one or more Klebsiella bacteria selected from the group consisting of Klebsiella variicola and Klebsiella pneumoniae in a sample, the method comprising the steps of: detecting the presence of Klebsiella variicola using a primer set of SEQ ID NOs: 7 and 8; and detecting the presence of Klebsiella pneumoniae using a primer set of SEQ ID NOs: 10 and 11.

[0053] It is preferable to combine a primer set with a probe. For example, by using the primers of SEQ ID NO: 7 and 8 in combination with the probe of SEQ ID NO: 9, Klebsiella variicola can be differentiated from other Klebsiella bacteria with high accuracy. A primer set and a probe for detecting Klebsiella pneumoniae may also be used in combination.

[0054] In order to distinguish Klebsiella pneumoniae from other Klebsiella bacteria with high accuracy, it is preferable to use, for example, the primers of SEQ ID NOs: 10 and 11 in combination with the probe of SEQ ID NO: 12. A primer set and a probe for detecting Klebsiella variicola may also be used in combination.

[0055] The samples may be further subjected to whole genome analysis and ANI (Average Nucleotide Identity) analysis to determine similarities and similarities between the detected bacteria.

[0056] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. EXAMPLES

[0057] Example 1: Detection of K. variicola and K. pneumoniae by real-time PCR targeting genomic DNA

[0058] Primer-probe design Primers and probes shown in SEQ ID NOs: 7 to 12 were designed based on the results of multiple alignment of portions of the gyrB genes of K. variicola strains DSM15968, MGH20, and X39 (sequence numbers 1, 2, and 3) and K. pneumoniae strains NU-CRE047, CHS105, and KPNIH33 (sequence numbers 4, 5, and 6) shown in Figure 1. [Table 1]

[0059] Nucleic acid amplification and melting curve analysis K. variicola JCM12419, K. pneumoniae JCM1662T Using genomic DNA extracted from the strain as a template, real-time PCR amplification was performed using primers of SEQ ID NOs: 7, 8, 10, and 11. The reagents used for amplification were TaKaRa Multiplex Assay Kit Ver.2 (Takara Bio Inc.), and EvaGreen (biotium) was used as an intercalator dye. Each primer was added to a final concentration of 0.2 μM, and template genomic DNA was added to 10 μg / L. 1× 2× Reaction buffer and 1U Multiplex Enzyme Mix in TaKaRa Multiplex Assay Kit Ver.2 were used, and the template DNA and other components were mixed to prepare a PCR reaction solution. A Light Cycler 480 System II (Roche Diagnostics) was used as the measurement device.

[0060] The amplification reaction conditions used were as follows: 94℃: 30 seconds 60℃: 60 seconds -> 94℃: 30 seconds (45 cycles) 72℃: 600 seconds

[0061] Furthermore, melting curve analysis of the amplified products was performed under the following conditions. 95℃: 30 seconds 60℃->95℃: Heating at 0.11℃ / sec

[0062] The results of the melting curve analysis are shown in Figure 2. The melting temperatures of the amplification products of K. variicola and K. pneumoniae were 84.7°C and 83.8°C, respectively, with a difference of approximately 1°C. From the above, it was found that the use of primers of SEQ ID NOs: 7, 8, 10, and 11 allows the amplification of the targeted nucleic acid component, and further allows the identification of the amplification products based on their melting temperatures.

[0063] It is worth noting that this example does not use the expensive TaqMan Probe that is often used for luminescent substances in this field, but instead allows identification using an inexpensive intercalator dye, making it superior in terms of the cost required for the experiment.

[0064] Example 2: On-substrate detection using DNA probes targeting genomic DNA of K. variicola and K. pneumoniae

[0065] Nucleic Acid Amplification and Detection K. variicola JCM12419, K. pneumoniae JCM1662 T Using genomic DNA extracted from the strain as a template, PCR amplification, hybridization reaction on a substrate, labeling, and fluorescence measurement were performed using a primer set of SEQ ID NOs: 7, 8, 10, and 11. Primers of SEQ ID NOs: 8 and 11 were modified with biotin at the 5' end via a linker.

[0066] For amplification, TaKaRa Multiplex Assay Kit Ver.2 (Takara Bio) was used. Each primer was added to a final concentration of 0.2 μM, and template genomic DNA was added to 10 μg / L. 1× 2× Reaction buffer and 1U Multiplex Enzyme Mix from the TaKaRa Multiplex Assay Kit Ver.2 were used to mix with template DNA and other components to prepare a PCR reaction solution. A T100 Thermal Cycler (Bio-Rad) was used as the nucleic acid amplifier. After the amplification reaction was completed, a thermal denaturation reaction was performed using the same device before subjecting the mixture to a hybridization reaction on the substrate.

[0067] The reagents used for the hybridization reaction on the substrate were a mixture of a solution containing substrate 1 on which the probe of sequence number 9 was immobilized and substrate 2 on which the probe of sequence number 12 was immobilized, saline-sodium-phosphate-EDTA buffer (SSPE-buffer), and a PCR reaction solution that had been thermally denatured.

[0068] Streptavidin-Phycoerythrin (PlexBio) was used as a reagent for labeling. The hybridization reaction and labeling on the substrate were performed using an IntelliPlex 1000 πCode Processor (PlexBio). The fluorescence measurement device used was a PlexBio (registered trademark) 100 Fluorescent Analayzer (PlexBio).

[0069] The reaction conditions used were as follows: Amplification reaction conditions 94℃: 30 seconds 60℃: 60 seconds -> 94℃: 30 seconds (30 cycles) 72℃: 600 seconds

[0070] Heat denaturation conditions 95℃: 5 minutes -> Rapid cooling to 4℃

[0071] Hybridization conditions Incubate at 37℃ for 20 minutes

[0072] Labeling conditions Incubate at 37℃ for 10 minutes

[0073] The measurement results are shown in Figure 3. When K. variicola JCM12419 strain was used as the template, significant fluorescence was observed on substrate 1, but almost no fluorescence was observed on substrate 2. In addition, when K. pneumoniae JCM1662 T When the strain was used as a template, weak fluorescence was observed on Substrate 1, while significant fluorescence was observed on Substrate 2. When water was used as a template as a negative control, weak fluorescence was observed on Substrate 1, while almost no fluorescence was observed on Substrate 2.

[0074] From this, it can be determined that the background value of Substrate 1 is high, and the results obtained by subtracting the value of the negative control from the results of FIG. 3 are shown in FIG.

[0075] By subtracting the negative control, K. pneumoniae JCM1662T When the strain was used as a template, almost no fluorescence was observed on substrate 1, whereas significant fluorescence was observed on substrate 2.

[0076] From the above, it is expected that by using the primers of SEQ ID NOs: 7, 8, 10, and 11 and the probes of SEQ ID NOs: 9 and 12, it will be possible to distinguish between K. variicola and K. pneumoniae, which were previously impossible to distinguish between.

[0077] Example 3: Detection on a substrate using DNA probes targeting five clinical specimens (blood culture fluid)

[0078] Nucleic Acid Amplification and Detection In the culture identification method, specimens 1 and 2 identified as K. pneumoniae, specimen 3 identified as K. aerogenes, specimen 4 identified as K. oxytoca, and specimen 5 identified as K. planticola were subjected to PCR amplification, hybridization reaction on a substrate, labeling, and fluorescence measurement using the primer set of SEQ ID NOs: 7, 8, 10, and 11, with genomic DNA extracted according to the standard protocol attached to the reagent as a template, using the QIAamp BiOstic Bacteremia DNA Kit (QIAGEN). At this time, primers of SEQ ID NOs: 8 and 11 modified with biotin at the 5' end via a linker were used.

[0079] For amplification, TaKaRa Multiplex Assay Kit Ver.2 (Takara Bio) was used. Each primer was added to a final concentration of 0.2 μM, and template genomic DNA was added to 10 μg / L. 1× 2× Reaction buffer and 1U Multiplex Enzyme Mix from the TaKaRa Multiplex Assay Kit Ver.2 were used to mix with template DNA and other components to prepare a PCR reaction solution. A T100 Thermal Cycler (Bio-Rad) was used as the nucleic acid amplifier. After the amplification reaction was completed, a thermal denaturation reaction was performed using the same device before subjecting the mixture to a hybridization reaction on the substrate.

[0080] The reagents used for the hybridization reaction on the substrate were a mixture of a solution containing substrate 1 on which the probe of sequence number 9 was immobilized and substrate 2 on which the probe of sequence number 12 was immobilized, saline-sodium-phosphate-EDTA buffer (SSPE-buffer), and a PCR reaction solution that had been thermally denatured.

[0081] Streptavidin-Phycoerythrin (PlexBio) was used as a reagent for labeling. The hybridization reaction and labeling on the substrate were performed using an IntelliPlex 1000 πCode Processor (PlexBio). The fluorescence measurement device used was a PlexBio (registered trademark) 100 Fluorescent Analayzer (PlexBio).

[0082] The reaction conditions used were as follows: Amplification reaction conditions 94℃: 30 seconds 60℃: 60 seconds -> 94℃: 30 seconds (30 cycles) 72℃: 600 seconds

[0083] Heat denaturation conditions 95℃: 5 minutes -> Rapid cooling to 4℃

[0084] Hybridization conditions Incubate at 37℃ for 20 minutes

[0085] Labeling conditions Incubate at 37℃ for 10 minutes

[0086] The measurement results are shown in Figure 5. In Example 2, since the background value of Substrate 1 was high, the value in Figure 5 was subtracted from the value when water was used as a template as a negative control.

[0087] When sample 1 was used as a template, fluorescence was observed on substrate 1, and when sample 2 was used as a template, fluorescence was observed on substrate 2. From this, in view of the results of Example 1, it was suggested that sample 1 may be K. variiicola. In verification 1 described later, whole genome analysis using a next-generation sequencer and bacterial species identification by ANI were performed on sample 1, and as a result, it was found that sample 1 was classified as K. variiicola. In addition to Example 1, it was revealed that by using the primer set of SEQ ID NOs: 7, 8, 10, and 11 and the probes of SEQ ID NOs: 9 and 12, it is possible to distinguish between K. variiicola and K. pneumoniae, which could not be distinguished in the past.

[0088] Furthermore, since no fluorescence was observed from either substrate for samples 3, 4, and 5, it was found that using the primer sets of sequence numbers 7, 8, 10, and 11 and the probes of sequence numbers 9 and 12 would not erroneously detect other Klebsiella bacteria, such as K. aerogenes, K. oxytoca, and K. planticola.

[0089] Verification 1: Whole genome analysis of sample 1 using a next-generation sequencer and bacterial species identification by ANI The DDH (DNA-DNA Hybridization) method has traditionally been used to identify bacterial species using gene sequence information, but this has now been replaced by the ANI (Average nucleotide Identity) method as the standard method. ANI is a method for identifying bacterial species based on the calculation results of which reference sequence registered in a database has the highest sequence similarity to the sequence of the entire genome being analyzed.

[0090] i) Picking bacteria from isolated colonies and extracting genomes Bacteria were picked from isolated colonies and treated with phenol / chloroform / isoamyl alcohol (Nihon Genetics). The supernatant was then used to extract the genome using a FastGene Gel / PCR Extraction Kit (Nihon Genetics) according to the standard protocol provided with the reagent.

[0091] ii) Library Preparation Libraries were prepared using the QIAseq FX DNA Library kit (QIAGEN) according to the standard protocol provided with the reagent.

[0092] iii) Sequencing reaction Using the MiSeq Reagent kit v3 600 cycles, 300 bp x 2 paired-end sequencing reactions were performed using a MiSeq (Illumina) instrument in accordance with the standard protocol provided with the reagent.

[0093] iiii) Bacterial species identification by ANI The raw data obtained from the instrument were trimmed using Trimmomatic version 0.38, and the whole genome was sequenced using SPAdes version 3.13.0 for de novo assembly. The database used was the NCBI RefSeq from the National Center for Biotechnology Information, and species identification was performed using ANI with BBTools.

[0094] Sample 1 showed the highest similarity (ANI Value: 99.15%) with K. variiicola in the database, followed by K. pneumoniae (ANI Value: 95.89%). In general, the threshold for determining that the species is the same in species identification by ANI is 95 to 96% (Kim, M, HS Oh, SC Park, J Chun. 2014. Towards a taxonomic coherence between average gyrBleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes. Int J Syst Evol Microbiol 64: 346-351.), and sample 1 was identified as K. variiicola based on this threshold. From the above, the validity of the results of Example 2 was supported by the whole genome analysis and species identification by ANI.

[0095] On-substrate detection using DNA probes targeting genomic DNA of K. variicola and K. pneumoniae

[0096] Primer and probe design The results of multiple alignment of parts of the gyrB genes of K. variicola strains DSM15968, MGH20, and X39 (SEQ ID NOs: 1, 2, and 3) shown in FIG. 1 are shown as SEQ ID NOs: 7 to 9; the results of multiple alignment of parts of the gyrB genes of K. variicola strains DSM15968, MGH20, and X39 (SEQ ID NOs: 1, 2, and 3) shown in FIG. 6 are shown as SEQ ID NOs: 13 to 15; the results of multiple alignment of parts of the gyrB genes of K. variicola strains DSM15968, MGH20, and X39 (SEQ ID NOs: 1, 2, and 3) shown in FIG. 7 are shown as SEQ ID NOs: 16 to 18; the results of multiple alignment of parts of the gyrB genes of K. variicola strains DSM15968, MGH20, and X39 (SEQ ID NOs: 1, 2, and 3) shown in FIG. Primers and probes of SEQ ID NOs: 19 to 21 were designed based on the multiple alignment results of parts of the gyrB genes (SEQ ID NOs: 1, 2, and 3) of K. variicola strains DSM15968, MGH20, and X39, SEQ ID NOs: 22 to 24 were designed based on the multiple alignment results of parts of the gyrB genes (SEQ ID NOs: 1, 2, and 3) of K. variicola strains DSM15968, MGH20, and X39 shown in Figure 9, and primers and probes of SEQ ID NOs: 25 to 27 were designed based on the multiple alignment results of parts of the gyrB genes (SEQ ID NOs: 1, 2, and 3) of K. variicola strains DSM15968, MGH20, and X39 shown in Figure 10. These primers and probes were designated as assay sets 1 to 6, respectively.

[0097] [Table 2]

[0098] Nucleic Acid Amplification and Detection K. variicola JCM12419, K. pneumoniae JCM1662 T Using genomic DNA extracted from the strain as a template, PCR amplification, hybridization reaction on a substrate, labeling, and fluorescence measurement were performed using a primer set of SEQ ID NOs: 7, 8, 10, 11, 13, 14, 10, 11, 16, 17, 10, 11, 19, 20, 10, 11, 22, 23, 10, 11, or 25, 26, 10, 11. Primers of SEQ ID NOs: 8, 11, 14, 17, 20, 23, and 26 were modified with biotin via a linker at the 5' end.

[0099] For amplification, TaKaRa Multiplex Assay Kit Ver.2 (Takara Bio) was used. Each primer was added to a final concentration of 0.2 μM, and template genomic DNA was added to 10 μg / L. 1× 2× Reaction buffer and 1U Multiplex Enzyme Mix from the TaKaRa Multiplex Assay Kit Ver.2 were used to mix with template DNA and other components to prepare a PCR reaction solution. A T100 Thermal Cycler (Bio-Rad) was used as the nucleic acid amplifier. After the amplification reaction was completed, a thermal denaturation reaction was performed using the same device before subjecting the mixture to a hybridization reaction on the substrate.

[0100] The reagents used for the hybridization reaction on the substrate were a mixture of a solution containing substrate 1 on which the probe of SEQ ID NO:9 was immobilized, substrate 3 on which the probe of SEQ ID NO:15 was immobilized, substrate 4 on which the probe of SEQ ID NO:18 was immobilized, substrate 5 on which the probe of SEQ ID NO:21 was immobilized, and substrate 6 on which the probe of SEQ ID NO:24 was immobilized, saline-sodium-phosphate-EDTA buffer (SSPE-buffer), and a PCR reaction solution that had been thermally denatured.

[0101] Streptavidin-Phycoerythrin (PlexBio) was used as a reagent for labeling. The hybridization reaction and labeling on the substrate were performed using an IntelliPlex 1000 πCode Processor (PlexBio). The fluorescence measurement device used was a PlexBio (registered trademark) 100 Fluorescent Analayzer (PlexBio).

[0102] The reaction conditions used were as follows: Amplification reaction conditions 94℃: 30 seconds 60℃: 60 seconds -> 94℃: 30 seconds (30 cycles) 72℃: 600 seconds

[0103] Heat denaturation conditions 95℃: 5 minutes -> Rapid cooling to 4℃

[0104] Hybridization conditions Incubate at 37℃ for 20 minutes

[0105] Labeling conditions Incubate at 37℃ for 10 minutes

[0106] The measurement results are shown in FIG. 11. In addition, the fluorescence value when K. variicola JCM12419 strain was used as a template was used as a signal, and the fluorescence value when K. pneumoniae JCM1662 T Table 3 shows the signal / noise ratios calculated for each assay set, with the fluorescence value when the strain was used as a template being taken as noise.

[0107] [Table 3]

[0108] The higher the signal / noise value, the higher the discrimination ability of the assay set, and from this, it was found that the assay set with the highest discrimination ability was assay set 1, followed by assay set 5 and thirdly assay set 3. The closer the signal / noise value is to 1, the lower the discrimination ability of the assay set, or the assay set cannot be discriminated at all, and it was found that assay sets 2, 4, and 6 had low discrimination ability.

[0109] From the above, it was found that of the assay sets 1 to 6 created in six regions within the gyrB gene, the primers of sequence numbers 7 and 8 and the probe of sequence number 9 in assay set 1 have the highest ability to distinguish between K. variicola and K. pneumoniae.

Claims

1. A primer set for detecting the presence of one or more Klebsiella bacteria selected from the group consisting of Klebsiella variicola and Klebsiella pneumoniae in a sample, comprising: a first primer and a second primer targeting the gyrB gene of a Klebsiella bacterium; In the case of detecting Klebsiella variicola, the first primer and the second primer have the nucleotide sequences set forth in SEQ ID NOs: 7 and 8, respectively; A primer set for detecting Klebsiella pneumoniae, wherein a first primer and a second primer have the nucleotide sequences set forth in SEQ ID NOs: 10 and 11, respectively.

2. A primer set for detecting the presence of Klebsiella variicola, the primer set comprising a first primer having a base sequence set forth in SEQ ID NO: 7 and a second primer having a base sequence set forth in SEQ ID NO:

8.

3. The primer set according to claim 2, further comprising a third primer and a fourth primer targeting the gyrB gene of Klebsiella pneumoniae, for detecting the presence of Klebsiella variicola.

4. The primer set according to claim 3 , wherein the third primer has the base sequence set forth in SEQ ID NO:

10.

5. The primer set according to claim 3 or 4, wherein the fourth primer has the base sequence set forth in SEQ ID NO:

11.

6. A primer set for detecting the presence of Klebsiella pneumoniae, the primer set comprising: a first primer having a nucleotide sequence set forth in SEQ ID NO: 10; and a second primer having a nucleotide sequence set forth in SEQ ID NO:

11.

7. The primer set according to claim 6 , further comprising a third primer and a fourth primer targeting the gyrB gene of Klebsiella variicola.

8. The primer set according to claim 7 , wherein the third primer has the base sequence set forth in SEQ ID NO:

7.

9. The primer set according to claim 7 or 8, wherein the fourth primer has the base sequence set forth in SEQ ID NO:

8.

10. The primer set according to any one of claims 1 to 9, wherein the primers are labeled with biotin.

11. A probe for detecting the presence of one or more Klebsiella bacteria selected from the group consisting of Klebsiella variicola and Klebsiella pneumoniae in a sample, comprising: The probe targets the gyrB gene of Klebsiella sp. In the case of detecting Klebsiella variicola, the probe has the nucleotide sequence set forth in SEQ ID NO:9, A probe for detecting Klebsiella pneumoniae, the probe having the nucleotide sequence set forth in SEQ ID NO:

12.

12. A kit comprising the primer set according to any one of claims 1 to 10 and the probe according to claim 11.

13. A method for detecting the presence of Klebsiella variikola in a sample, comprising using a primer set according to any one of claims 2 to 5.

14. The method according to claim 13, comprising a step of distinguishing Klebsiella variicola from other Klebsiella bacteria using the first and second primers.

15. The method according to claim 13 or 14, further comprising the use of a probe according to claim 11.

16. The Klebsiella genus bacteria that can be differentiated from Klebsiella variicola include Klebsiella pneumoniae, Klebsiella michiganensis, Klebsiella quasipneumoniae, Klebsiella aerogenes, Klebsiella planticola, Klebsiella granulomatis, Klebsiella grimonti, Klebsiella pneumoniae ... The method according to any one of claims 13 to 15, wherein the bacterium is one or more selected from the group consisting of: Bacillus subtilis; Bacillus niger; and Klebsiella oxytoca.

17. The method according to any one of claims 13 to 16, wherein the Klebsiella bacterium to be differentiated from Klebsiella variicola is Klebsiella pneumoniae, and a third primer and a fourth primer and / or a probe targeting the gyrB gene of Klebsiella pneumoniae are further used.

18. A method for detecting the presence of Klebsiella pneumoniae in a sample, comprising using the primer set according to any one of claims 6 to 9.

19. The method according to claim 18, comprising a step of distinguishing Klebsiella pneumoniae from other Klebsiella bacteria using the first and second primers.

20. 20. The method of claim 19, further comprising the use of a probe according to claim 11.

21. The method according to claim 19 or 20, wherein the Klebsiella bacterium to be identified as Klebsiella pneumoniae is one or more species selected from the group consisting of Klebsiella variicola; Klebsiella michiganensis; Klebsiella cassipneumoniae; Klebsiella aerogenes; Klebsiella planticola; Klebsiella granulomatis; Klebsiella grimonti; and Klebsiella oxytoca.

22. The method according to any one of claims 19 to 21, further comprising using a third primer and a fourth primer and / or a probe targeting the gyrB gene of Klebsiella variicola, wherein the Klebsiella bacterium to be differentiated from Klebsiella pneumoniae is Klebsiella variicola.

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