WO2014178401A1 - DETECTION METHOD FOR CARBAPENEM-BASED DRUG-RESISTANT BACTERIUM (KPC β-LACTAMASES PRODUCING BACTERIUM) - Google Patents

DETECTION METHOD FOR CARBAPENEM-BASED DRUG-RESISTANT BACTERIUM (KPC β-LACTAMASES PRODUCING BACTERIUM) Download PDF

Info

Publication number
WO2014178401A1
WO2014178401A1 PCT/JP2014/061980 JP2014061980W WO2014178401A1 WO 2014178401 A1 WO2014178401 A1 WO 2014178401A1 JP 2014061980 W JP2014061980 W JP 2014061980W WO 2014178401 A1 WO2014178401 A1 WO 2014178401A1
Authority
WO
WIPO (PCT)
Prior art keywords
kpc
seq
lactamase
lamp
bacterium
Prior art date
Application number
PCT/JP2014/061980
Other languages
French (fr)
Japanese (ja)
Inventor
竜一 中野
康雄 斧
Original Assignee
学校法人帝京大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 学校法人帝京大学 filed Critical 学校法人帝京大学
Priority to JP2015514866A priority Critical patent/JP6358753B2/en
Publication of WO2014178401A1 publication Critical patent/WO2014178401A1/en

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism

Definitions

  • the present invention relates to a method for detecting a KPC ⁇ -lactamase-producing bacterium that is a carbapenem-resistant bacterium causing a hospital infection or the like.
  • KPC Klebsiella pneumoniae carbapenemase
  • Non-patent Document 3 KPC-producing bacteria have recently appeared in various parts of the world, and are particularly prevalent in the United States, Puerto Rico, Colombia, Greece, Israel, and China (Non-Patent Documents 2 and 4). It has been reported in many European countries and South Africa, and relatively few in Asian countries other than China and Africa. In Taiwan, Japan, etc., it has been reported that most of the carbapenem-resistant bacteria are IMP-type ⁇ -lactamase producing bacteria (Non-patent Documents 2 and 5).
  • Non-Patent Documents 6 and 7 The fatality rate due to infection with KPC-producing bacteria ranges from 27.5% to 57% (Non-Patent Documents 6 and 7). Such high fatality rates are due in part to inappropriate and inefficient antibacterial treatment.
  • the Detection of carbapenem-resistant bacteria using current susceptibility testing methods is often ineffective because it is ineffective (Non-patent Document 8).
  • the detection method of KPC-producing bacteria is limited to Hodge test and the like improved from the sensitivity test method (Non-patent Document 9). Because it is different from the detection method for other carbapenemases such as IMP type ⁇ -lactamase (Class B) and OXA-48 type ⁇ -lactamase (Class D), there are cases where missed KPC-producing bacteria may be mistreated.
  • KPC ⁇ -lactamase gene (KPC-2 ⁇ -lactamase gene) was identified from several species of Gram-negative bacilli including Enterobacteriaceae (Non-patent Document 10), and KPC ⁇ -lactamase targeting this gene Detection of producing bacteria is being studied.
  • KPC-3 to KPC-17 mutants having structural mutations have been reported all over the world in the KPC ⁇ -lactamase gene (Non-patent Documents 11-13), and all types of KPC ⁇ -lactamase are reported. No means has been found for rapidly detecting producers.
  • LAMP (loop-mediated isothermal amplification) method is known as a DNA amplification method in a short time (Patent Document 1, Non-Patent Documents 14-16).
  • This LAMP method is a method for amplifying a target DNA sequence under isothermal conditions (60 to 69 ° C., 30 to 60 minutes), and has the advantage that the temperature control that is essential for the PCR method is unnecessary. Moreover, it has high specificity and sensitivity when amplifying the target DNA sequence, and the result can be determined even with the naked eye (Non-patent Document 17).
  • Non-patent Document 18 Although the PCR method is known to be suppressed by serum and plasma heparin (Non-patent Document 18), the LAMP method is not affected by these; LAMP The sensitivity of the method is not affected by the presence of non-target DNA (Non-Patent Document 13); it does not require DNA purification from the sample (Non-Patent Document 19).
  • detection of ⁇ -lactamase gene by LAMP method detection of IMP type ⁇ -lactamase gene (Patent Document 2) and detection of NDM type ⁇ -lactamase gene (Non-Patent Document 20) are known.
  • nucleic acid microarrays to perform molecular epidemiology and detect novel ⁇ -lactamases: a snapshot of extended-spectrum ⁇ -lactamases throughout the world.
  • J Clin Microbiol 50, 1632-1639 Rapp, R. P. & Urban, C. (2012). Klebsiella pneumoniae carbapenemases in Enterobacteriaceae: history, evolution, and microbiology concerns.
  • Tzouvelekis, L. S. Markogiannakis, A., Psichogiou, M., Tassios, P. T. & Daikos, G. L. (2012).
  • Loop-mediated isothermal amplification technology towards point of care diagnostics.
  • loop-mediated isothermal amplification of the IS900 sequence for rapid detection of cultured Mycobacterium avium subsp. Paratuberculosis. J Clin Microbiol 41, 4359-4365. Kaneko, H., Kawana, T., Fukushima E. & T. Suzutani (2007). Tolerance of loop-mediated isothermal amplification to a culture medium and biological substances. J Biochem Biophys Methods 70, 499-501.
  • KPC ⁇ -lactamase producing bacteria differ from IMP ⁇ -lactamase producing bacteria and NDM ⁇ -lactamase producing bacteria in the mechanism of acquiring drug resistance, the epidemic pattern, and the coping method. In order to accurately monitor, prevent and appropriately take the appearance of drug-resistant bacteria, it is necessary to accurately and quickly screen each different drug-resistant bacterium. Establishing a means of detecting KPC ⁇ -lactamase producing bacteria, whose epidemic is regarded as a problem, is an important issue.
  • KPC ⁇ -lactamase genes there are at least 16 types of KPC ⁇ -lactamase genes, and the LAMP method targeting a single KPC ⁇ -lactamase gene cannot detect KPC ⁇ -lactamase-producing bacteria having other genes.
  • the present invention has been made in view of the circumstances as described above, and is a method for accurately and rapidly detecting a KPC ⁇ -lactamase-producing bacterium that has not been established so far.
  • the present invention is a method for detecting Klebsiella pneumoniae carbapenemase (KPC) ⁇ -lactamase producing bacteria as a means for solving the above-mentioned problem, wherein LAMP (loop-mediated isothermal amplification using bacterial DNA contained in a sample as a template) )
  • LAMP loop-mediated isothermal amplification using bacterial DNA contained in a sample as a template
  • the nucleotide sequence of SEQ ID NO: 1 is amplified by the method, and when the nucleotide sequence is amplified, it is determined in 15 to 40 minutes that the bacteria contained in the sample are KPC ⁇ -lactamase producing bacteria.
  • the bacteria in the sample are Gram-negative bacilli including Enterobacteriaceae
  • the detected KPC ⁇ -lactamase producing bacterium is any ⁇ -lactamase producing bacterium from KPC-2 to KPC-17 This is a preferred embodiment.
  • the primer used in the LAMP method is a synthetic oligonucleotide consisting of each nucleotide sequence of SEQ ID NO: 2-5, or a synthetic oligonucleotide consisting of each nucleotide sequence of SEQ ID NO: 2-6. It is another preferred embodiment.
  • the primers used in the LAMP method are synthetic oligonucleotides consisting of the nucleotide sequences of SEQ ID NOs: 7, 8, 4 and 5, SEQ ID NOs: 7, 8, 4, 5 and SEQ ID NO: 9 or 10, or SEQ ID NO: 7 , 8, 4, 5, 9 and 10 are synthetic oligonucleotides each having a nucleotide sequence.
  • the present invention also provides a primer set for detecting a KPC ⁇ -lactamase-producing bacterium by the LAMP method, comprising a synthetic oligonucleotide consisting of each nucleotide sequence of SEQ ID NOs: 2-5, or SEQ ID NO: 2-
  • a primer set comprising a synthetic oligonucleotide consisting of 6 respective nucleotide sequences is provided.
  • This primer set is also a primer set comprising a synthetic oligonucleotide consisting of each nucleotide sequence of SEQ ID NO: 7, 8, 4 and 5, or SEQ ID NO: 7, 8, 4, 5 and SEQ ID NO: 9 or 10, or a sequence It may be a primer set including a synthetic oligonucleotide consisting of the nucleotide sequences of Nos. 7, 8, 4, 5, 9 and 10.
  • the present invention provides a detection kit for KPC ⁇ -lactamase-producing bacteria comprising the primer set, DNA polymerase and LAMP reaction solution.
  • a KPC ⁇ -lactamase-producing bacterium expressing any one of at least 16 different KPC ⁇ -lactamase genes can be detected quickly (15 to 40 minutes) and accurately.
  • the LAMP method shows a highly sensitive characteristic indicating a detection limit of 10 0 CFU / ml even for a sample whose detection limit is 10 4 CFU / ml in the PCR method.
  • the present invention contributes to the rapid selection of appropriate antibiotic treatments in clinical settings and to avoid the selection of inappropriate antibiotics.
  • the short-time detection of KPC-producing bacteria contributes to the promotion of epidemiological studies of KPC-producing bacteria and the monitoring system for their appearance.
  • Example 1 it is the LAMP primer used for the detection of the KPC ⁇ -lactamase gene.
  • (a) The nucleotide sequence of the homologous region of the KPC ⁇ -lactamase gene used for designing the LAMP primer. The sequence of the LAMP primer is indicated by an arrow. The numbers on the right are the number of nucleotides when A of the start codon (ATG) of each gene is 1.
  • (b) Five primer sequences used for the LAMP reaction. F1c and B2c sequences represent sequences complementary to the F1 and B2 regions. The result of having examined the optimal temperature in the LAMP reaction for the rapid detection of a KPC (beta) -lactamase gene in Example 1.
  • the purified plasmid DNA of Klebsiella pneumoniae ATCC BAA-1705 strain was used as a template for LAMP reaction.
  • the LAMP reaction was assessed at a temperature range of 60 ° C. to 69 ° C. for 50 minutes.
  • Lane M is 100-bp stepwise marker; Lanes 1-8 are 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 1 , 10 for each reaction of template DNA purified from Klebsiella pneumoniae ATCC BAA-1705 strain. 0 and 0 CFU are shown, respectively.
  • Example 1 the measurement result of the detection sensitivity of KPC beta-lactamase gene by LAMP method is measured. Amplification was performed at 68 ° C. for 50 minutes. DNA was extracted from serially diluted culture (10 6 to 10 0 CFU) of Klebsiella pneumoniae ATCC BAA-1705 strain and used as a template for LAMP reaction. The curve shows the decreasing concentration of bacteria (from 10 6 to 10 0 CFU per reaction) from left to right. The result of having measured the sensitivity of the direct detection from the sample by LAMP method in Example 1. FIG. Amplification was performed at 68 ° C. for 50 minutes.
  • Klebsiella pneumoniae ATCC serial dilutions of BAA-1705 strain (10 7, 10 5, 10 3, 10 1, 10 0 CFU / ml) , respectively from the specimen was added was used to extract DNA directly.
  • the results for sputum (a), urine (b), stool (c), and blood (d) are shown.
  • FIG. Klebsiella pneumoniae ATCC serial dilutions of BAA-1705 strain (10 7 ⁇ 10 0, 0 CFU / ml) , respectively from the specimen was added was used to extract DNA directly.
  • the results for sputum (a), urine (b), stool (c), and blood (d) are shown.
  • (A) is the result of measuring the detection sensitivity of the KPC ⁇ -lactamase gene by the LAMP method in Example 2. Amplification was performed at 68 ° C. for 50 minutes. DNA was extracted from serially diluted culture (10 6 to 10 0 CFU) of Klebsiella pneumoniae ATCC BAA-1705 strain and used as a template for LAMP reaction. The curve shows the decreasing concentration of bacteria (from 10 6 to 10 0 CFU per reaction) from left to right.
  • (B) shows six primer sequences used in the LAMP reaction. F1c and B2c sequences are complementary to F1 and B2 regions
  • the method for detecting a KPC ⁇ -lactamase-producing bacterium comprises amplifying the nucleotide sequence of SEQ ID NO: 1 with LAMP using bacterial DNA contained in a sample as a template, and when this nucleotide sequence is amplified, It is determined that the bacteria contained are KPC ⁇ -lactamase producing bacteria.
  • KPC-2 GenBank / AY034847
  • KCP-3 AB557734
  • KPC-4 FJ473382
  • KPC-5 EU400222
  • KPC-6 EU555534
  • KPC-7 EU729727
  • KPC-8 FJ234412
  • KPC-9 FJ624872
  • KPC-10 GQ140348
  • KPC-11 HM066995
  • KPC-12 HQ641421
  • KPC-13 HQ342890
  • KPC -14 JX524191
  • KPC-15 KPC-16
  • KPC-17 KPC-17
  • nucleotide sequence region of SEQ ID NO: 1 is a homologous region common to all these KPC ⁇ -lactamase genes, and completed the present invention. That is, the nucleotide sequence of SEQ ID NO: 1 is the sequence from No. 46 to No. 245 of the KPC-2 ⁇ -lactamase gene (GenBank / AY034847). The nucleotide sequence of SEQ ID NO: 1 is also present in other KPC-3-17 ⁇ ⁇ -lactamase genes.
  • Amplification of the nucleotide sequence region of SEQ ID NO: 1 can be performed according to a known LAMP method (Patent Documents 1 and 2, Non-Patent Documents 14-16). That is, the LAMP method is a method of amplifying a target DNA at a constant temperature ranging from 60 ° C. to 69 ° C. for 30 minutes to 60 minutes using four kinds of primer sets and a DNA polymerase having strand displacement activity. Of the 4 types of primer sets, 2 types of primers are the outer primer F3 that anneals to the 5 'side region (F3) of the target DNA, and the outer primer B3 that anneals to the 3' side region (B3).
  • the types of primers are an inner primer FIP that recognizes two regions (F1, F2) on the 5 ′ side of the target DNA and an inner primer BIP that recognizes two regions (B1, B2) on the 3 ′ side.
  • the sequence of the 5 'side of these inner primers is set to anneal within the complementary strand region synthesized by the extension reaction from the 3' side, and the amplification reaction is self-extension from the stem loop structure generated from this inner primer. It proceeds by repeating the reaction and the strand displacement synthesis reaction from the inner primer newly annealed to the loop part.
  • loop primer (LB) (LF)
  • LB and LF loop primers
  • the loop primers LB and LF are designed for the region of the central portion of the target DNA where the primer does not anneal. By using this loop primer, the reaction time can be shortened.
  • Primer Explorer version V4 software uses the nucleotide sequence of SEQ ID NO: 1 as the target DNA for the primer set used in the LAMP method for detecting KPC ⁇ -lactamase producing bacteria. .html) and other known primer designing means.
  • the present invention provides the following primer set (A) as an example.
  • FIP 5'-AGCCGCCAAAGTCCTGTTCGAG CCACCGCGCTGACCAA- 3 '(F1c + F2): SEQ ID NO: 2
  • BIP 5′-CATCGGTGTGTACGCGAT GAGCTGCACAGTGGGAAG -3 ′
  • B1 + B2c SEQ ID NO: 3
  • F3 5′-CGCTGGCTGGCTTTTCTG-3 ′: SEQ ID NO: 4
  • primers correspond to each region (F3-1, LB, B1-3) of the homologous region (SEQ ID NO: 1) of 16 kinds of KPCK ⁇ -lactamase genes as shown in FIG. 1 (a). Yes.
  • the underlined portion of the FIP primer corresponds to the F2 region
  • the underlined portion of the BIP primer corresponds to the B2c region.
  • a set of four primers FIP, BIP, F3 and B3 may be used.
  • KPC ⁇ -lactamase producing bacteria can also be detected by the following primer set (B).
  • FIP 5′-GGAGCCGCCAAAGTCCTGTTCG CCACCGCGCTGACCAA- 3 ′
  • F1c + F2 SEQ ID NO: 7
  • BIP 5′-ATACCGGCTCAGGCGCAAC GAGCTGCACAGTGGGAAG -3 ′
  • B1 + B2c SEQ ID NO: 8
  • F3 5′-CGCTGGCTGGCTTTTCTG-3 ′: SEQ ID NO: 4
  • LB 5′-TGTAAGTTACCGCGCTGAGG-3 ′: SEQ ID NO: 10
  • the F2 region (underlined part) of the FIP primer (SEQ ID NO: 7) and the B2c region (underlined part) of the BIP primer (SEQ ID NO: 8) have the same sequence as those of the primer set (A) (each underlined part). is there.
  • the F3 primer and the B3 primer are the same as the primer set (A).
  • the F1c region of the FIP primer (sequence 7) is the region of nucleotide numbers 108-129 in FIG.
  • the B1 region of the BIP primer (sequence number 9) is the 149-167 region
  • the LF primer (sequence number 9) is 81 -97 region and LB primer (SEQ ID NO: 10) correspond to 168-187 region, respectively.
  • KPC ⁇ -lactamase-producing bacteria can be detected with a set of 4 primers, FIP, BIP, F3 and B3. However, use an additional LF primer and / or LB primer. Is preferred.
  • the LAMP reaction can be carried out using the above primer set, DNA polymerase (for example, Bst DNA polymerase) and a known LAMP reaction solution using DNA in the sample as a template.
  • the composition of the LAMP reaction solution is, for example, Tris-HCl (pH 8.8) 40 mM; KCl 20 mM; MgSO 4 16 mM; (NH 4 ) 2 SO 4 20 mM; Tween 20 0.2%; Betaine 1.6 M; mM and the like.
  • the present invention provides a kit for detecting a KPC ⁇ -lactamase-producing bacterium comprising such a LAMP reaction solution, the above primer set, and a DNA polymerase.
  • the specimen that is the target for detecting the presence of KPC ⁇ -lactamase-producing bacteria is a specimen derived from a living body of a human or other animal suspected of having a bacterial infection, such as a patient catheter wiped specimen, sputum, bronchoalveolar lavage fluid.
  • These specimens can be used in an unpurified state, but can also be used after pretreatment such as separation, extraction, concentration, and purification.
  • tissue cell-derived protein can be decomposed with a proteolytic enzyme and then purified by extraction with phenol and chloroform, or purified using a commercially available extraction kit to increase the purity of DNA.
  • Example 1 Detection of KPC ⁇ -lactamase producing bacteria by LAMP primer set (A) Method (1) Strain K. pneumoniae ATCC BAA-1705 strain producing KPC-2 was purchased from American Type Culture Collection (Manassas, VA, USA) and used as a reference strain. Fifteen strains were used to evaluate the sensitivity of LAMP method to detect KPC ⁇ -lactamase gene. Of these, 4 strains (Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa and Acinetobacter baumannii) producing KPC-2 ⁇ -lactamase were used as positive controls. In addition, 11 strains (E. coli, K. pneumoniae, A. baumannii and P.
  • class A CX-M-2, CTX- M-9, TEM-1 and SHV-1
  • CTX-M-2 CX-M-9, TEM-1 and SHV-1
  • Class B IMP-1, NDM-1 and VIM-2
  • Class C CMY-4, MOX-1 and DHA-1 (Nakano, R., Okamoto, R., Nagano N.
  • plasmid DNA was extracted from K. pneumoniae ATCC BAA-1705 strain using Genopure Plasmid Midi Kit (Roche, Germany). To test the specificity and sensitivity of the LAMP method, bacterial total DNA, including genomic DNA and plasmid DNA, was extracted by heating at 90 ° C. for 10 minutes.
  • the LAMP method was performed using a Loopamp DNA amplification kit (Eiken Chemical Co. Ltd., Tokyo, Japan) in a 25 ⁇ l volume of reaction solution (Non-patent Documents 14 and 15).
  • the composition of the reaction solution is as follows: a set of 5 primers: 2 inner primers (FIP and BIP, 40 pmol each), 2 outer primers (F3 and B3, 5 pmol each) and loop Primer (LB, 20 pmol); 1 ⁇ l Bst DNA polymerase (8 units); 2 ⁇ l template DNA; and 12.5 ⁇ l reaction solution supplied with the kit.
  • the LAMP reaction was performed for 50 minutes under isothermal conditions ranging from 60 ° C to 69 ° C while measuring 650 nm turbidity every 6 seconds under a Loopamp real-time turbidimeter (LoopampEXIA; Eiken Chemical). The reaction was stopped at 80 ° C.
  • LoopampEXIA Loopamp real-time turbidimeter
  • Primer design LAMP primer set candidates are the nucleotide sequences of KPC-2 to KPC-17 ⁇ -lactamase genes (GenBank / AY034847, AB557734, FJ473382, EU400222, EU555534, EU729727, FJ234412, FJ624872, GQ140348, HM066995, Designed using Primer Explorer version V4 software (https://primerexplorer.jp/lamp4.0.0/index.html) based on HQ641421, HQ342890, JX524191, KC433553, KC465199 and KC465200).
  • an appropriate primer designed from the homologous region of the KPC-2 to KPC-17 ⁇ -lactamase gene and capable of amplifying the target gene in the shortest time was selected. Initial standardization and optimization of the LAMP assay was performed under identical reaction conditions.
  • the selected primer set (A) is shown in FIG.
  • the nucleotide sequence of each primer is SEQ ID NO: 2-6.
  • KPC-2 producing K. pneumoniae ATCC BAA-1705 is added to clinical samples (spider, urine, feces, blood) at 10 7 to 10 0 and 0 CFU / ml
  • the LAMP method was tried using as a sample. As a result, it was possible to detect a KPC-2 producing strain from any sample (FIG. 5). Even 10 0 CFU / ml production bacteria were both detectable within 40 minutes.
  • the detection limit was 10 4 CFU / ml. It was found that the direct detection from the specimen by the LAMP method has very high sensitivity and rapidity compared with the PCR method.
  • Example 2 Detection of KPC ⁇ -lactamase-producing bacteria using LAMP primer set (B) According to the same method as in Example 1 except that six primer sets (B) shown in Fig. 7 (lower) were used. The sensitivity of the LAMP method was measured for DNA extracted from the strain. The results are as shown in FIG. 7 (upper row), and it was confirmed that it was possible to detect a bacterial target gene having a concentration of 10 0 CFU even when the primer set (B) was used. Further, clinical samples (sputum, urine, feces, blood) also intended for, it was possible to detect 10 0 CFU concentration of KPC beta-lactamase producing bacteria.
  • the method of the present invention is a rapid, inexpensive and accurate method for detecting KPC- ⁇ -lactamase producing bacteria, and this method can be applied to point-of-care tests or outpatient clinics. Given the horizontal and vertical infectivity of this KPC ⁇ -lactamase producing bacterium, its rapid and accurate detection is an important means of epidemiological investigation and helps to select an appropriate antibiotic treatment And minimizes the spread of these resistance determinants and provides a means to control nosocomial infections.

Abstract

A detection method for carbapenem-based drug-resistant bacterium (KPC β-lactamases producing bacterium), whereby: a SEQ NO. 1 nucleotide sequence is amplified using the loop-mediated isothermal amplification (LAMP) method whereby genome DNA for bacterium included in a specimen is used as a template; and, if the nucleotide sequence has been amplified, whether or not the bacterium contained in the specimen is KPC β-lactamases producing bacterium is determined in 15-40 minutes. Any β-lactamases producing bacterium between KPC-2 and KPC-17 can be targeted. The precision of this method has high-sensitivity characteristics whereby both detection of 100 CFU with high specificity without false positives for other resistant genes, and direct detection from a specimen are possible.

Description

カルバペネム系薬耐性菌（ＫＰＣ　β－ラクタマーゼ産生菌）の検出方法Carbapenem-resistant bacteria (KPC β-lactamase producing bacteria) detection method
　本願発明は、院内感染等の原因となるカルバペネム系薬耐性菌であるKPC β-ラクタマーゼ産生菌を検出する方法に関する。 The present invention relates to a method for detecting a KPC β-lactamase-producing bacterium that is a carbapenem-resistant bacterium causing a hospital infection or the like.
（１）KPC β-ラクタマーゼについて
　カルバペネム系薬（carbapenem）耐性グラム陰性桿菌が世界的規模で報告されている。この耐性は主に、カルバペネマーゼ（carbapenemase）遺伝子の獲得の結果として生じる（非特許文献１、２）。多種類のカルバペネマーゼが同定されているが、これらは3クラスのβ-ラクタマーゼ（β-lactamases）に分類される：アンブラー（Ambler）クラスA、BおよびDのβ-ラクタマーゼである（非特許文献３）。カルバペネマーゼは抗生物質ペニシリン系とセファロスポリン系のみならずカルバペネム系も加水分解して、その抗菌効果を無効にする。特効薬であるカルバペネム系薬まで無効にするため、カルバペネマーゼ産生菌の出現は感染症治療において脅威となっている。 (1) About KPC β-lactamase Carbapenem-resistant gram-negative bacilli have been reported on a global scale. This resistance mainly occurs as a result of acquisition of the carbapenemase gene (Non-Patent Documents 1 and 2). Many types of carbapenemases have been identified, but these are classified into three classes of β-lactamases: Ambler classes A, B and D β-lactamases (Non-patent Document 3) ). Carbapenemase hydrolyzes the carbapenems as well as the antibiotic penicillins and cephalosporins, rendering their antibacterial effects ineffective. The emergence of carbapenemase-producing bacteria is a threat in the treatment of infectious diseases in order to invalidate even the carbapenem series, which is a specific medicine.
　Klebsiella pneumoniae carbapenemase（KPC）は機能性グループf2に属するクラスAのセリンβ-ラクタマーゼである（非特許文献３）。KPC産生菌は近年になって世界各地に出現しており、特にアメリカ合衆国、プエルトリコ、コロンビア、ギリシャ、イスラエルおよび中国で流行している（非特許文献２、４）。多くの欧州諸国および南アフリカでも報告されており、中国以外のアジア諸国やアフリカでは比較的少ない。台湾や日本などでは、カルバペネム系薬耐性菌のほとんどがIMP型β-ラクタマーゼ産生菌であることが報告されている（非特許文献２、５）。KPC産生菌の感染による致死率は27.5％～57％であるが（非特許文献６、７）、このような高い致死率は、一部には不適切、不効率な抗菌治療が原因とされる。現在の感受性試験法を用いたカルバペネム耐性菌の検出では非効果的なために見逃すことがしばしばある（非特許文献８）。
　KPC産生菌の検出法は感受性試験法を改良したHodge testなどに限られている（非特許文献９）。その他のカルバペネマーゼであるIMP型β-ラクタマーゼ（クラスB）やOXA-48型β-ラクタマーゼ（クラスD）などに対する検出法とは異なるため、KPC産生菌を見逃すことで誤った治療を施すことがある。日本など検出率の低い地域ではKPC産生菌を見逃すことで耐性菌の蔓延を招く可能性がある。臨床現場でのKPC産生菌の迅速検出法の開発は感染症治療と感染制御の上で重要である。腸内細菌科を含むグラム陰性桿菌の幾つかの菌種からKPC β-ラクタマーゼ遺伝子（KPC-2 β-ラクタマーゼ遺伝子）が同定され（非特許文献１０）、この遺伝子を標的とするKPC β-ラクタマーゼ産生菌の検出が検討されている。ただし、KPC β-ラクタマーゼ遺伝子には構造的変異を有する変異体（KPC-3～KPC-17）が世界中で報告されており（非特許文献１１－１３）、全ての種類のKPC β-ラクタマーゼ産生菌を迅速に検出する手段は見出されていない。 Klebsiella pneumoniae carbapenemase (KPC) is a class A serine β-lactamase belonging to the functional group f2 (Non-patent Document 3). KPC-producing bacteria have recently appeared in various parts of the world, and are particularly prevalent in the United States, Puerto Rico, Colombia, Greece, Israel, and China (Non-Patent Documents 2 and 4). It has been reported in many European countries and South Africa, and relatively few in Asian countries other than China and Africa. In Taiwan, Japan, etc., it has been reported that most of the carbapenem-resistant bacteria are IMP-type β-lactamase producing bacteria (Non-patent Documents 2 and 5). The fatality rate due to infection with KPC-producing bacteria ranges from 27.5% to 57% (Non-Patent Documents 6 and 7). Such high fatality rates are due in part to inappropriate and inefficient antibacterial treatment. The Detection of carbapenem-resistant bacteria using current susceptibility testing methods is often ineffective because it is ineffective (Non-patent Document 8).
The detection method of KPC-producing bacteria is limited to Hodge test and the like improved from the sensitivity test method (Non-patent Document 9). Because it is different from the detection method for other carbapenemases such as IMP type β-lactamase (Class B) and OXA-48 type β-lactamase (Class D), there are cases where missed KPC-producing bacteria may be mistreated. . In areas with low detection rates, such as Japan, missing KPC-producing bacteria can lead to the spread of resistant bacteria. Development of a rapid detection method for KPC-producing bacteria in clinical settings is important for infectious disease treatment and infection control. KPC β-lactamase gene (KPC-2 β-lactamase gene) was identified from several species of Gram-negative bacilli including Enterobacteriaceae (Non-patent Document 10), and KPC β-lactamase targeting this gene Detection of producing bacteria is being studied. However, mutants having structural mutations (KPC-3 to KPC-17) have been reported all over the world in the KPC β-lactamase gene (Non-patent Documents 11-13), and all types of KPC β-lactamase are reported. No means has been found for rapidly detecting producers.
（２）LAMP法について
　短時間でのDNA増幅法としてLAMP（loop-mediated isothermal amplification）法が知られている（特許文献１、非特許文献１４－１６）。このLAMP法は等温条件下（60～69℃、30～60分間）で標的DNA配列を増幅する方法であり、PCR法では不可欠な温度制御が不要であるという利点を有している。また、標的DNA配列の増幅に際して高い特異性と感度を有しており、結果は裸眼でも判定可能である（非特許文献１７）。さらに、以下のような利点をも有している：PCR法は血清および血漿のへパリンによって抑制されることが知られているが（非特許文献１８）、LAMP法はこれらによって影響されない；LAMP法の感度は非標的DNAの存在によって影響を受けない（非特許文献１３）；サンプルからのDNA精製を必要としない（非特許文献１９）。LAMP法によるβ-ラクタマーゼ遺伝子の検出としては、IMP型β-ラクタマーゼ遺伝子の検出（特許文献２）や、NDM型 β-ラクタマーゼ遺伝子の検出（非特許文献２０）が知られている。 (2) LAMP method LAMP (loop-mediated isothermal amplification) method is known as a DNA amplification method in a short time (Patent Document 1, Non-Patent Documents 14-16). This LAMP method is a method for amplifying a target DNA sequence under isothermal conditions (60 to 69 ° C., 30 to 60 minutes), and has the advantage that the temperature control that is essential for the PCR method is unnecessary. Moreover, it has high specificity and sensitivity when amplifying the target DNA sequence, and the result can be determined even with the naked eye (Non-patent Document 17). In addition, it has the following advantages: Although the PCR method is known to be suppressed by serum and plasma heparin (Non-patent Document 18), the LAMP method is not affected by these; LAMP The sensitivity of the method is not affected by the presence of non-target DNA (Non-Patent Document 13); it does not require DNA purification from the sample (Non-Patent Document 19). As detection of β-lactamase gene by LAMP method, detection of IMP type β-lactamase gene (Patent Document 2) and detection of NDM type β-lactamase gene (Non-Patent Document 20) are known.
国際公開第00/28082号パンフレットInternational Publication No. 00/28082 Pamphlet 特開2007-195421号公報JP 2007-195421 A
　種々のβ-ラクタマーゼ遺伝子が知られており、また細菌の特定遺伝子を検出する手段としてLAMP法は優れた方法である。実際に、IMP型β-ラクタマーゼ遺伝子やNDM 型β-ラクタマーゼ遺伝子をLAMP法によって検出する技術が知られている（非特許文献２、２０）。しかしながら、KPC β-ラクタマーゼ産生菌は、IMP型β-ラクタマーゼ産生菌やNDM 型β-ラクタマーゼ産生菌とは薬剤耐性の獲得機序やその流行パターン、対処方法などが異なる。薬剤耐性菌の出現を正確に監視し、予防し、適切な処置を行うためには、個々に異なる薬剤耐性菌をそれぞれ正確かつ迅速にスクリーニングすることが求められる。その流行が問題視されているKPC β-ラクタマーゼ産生菌の検出手段の確立は重要な課題である。 Various β-lactamase genes are known, and the LAMP method is an excellent method for detecting bacterial specific genes. Actually, a technique for detecting an IMP-type β-lactamase gene and an NDM-type β-lactamase gene by the LAMP method is known (Non-patent Documents 2 and 20). However, KPC β-lactamase producing bacteria differ from IMP β-lactamase producing bacteria and NDM β-lactamase producing bacteria in the mechanism of acquiring drug resistance, the epidemic pattern, and the coping method. In order to accurately monitor, prevent and appropriately take the appearance of drug-resistant bacteria, it is necessary to accurately and quickly screen each different drug-resistant bacterium. Establishing a means of detecting KPC β-lactamase producing bacteria, whose epidemic is regarded as a problem, is an important issue.
　また、KPC β-ラクタマーゼ遺伝子は少なくとも16種が存在し、単一のKPC β-ラクタマーゼ遺伝子を標的としたLAMP法では他の遺伝子を有するKPC β-ラクタマーゼ産生菌を検出することができない。 In addition, there are at least 16 types of KPC β-lactamase genes, and the LAMP method targeting a single KPC β-lactamase gene cannot detect KPC β-lactamase-producing bacteria having other genes.
　本願発明は、以上のとおりの事情に鑑みてなされたものであり、これまで確立されていなかったKPC β-ラクタマーゼ産生菌を正確かつ迅速に検出する方法である。特に、少なくとも16種のKPC β-ラクタマーゼ遺伝子のいずれを有する菌体であっても正確かつ迅速にそれを検出することのできる方法を提供することを課題としている。 The present invention has been made in view of the circumstances as described above, and is a method for accurately and rapidly detecting a KPC β-lactamase-producing bacterium that has not been established so far. In particular, it is an object of the present invention to provide a method capable of accurately and rapidly detecting a microbial cell having any of at least 16 types of KPC β-lactamase genes.
　本願発明は、前記の課題を解決する手段として、Klebsiella pneumoniae carbapenemase（KPC） β-ラクタマーゼ産生菌の検出方法であって、検体中に含まれる細菌のDNAを鋳型とするLAMP（loop-mediated isothermal amplification）法によって配列番号１のヌクレオチド配列を増幅し、前記ヌクレオチド配列が増幅された場合に、検体中に含まれる細菌がKPC β-ラクタマーゼ産生菌であることを15分から40分で判定することを特徴とする方法を提供する。 The present invention is a method for detecting Klebsiella pneumoniae carbapenemase (KPC) β-lactamase producing bacteria as a means for solving the above-mentioned problem, wherein LAMP (loop-mediated isothermal amplification using bacterial DNA contained in a sample as a template) ) The nucleotide sequence of SEQ ID NO: 1 is amplified by the method, and when the nucleotide sequence is amplified, it is determined in 15 to 40 minutes that the bacteria contained in the sample are KPC β-lactamase producing bacteria. To provide a method.
　この方法においては、検体中の細菌が腸内細菌科を含むグラム陰性桿菌であり、検出されるKPC β-ラクタマーゼ産生菌がKPC-2からKPC-17のいずれかのβ-ラクタマーゼ産生菌であることを好ましい態様としている。 In this method, the bacteria in the sample are Gram-negative bacilli including Enterobacteriaceae, and the detected KPC β-lactamase producing bacterium is any β-lactamase producing bacterium from KPC-2 to KPC-17 This is a preferred embodiment.
　またこの方法においては、LAMP法に使用するプライマーが、配列番号２－５のそれぞれのヌクレオチド配列からなる合成オリゴヌクレオチドであるか、または配列番号２－６のそれぞれのヌクレオチド配列からなる合成オリゴヌクレオチドであることを別の好ましい態様としている。あるいは、LAMP法に使用するプライマーが、配列番号７、８、４および５のそれぞれのヌクレオチド配列からなる合成オリゴヌクレオチド、配列番号７、８、４、５と配列番号９または１０、もしくは配列番号７、８、４、５、９および１０のそれぞれのヌクレオチド配列からなる合成オリゴヌクレオチドであることを好ましい態様としている。 In this method, the primer used in the LAMP method is a synthetic oligonucleotide consisting of each nucleotide sequence of SEQ ID NO: 2-5, or a synthetic oligonucleotide consisting of each nucleotide sequence of SEQ ID NO: 2-6. It is another preferred embodiment. Alternatively, the primers used in the LAMP method are synthetic oligonucleotides consisting of the nucleotide sequences of SEQ ID NOs: 7, 8, 4 and 5, SEQ ID NOs: 7, 8, 4, 5 and SEQ ID NO: 9 or 10, or SEQ ID NO: 7 , 8, 4, 5, 9 and 10 are synthetic oligonucleotides each having a nucleotide sequence.
　また本願発明は、KPC β-ラクタマーゼ産生菌をLAMP法で検出するためのプライマーセットであって、配列番号２－５のそれぞれのヌクレオチド配列からなる合成オリゴヌクレオチドを含むプライマーセット、または配列番号２－６のそれぞれのヌクレオチド配列からなる合成オリゴヌクレオチドを含むプライマーセットを提供する。このプライマーセットは、また、配列番号７、８、４および５のそれぞれのヌクレオチド配列からなる合成オリゴヌクレオチドを含むプライマーセット、あるいは配列番号７、８、４、５と配列番号９または１０、もしくは配列番号７、８、４、５、９および１０のそれぞれのヌクレオチド配列からなる合成オリゴヌクレオチドを含むプライマーセットであってもよい。 The present invention also provides a primer set for detecting a KPC β-lactamase-producing bacterium by the LAMP method, comprising a synthetic oligonucleotide consisting of each nucleotide sequence of SEQ ID NOs: 2-5, or SEQ ID NO: 2- A primer set comprising a synthetic oligonucleotide consisting of 6 respective nucleotide sequences is provided. This primer set is also a primer set comprising a synthetic oligonucleotide consisting of each nucleotide sequence of SEQ ID NO: 7, 8, 4 and 5, or SEQ ID NO: 7, 8, 4, 5 and SEQ ID NO: 9 or 10, or a sequence It may be a primer set including a synthetic oligonucleotide consisting of the nucleotide sequences of Nos. 7, 8, 4, 5, 9 and 10.
　さらに本願発明は、前記のプライマーセット、DNAポリメラーゼおよびLAMP反応溶液を含むKPC β-ラクタマーゼ産生菌の検出キットを提供する。 Furthermore, the present invention provides a detection kit for KPC β-lactamase-producing bacteria comprising the primer set, DNA polymerase and LAMP reaction solution.
　本願発明によって、少なくとも16種の異なったKPC β-ラクタマーゼ遺伝子のいずれかを発現するKPC β-ラクタマーゼ産生菌を迅速（15分から40分）かつ正確に検出することが可能となる。PCR法では104CFU/mlが検出限界である検体に対しても、LAMP法では100 CFU/mlの検出限界を示す高感度な特性を示す。本願発明は、臨床場面において適切な抗生物質治療を迅速に選択し、不適切な抗生物質の選択を回避することに貢献する。さらに、KPC産生菌の短時間の検出は、KPC産生菌の疫学的な研究の促進やその出現の監視体制に貢献する。 According to the present invention, a KPC β-lactamase-producing bacterium expressing any one of at least 16 different KPC β-lactamase genes can be detected quickly (15 to 40 minutes) and accurately. The LAMP method shows a highly sensitive characteristic indicating a detection limit of 10 0 CFU / ml even for a sample whose detection limit is 10 4 CFU / ml in the PCR method. The present invention contributes to the rapid selection of appropriate antibiotic treatments in clinical settings and to avoid the selection of inappropriate antibiotics. In addition, the short-time detection of KPC-producing bacteria contributes to the promotion of epidemiological studies of KPC-producing bacteria and the monitoring system for their appearance.
実施例１において、KPC β-ラクタマーゼ遺伝子の検出に用いたLAMPプライマーである。(a)LAMPプライマーの設計に使用したKPC β-ラクタマーゼ遺伝子の相同領域のヌクレオチド配列。LAMPプライマーの配列は矢印で示した。右側の数字は各遺伝子の開始コドン（ATG）のAを１とした時のヌクレオチド数である。(b)LAMP反応に使用した5個のプライマー配列。F1cおよびB2c配列はF1およびB2領域に相補的な配列を示す。In Example 1, it is the LAMP primer used for the detection of the KPC β-lactamase gene. (a) The nucleotide sequence of the homologous region of the KPC β-lactamase gene used for designing the LAMP primer. The sequence of the LAMP primer is indicated by an arrow. The numbers on the right are the number of nucleotides when A of the start codon (ATG) of each gene is 1. (b) Five primer sequences used for the LAMP reaction. F1c and B2c sequences represent sequences complementary to the F1 and B2 regions. 実施例１において、KPC β-ラクタマーゼ遺伝子の迅速検出のためのLAMP反応における至適温度を検討した結果。Klebsiella pneumoniae ATCC BAA-1705株の精製プラスミドDNAをLAMP反応の鋳型として用いた。LAMP反応は、60℃から69℃の温度範囲、50分間で査定した。The result of having examined the optimal temperature in the LAMP reaction for the rapid detection of a KPC (beta) -lactamase gene in Example 1. FIG. The purified plasmid DNA of Klebsiella pneumoniae ATCC BAA-1705 strain was used as a template for LAMP reaction. The LAMP reaction was assessed at a temperature range of 60 ° C. to 69 ° C. for 50 minutes. 実施例１において、KPC β-ラクタマーゼ遺伝子の（a）LAMP産物および(b)PCR産物を電気泳動分析した結果。レーンMは100-bp段階的マーカー；レーン1-8はKlebsiella pneumoniae ATCC BAA-1705株から精製した鋳型DNAの反応毎に106、105、104、103、102、101、100および0 CFUをそれぞれ示す。In Example 1, (a) LAMP product and (b) PCR product of KPC β-lactamase gene were subjected to electrophoretic analysis. Lane M is 100-bp stepwise marker; Lanes 1-8 are 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 1 , 10 for each reaction of template DNA purified from Klebsiella pneumoniae ATCC BAA-1705 strain. 0 and 0 CFU are shown, respectively. 実施例１において、LAMP法によるKPC β-ラクタマーゼ遺伝子の検出感度を測定した結果。増幅は68℃、50分間で行った。DNAは、Klebsiella pneumoniae ATCC BAA-1705株の段階希釈培養物（106から100 CFU）から抽出し、LAMP反応の鋳型として使用した。カーブは、左から右に細菌の減少濃度（反応毎に106から100 CFU）を示す。In Example 1, the measurement result of the detection sensitivity of KPC beta-lactamase gene by LAMP method is measured. Amplification was performed at 68 ° C. for 50 minutes. DNA was extracted from serially diluted culture (10 6 to 10 0 CFU) of Klebsiella pneumoniae ATCC BAA-1705 strain and used as a template for LAMP reaction. The curve shows the decreasing concentration of bacteria (from 10 6 to 10 0 CFU per reaction) from left to right. 実施例１において、LAMP法による検体からの直接検出の感度を測定した結果。増幅は68℃、50分間で行った。Klebsiella pneumoniae ATCC BAA-1705株の段階希釈（107、105、103、101、100 CFU/ml）を添加した検体よりそれぞれDNAを直接抽出して用いた。喀痰(a)、尿(b)、便(c)、血液(d)に対する結果をそれぞれ示す。The result of having measured the sensitivity of the direct detection from the sample by LAMP method in Example 1. FIG. Amplification was performed at 68 ° C. for 50 minutes. Klebsiella pneumoniae ATCC serial dilutions of BAA-1705 strain (10 7, 10 5, 10 3, 10 1, 10 0 CFU / ml) , respectively from the specimen was added was used to extract DNA directly. The results for sputum (a), urine (b), stool (c), and blood (d) are shown. 実施例１において、PCR法による検体からの直接検出の感度を測定した結果。Klebsiella pneumoniae ATCC BAA-1705株の段階希釈（107～100、0 CFU/ml）を添加した検体よりそれぞれDNAを直接抽出して用いた。喀痰(a)、尿(b)、便(c)、血液(d)に対する結果をそれぞれ示す。7、6、5、4、3、2、1、NCはそれぞれ106、105、104、103、102、101、100、0 CFU/mlを示す。The result of having measured the sensitivity of the direct detection from the sample by PCR method in Example 1. FIG. Klebsiella pneumoniae ATCC serial dilutions of BAA-1705 strain (10 7 ~ 10 0, 0 CFU / ml) , respectively from the specimen was added was used to extract DNA directly. The results for sputum (a), urine (b), stool (c), and blood (d) are shown. 7, 6 , 5 , 4 , 3 , 2 , 1 , and NC represent 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 1 , 10 0 , and 0 CFU / ml, respectively. （a）は、実施例２においてLAMP法によるKPC β-ラクタマーゼ遺伝子の検出感度を測定した結果。増幅は68℃、50分間で行った。DNAは、Klebsiella pneumoniae ATCC BAA-1705株の段階希釈培養物（106から100 CFU）から抽出し、LAMP反応の鋳型として使用した。カーブは、左から右に細菌の減少濃度（反応毎に106から100 CFU）を示す。（b）は、LAMP反応に使用した６個のプライマー配列。F1cおよびB2c配列はF1およびB2領域に相補的な配列を示す(A) is the result of measuring the detection sensitivity of the KPC β-lactamase gene by the LAMP method in Example 2. Amplification was performed at 68 ° C. for 50 minutes. DNA was extracted from serially diluted culture (10 6 to 10 0 CFU) of Klebsiella pneumoniae ATCC BAA-1705 strain and used as a template for LAMP reaction. The curve shows the decreasing concentration of bacteria (from 10 6 to 10 0 CFU per reaction) from left to right. (B) shows six primer sequences used in the LAMP reaction. F1c and B2c sequences are complementary to F1 and B2 regions
　本願発明のKPC β-ラクタマーゼ産生菌検出方法は、検体中に含まれる細菌のDNAを鋳型とするLAMPによって配列番号１のヌクレオチド配列を増幅し、このヌクレオチド配列が増幅された場合に、検体中に含まれる細菌がKPC β-ラクタマーゼ産生菌であると判定する。 The method for detecting a KPC β-lactamase-producing bacterium according to the present invention comprises amplifying the nucleotide sequence of SEQ ID NO: 1 with LAMP using bacterial DNA contained in a sample as a template, and when this nucleotide sequence is amplified, It is determined that the bacteria contained are KPC β-lactamase producing bacteria.
　配列番号１のヌクレオチド配列は、KPC β-ラクタマーゼ遺伝子の16種（KPC-2からKPC-17）の全てに共通して存在する相同領域である。16種のKPC β-ラクタマーゼ遺伝子は公知である： KPC-2（GenBank/AY034847）、KCP-3（AB557734）、KPC-4（FJ473382）、KPC-5（EU400222）、KPC-6（EU555534）、KPC-7（EU729727）、KPC-8（FJ234412）、KPC-9（FJ624872）、KPC-10（GQ140348）、KPC-11（HM066995）、KPC-12（HQ641421）、KPC-13（HQ342890）、KPC-14（JX524191）、KPC-15（KC433553）、KPC-16（KC465199）およびKPC-17（KC465200）。本願発明者らは、配列番号１のヌクレオチド配列領域（図１a）がこれら全てのKPC β-ラクタマーゼ遺伝子に共通する相同領域であることを見出して、本願発明を完成させた。すなわち、配列番号１のヌクレオチド配列は、KPC-2 β-ラクタマーゼ遺伝子（GenBank/AY034847）の46番から245番までの配列である。他のKPC-3～17 β-ラクタマーゼ遺伝子にもこの配列番号１のヌクレオチド配列が存在する。なお、KPC-2～KPC-17とは別の新たなKPC β-ラクタマーゼ遺伝子を保有する新規な細菌が存在するとしても、その遺伝子が配列番号１のヌクレオチド配列を有するものである限り、検出可能である。 The nucleotide sequence of SEQ ID NO: 1 is a homologous region that is common to all 16 types of KPCKβ-lactamase genes (KPC-2 to KPC-17). Sixteen KPC β-lactamase genes are known: KPC-2 (GenBank / AY034847), KCP-3 (AB557734), KPC-4 (FJ473382), KPC-5 (EU400222), KPC-6 (EU555534), KPC-7 (EU729727), KPC-8 (FJ234412), KPC-9 (FJ624872), KPC-10 (GQ140348), KPC-11 (HM066995), KPC-12 (HQ641421), KPC-13 (HQ342890), KPC -14 (JX524191), KPC-15 (KC433553), KPC-16 (KC465199) and KPC-17 (KC465200). The present inventors have found that the nucleotide sequence region of SEQ ID NO: 1 (FIG. 1a) is a homologous region common to all these KPC β-lactamase genes, and completed the present invention. That is, the nucleotide sequence of SEQ ID NO: 1 is the sequence from No. 46 to No. 245 of the KPC-2 β-lactamase gene (GenBank / AY034847). The nucleotide sequence of SEQ ID NO: 1 is also present in other KPC-3-17 ～ β-lactamase genes. In addition, even if there is a new bacterium having a new KPC ラ ク β-lactamase gene different from KPC-2 to KPC-17, it can be detected as long as the gene has the nucleotide sequence of SEQ ID NO: 1. It is.
　この配列番号１のヌクレオチド配列領域の増幅は、公知のLAMP法（特許文献１、２、非特許文献１４－１６）に従って行うことができる。すなわち、LAMP法は４種類のプライマーセットと鎖置換活性を持つDNAポリメラーゼを用いて、30分から60分間、60℃から69℃の範囲の一定温度で標的DNAを増幅する方法である。４種類のプライマーセットのうち、２種類のプライマーは標的DNAの5’側領域（F3）にアニールするアウタープライマーF3と、3’側領域（B3）にアニールするアウタープライマーB3であり、他の2種類のプライマーは、標的DNAの5’側の２つの領域（F1、F2）を認識するインナープライマーFIPと3’側の２つの領域（B1、B2）を認識するインナープライマーBIPである。これらインナープライマーの5’側の配列はその3’側からの伸長反応で合成した相補鎖領域内にアニールするよう設定されており、増幅反応はこのインナープライマーから生成するステムループ構造からの自己伸長反応と、ループ部分に新たにアニールしたインナープライマーからの鎖置換合成反応を繰り返すことによって進行する。 Amplification of the nucleotide sequence region of SEQ ID NO: 1 can be performed according to a known LAMP method ( Patent Documents 1 and 2, Non-Patent Documents 14-16). That is, the LAMP method is a method of amplifying a target DNA at a constant temperature ranging from 60 ° C. to 69 ° C. for 30 minutes to 60 minutes using four kinds of primer sets and a DNA polymerase having strand displacement activity. Of the 4 types of primer sets, 2 types of primers are the outer primer F3 that anneals to the 5 'side region (F3) of the target DNA, and the outer primer B3 that anneals to the 3' side region (B3). The types of primers are an inner primer FIP that recognizes two regions (F1, F2) on the 5 ′ side of the target DNA and an inner primer BIP that recognizes two regions (B1, B2) on the 3 ′ side. The sequence of the 5 'side of these inner primers is set to anneal within the complementary strand region synthesized by the extension reaction from the 3' side, and the amplification reaction is self-extension from the stem loop structure generated from this inner primer. It proceeds by repeating the reaction and the strand displacement synthesis reaction from the inner primer newly annealed to the loop part.
　さらに、これら4種のプライマーに「ループプライマー（LB）（LF）」のいずれか一方または両方を追加して使用することもできる。ループプライマーLB、LFは、標的DNAの中央部分の、前記プライマーがアニールしない領域を対象として設計する。このループプライマーの使用によって反応時間を短縮することができる。 Furthermore, one or both of “loop primer (LB) (LF)” can be added to these four types of primers. The loop primers LB and LF are designed for the region of the central portion of the target DNA where the primer does not anneal. By using this loop primer, the reaction time can be shortened.
　KPC β-ラクタマーゼ産生菌を検出するためのLAMP法に使用するプライマーセットは、配列番号１のヌクレオチド配列を標的DNAとして、Primer Explorer version V4 software (https://primerexplorer.jp/lamp4.0.0/index.html)などの公知のプライマー設計手段を用いて設計することができる。本願発明は、その一例として以下のプライマーセット（Ａ）を提供する。
FIP：5′-AGCCGCCAAAGTCCTGTTCGAGCCACCGCGCTGACCAA-3′(F1c+F2)：配列番号２
BIP：5′-CATCGGTGTGTACGCGATGAGCTGCACAGTGGGAAG-3′(B1+B2c)：配列番号３
F3： 5′-CGCTGGCTGGCTTTTCTG-3′：配列番号４
B3： 5′-CACAGCGGCAGCAAGAA-3′：配列番号５
LB： 5′-GGCGCAACTGTAAGTTACCG-3′：配列番号６ Primer Explorer version V4 software (https://primerexplorer.jp/lamp4.0.0/index) uses the nucleotide sequence of SEQ ID NO: 1 as the target DNA for the primer set used in the LAMP method for detecting KPC β-lactamase producing bacteria. .html) and other known primer designing means. The present invention provides the following primer set (A) as an example.
FIP: 5'-AGCCGCCAAAGTCCTGTTCGAG CCACCGCGCTGACCAA- 3 '(F1c + F2): SEQ ID NO: 2
BIP: 5′-CATCGGTGTGTACGCGAT GAGCTGCACAGTGGGAAG -3 ′ (B1 + B2c): SEQ ID NO: 3
F3: 5′-CGCTGGCTGGCTTTTCTG-3 ′: SEQ ID NO: 4
B3: 5′-CACAGCGGCAGCAAGAA-3 ′: SEQ ID NO: 5
LB: 5′-GGCGCAACTGTAAGTTACCG-3 ′: SEQ ID NO: 6
　これらの各プライマーは、図１(a)に示したとおり、16種のKPC β-ラクタマーゼ遺伝子の相同領域（配列番号１）の各領域（F3-1、LB、B1-3）に対応している。なお、FIPプライマー（配列番号２）の下線部がF2領域に対応し、BIPプライマー（配列番号３）の下線部がB2c領域に対応している。KPC β-ラクタマーゼ産生菌を検出するには、FIP、BIP、F3およびB3の４プライマーのセットでもよいが、反応時間を短縮させるためにはLBプライマーを追加して使用することが好ましい。 These primers correspond to each region (F3-1, LB, B1-3) of the homologous region (SEQ ID NO: 1) of 16 kinds of KPCKβ-lactamase genes as shown in FIG. 1 (a). Yes. The underlined portion of the FIP primer (SEQ ID NO: 2) corresponds to the F2 region, and the underlined portion of the BIP primer (SEQ ID NO: 3) corresponds to the B2c region. In order to detect KPC β-lactamase producing bacteria, a set of four primers FIP, BIP, F3 and B3 may be used. However, in order to shorten the reaction time, it is preferable to additionally use an LB primer.
　あるはまた、以下のプライマーセット（Ｂ）によってもKPC β-ラクタマーゼ産生菌を検出することができる。
FIP：5′-GGAGCCGCCAAAGTCCTGTTCGCCACCGCGCTGACCAA-3′(F1c+F2)：配列番号７
BIP：5′-ATACCGGCTCAGGCGCAACGAGCTGCACAGTGGGAAG-3′(B1+B2c)：配列番号８
F3： 5′-CGCTGGCTGGCTTTTCTG-3′：配列番号４
B3： 5′-CACAGCGGCAGCAAGAA-3′：配列番号５
LF： 5’-ATGGTTCCGCGACGAGG-3’：配列番号９
LB： 5′-TGTAAGTTACCGCGCTGAGG-3′：配列番号１０ Alternatively, KPC β-lactamase producing bacteria can also be detected by the following primer set (B).
FIP: 5′-GGAGCCGCCAAAGTCCTGTTCG CCACCGCGCTGACCAA- 3 ′ (F1c + F2): SEQ ID NO: 7
BIP: 5′-ATACCGGCTCAGGCGCAAC GAGCTGCACAGTGGGAAG -3 ′ (B1 + B2c): SEQ ID NO: 8
F3: 5′-CGCTGGCTGGCTTTTCTG-3 ′: SEQ ID NO: 4
B3: 5′-CACAGCGGCAGCAAGAA-3 ′: SEQ ID NO: 5
LF: 5′-ATGGTTCCGCGACGAGG-3 ′: SEQ ID NO: 9
LB: 5′-TGTAAGTTACCGCGCTGAGG-3 ′: SEQ ID NO: 10
　FIPプライマー（配列番号７）のF2領域（下線部）とBIPプライマー（配列番号８）のB2c領域（下線部）は、それぞれ前記のプライマーセット（Ａ）のそれら（各下線部）と同一配列である。F3プライマーおよびB3プライマーは前記プライマーセット（Ａ）と同一である。FIPプライマー（配列７）のF1c領域は、図１(a)のヌクレオチド番号108-129の領域、BIPプライマー（配列番号９）のB1領域は149-167領域、LFプライマー（配列番号９）は81-97領域、LBプライマー（配列番号１０）は168-187領域に、それぞれ対応している。このプライマーセット（Ｂ）においても、FIP、BIP、F3およびB3の４プライマーのセットでKPC β-ラクタマーゼ産生菌を検出することができるが、LFプライマーおよび／またはLBプライマーを追加して使用することが好ましい。 The F2 region (underlined part) of the FIP primer (SEQ ID NO: 7) and the B2c region (underlined part) of the BIP primer (SEQ ID NO: 8) have the same sequence as those of the primer set (A) (each underlined part). is there. The F3 primer and the B3 primer are the same as the primer set (A). The F1c region of the FIP primer (sequence 7) is the region of nucleotide numbers 108-129 in FIG. 1 (a), the B1 region of the BIP primer (sequence number 9) is the 149-167 region, and the LF primer (sequence number 9) is 81 -97 region and LB primer (SEQ ID NO: 10) correspond to 168-187 region, respectively. In this primer set (B), KPC β-lactamase-producing bacteria can be detected with a set of 4 primers, FIP, BIP, F3 and B3. However, use an additional LF primer and / or LB primer. Is preferred.
　LAMP反応は、検体中のDNAを鋳型として、前記のプライマーセット、DNAポリメラーゼ（例えばBst DNA ポリメラーゼ）および公知のLAMP反応溶液を用いて行うことができる。LAMP反応溶液の組成は、例えば、Tris－HCl（pH8.8） 40 mM；KCl 20 mM；MgSO4 16 mM；（NH4）2SO4 20 mM；Tween20 0.2 ％；Betaine 1.6 M；dNTPs 各2.8 mMなどとすることができる。本願発明は、このようなLAMP反応溶液と、前記のプライマーセットおよびDNAポリメラーゼを含むKPC β-ラクタマーゼ産生菌検出キットを提供する。 The LAMP reaction can be carried out using the above primer set, DNA polymerase (for example, Bst DNA polymerase) and a known LAMP reaction solution using DNA in the sample as a template. The composition of the LAMP reaction solution is, for example, Tris-HCl (pH 8.8) 40 mM; KCl 20 mM; MgSO 4 16 mM; (NH 4 ) 2 SO 4 20 mM; Tween 20 0.2%; Betaine 1.6 M; mM and the like. The present invention provides a kit for detecting a KPC β-lactamase-producing bacterium comprising such a LAMP reaction solution, the above primer set, and a DNA polymerase.
　また、KPC β-ラクタマーゼ産生菌の存在を検出する対象である検体は、細菌感染が疑われるヒトまたは他の動物の生体由来の検体であり、例えば、患者カテーテル拭き取り検体、喀痰、気管支肺胞洗浄液、鼻汁、鼻腔吸引液、鼻腔洗浄液、鼻腔拭い液、咽頭拭い液、うがい液、唾液、血液、血清、血漿、髄液、尿、糞便、組織等であり、あるいはこれらの検体から分離培養した発育コロニー、培養液等である。これらの検体は、未精製の状態でも使用できるが、さらに分離、抽出、濃縮、精製等の前処理をして使用することもできる。例えば、タンパク質分解酵素等による組織細胞由来タンパク質を分解後、フェノールおよびクロロホルム抽出により精製したり、市販の抽出キットを用いて精製してDNAの純度を高めて使用することができる。 The specimen that is the target for detecting the presence of KPC β-lactamase-producing bacteria is a specimen derived from a living body of a human or other animal suspected of having a bacterial infection, such as a patient catheter wiped specimen, sputum, bronchoalveolar lavage fluid. Nasal discharge, nasal aspirate, nasal wash, nasal wipe, pharyngeal wipe, gargle, saliva, blood, serum, plasma, spinal fluid, urine, stool, tissue, etc., or separated and cultured from these specimens Colonies, culture solutions, etc. These specimens can be used in an unpurified state, but can also be used after pretreatment such as separation, extraction, concentration, and purification. For example, tissue cell-derived protein can be decomposed with a proteolytic enzyme and then purified by extraction with phenol and chloroform, or purified using a commercially available extraction kit to increase the purity of DNA.
　以下、実施例を示して本願発明をさらに詳細かつ具体的に説明するが、本願発明は以下の例に限定されるものではない。 Hereinafter, the present invention will be described in more detail and specifically with reference to examples, but the present invention is not limited to the following examples.
実施例１：LAMPプライマーセット（Ａ）によるKPC β-ラクタマーゼ産生菌の検出
１．方法
（1）菌株
KPC-2を産生するK. pneumoniae ATCC BAA-1705株をAmerican Type Culture Collection (Manassas, VA, USA)から購入し、参考株として用いた。LAMP法のKPC β-ラクタマーゼ遺伝子検出感度を評価するため15の菌株を用いた。これらのうち、KPC-2 β-ラクタマーゼを産生する４菌株（Escherichia coli、Salmonella enterica、Pseudomonas aeruginosaおよび Acinetobacter baumannii）をポジティブコントロールとして用いた。また、以下の各β-ラクタマーゼを産生する11種の菌株（E. coli、K. pneumoniae、A. baumanniiおよびP. aeruginosa）をネガティブコントロールとして用いた：クラスA（CTX-M-2、CTX-M-9、TEM-1およびSHV-1）（Nakano, R., Nakano, A., Abe, M., Inoue M. & Okamoto R. (2012). Regional outbreak of CTX-M-2 β-lactamase-producing Proteus mirabilis in Japan. J Med Microbiol 61(Pt 12), 1727-1735）、クラスB（IMP-1、NDM-1およびVIM-2）（非特許文献３、table of contents）、クラスC（CMY-4、MOX-1およびDHA-1）（Nakano, R., Okamoto, R., Nagano N. & Inoue M. (2007). Resistance to gram-negative organisms due to high-level expression of plasmid-encoded ampC β-lactamase blaCMY-4 promoted by insertion sequence ISEcp1. J Infect Chemother 13, 18-23）、およびクラスD（OXA-48）（Nagano, N., Endoh, Y., Nagano, Y., Toyama, M., Matsui, M., Shibayama K. & Arakawa Y. (2013). First Report of OXA-48 Carbapenemase-Producing Klebsiella pneumoniae and Escherichia coli in Japan from a Patient Returned from Southeast Asia. Jpn J Infect Dis 66, 79-81)。 Example 1: Detection of KPC β-lactamase producing bacteria by LAMP primer set (A) Method (1) Strain
K. pneumoniae ATCC BAA-1705 strain producing KPC-2 was purchased from American Type Culture Collection (Manassas, VA, USA) and used as a reference strain. Fifteen strains were used to evaluate the sensitivity of LAMP method to detect KPC β-lactamase gene. Of these, 4 strains (Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa and Acinetobacter baumannii) producing KPC-2 β-lactamase were used as positive controls. In addition, 11 strains (E. coli, K. pneumoniae, A. baumannii and P. aeruginosa) producing the following β-lactamases were used as negative controls: class A (CTX-M-2, CTX- M-9, TEM-1 and SHV-1) (Nakano, R., Nakano, A., Abe, M., Inoue M. & Okamoto R. (2012). Regional outbreak of CTX-M-2 β-lactamase -producing Proteus mirabilis in Japan. J Med Microbiol 61 (Pt 12), 1727-1735), Class B (IMP-1, NDM-1 and VIM-2) (Non-Patent Document 3, table of contents), Class C ( CMY-4, MOX-1 and DHA-1) (Nakano, R., Okamoto, R., Nagano N. & Inoue M. (2007). Resistance to gram-negative organisms due to high-level expression of plasmid-encoded ampC β-lactamase bla CMY-4 promoted by insertion sequence ISEcp1. J Infect Chemother 13, 18-23) and Class D (OXA-48) (Nagano, N., Endoh, Y., Nagano, Y., Toyama, M., Matsui, M., Shibayama K. & Arakawa Y. (2013). First Report of OXA-48 Carbapenemase-Producing Klebsiella p neumoniae and Escherichia coli in Japan from a Patient Returned from Southeast Asia. Jpn J Infect Dis 66, 79-81).
（2）DNA抽出
　LAMP法の適切な温度を決定するため、Genopure Plasmid Midi Kit (Roche, Germany)を用いてK. pneumoniaeATCC BAA-1705株からプラスミドDNAを抽出した。LAMP法の特異性と感度を検定するため、ゲノムDNAおよびプラスミドDNAを含む細菌のトータルDNAを、90℃で10分間加熱することによって抽出した。 (2) DNA extraction In order to determine an appropriate temperature for the LAMP method, plasmid DNA was extracted from K. pneumoniae ATCC BAA-1705 strain using Genopure Plasmid Midi Kit (Roche, Germany). To test the specificity and sensitivity of the LAMP method, bacterial total DNA, including genomic DNA and plasmid DNA, was extracted by heating at 90 ° C. for 10 minutes.
（3）LAMP法
　LAMP法は、25μl容量の反応溶液中でLoopamp DNA amplification kit (Eiken Chemical Co. Ltd., Tokyo, Japan) を用いて行った（非特許文献１４、１５）。反応溶液の組成は以下のとおりである：５個のプライマーのセット、すなわち２個のインナープライマー（FIPおよびBIP、各40 pmol）、２個のアウタープライマー（F3およびB3、各5 pmol）およびループプライマー（LB、20 pmol）；1 μlのBst DNA ポリメラーゼ（8 units）；2 μlの鋳型DNA；およびキットに付属の12.5 μl 反応溶液。LAMP反応は、Loopamp real-time turbidimeter (LoopampEXIA; Eiken Chemical)のもとで6秒毎に650 nmの濁度計測を行いながら、60℃から69℃の範囲の等温条件で50分間行い、5分間80℃で反応を停止させた。 (3) LAMP method The LAMP method was performed using a Loopamp DNA amplification kit (Eiken Chemical Co. Ltd., Tokyo, Japan) in a 25 μl volume of reaction solution (Non-patent Documents 14 and 15). The composition of the reaction solution is as follows: a set of 5 primers: 2 inner primers (FIP and BIP, 40 pmol each), 2 outer primers (F3 and B3, 5 pmol each) and loop Primer (LB, 20 pmol); 1 μl Bst DNA polymerase (8 units); 2 μl template DNA; and 12.5 μl reaction solution supplied with the kit. The LAMP reaction was performed for 50 minutes under isothermal conditions ranging from 60 ° C to 69 ° C while measuring 650 nm turbidity every 6 seconds under a Loopamp real-time turbidimeter (LoopampEXIA; Eiken Chemical). The reaction was stopped at 80 ° C.
（4）プライマーの設計
　LAMPプライマーセットの候補は、KPC-2～KPC-17 β-ラクタマーゼ遺伝子のヌクレオチド配列（GenBank/AY034847、AB557734、FJ473382、EU400222、EU555534、EU729727、FJ234412、FJ624872、GQ140348、HM066995、HQ641421、HQ342890、JX524191、KC433553、KC465199およびKC465200）に基づき、Primer Explorer version V4 software (https://primerexplorer.jp/lamp4.0.0/index.html)を用いて設計した。３つのプライマーセットの候補うち、KPC-2～KPC-17 β-ラクタマーゼ遺伝子の相同領域から設計され、最短時間で標的遺伝子を増幅することのできる適切なプライマーを選択した。LAMPアッセイの最初の標準化と最適化は同一の反応条件下で行った。選択したプライマーセット（Ａ）を図１に示した。また、各プライマーのヌクレオチド配列はそれぞれ配列番号２－６である。 (4) Primer design LAMP primer set candidates are the nucleotide sequences of KPC-2 to KPC-17 β-lactamase genes (GenBank / AY034847, AB557734, FJ473382, EU400222, EU555534, EU729727, FJ234412, FJ624872, GQ140348, HM066995, Designed using Primer Explorer version V4 software (https://primerexplorer.jp/lamp4.0.0/index.html) based on HQ641421, HQ342890, JX524191, KC433553, KC465199 and KC465200). Among the three primer set candidates, an appropriate primer designed from the homologous region of the KPC-2 to KPC-17 β-lactamase gene and capable of amplifying the target gene in the shortest time was selected. Initial standardization and optimization of the LAMP assay was performed under identical reaction conditions. The selected primer set (A) is shown in FIG. The nucleotide sequence of each primer is SEQ ID NO: 2-6.
（5）LAMPアッセイの感度の決定
　LAMPアッセイによるKPC β-ラクタマーゼ遺伝子検出感度を決定するため、培養したK. pneumoniae ATCC BAA-1705株から抽出したDNAの段階希釈液（106-100CFU per reaction）を用いた。さらに、感度の比較対象として、文献（Poirel, L., Walsh, T. R., Cuvillier V. & Nordmann, P. (2011). Multiplex PCR for detection of acquired carbapenemase genes. Diagn Microbiol Infect Dis 70, 119-123）記載の方法に基づきPCR増幅を実施した。LAMP産物およびPCR産物は1.2％アガロースゲル電気泳動し、エチジウムブロマイドで染色し、UV光の下で可視化した。 (5) Determination of sensitivity of LAMP assay In order to determine the sensitivity of KPC β-lactamase gene detection by LAMP assay, a serial dilution of DNA extracted from cultured K. pneumoniae ATCC BAA-1705 strain (10 6 -10 0 CFU per reaction). Furthermore, as a comparison object of sensitivity, literature (Poirel, L., Walsh, TR, Cuvillier V. & Nordmann, P. (2011). Multiplex PCR for detection of acquired carbapenemase genes. Diagn Microbiol Infect Dis 70, 119-123) PCR amplification was performed based on the method described. LAMP and PCR products were subjected to 1.2% agarose gel electrophoresis, stained with ethidium bromide and visualized under UV light.
（6）検体からの直接検出
　KPC β-ラクタマーゼ産生菌を含む臨床検体より、培地にて産生菌を分離培養することなく直接LAMPアッセイにより検出できる感度を測定した。健常人から提供された検体（喀痰、尿、便、血液）にKPC-2産生K. pneumoniae ATCC BAA-1705を107～100、0 CFU/mlになるように添加したものをサンプルとして用いた。MORA-EXTRACT（極東製薬）を用いて、それぞれの検体からDNAを直接抽出し、LAMP法を試した。比較として同じサンプルを用いてPCR法を行った。 (6) Direct detection from specimens From clinical specimens containing KPC β-lactamase-producing bacteria, the sensitivity that can be directly detected by the LAMP assay was measured without separating the cultured bacteria in the medium. Samples provided from healthy individuals (spider, urine, feces, blood) with KPC-2 producing K. pneumoniae ATCC BAA-1705 added to 10 7 to 10 0 , 0 CFU / ml are used as samples. It was. Using MORA-EXTRACT (Kyokuto Pharmaceutical), DNA was directly extracted from each specimen and the LAMP method was tested. As a comparison, PCR was performed using the same sample.
（7）DNA配列決定
　LAMP反応の確度を確認するため、BigDye Terminator kit（Applied Biosystems）およびDNA Sequencer ABI Prism310（Applied Biosystems）を用いて、メーカーの指示のとおりにLAMP産物の配列決定を行った。 (7) DNA sequencing In order to confirm the accuracy of the LAMP reaction, LAMP products were sequenced according to the manufacturer's instructions using BigDye Terminator kit (Applied Biosystems) and DNA Sequencer ABI Prism310 (Applied Biosystems).
２．結果
　反応混合物を60℃から69℃でインキュベートすることによって、効果的な増幅のための至適温度を特定した（図２）。濁度測定により、増幅のための至適温度は68℃であることが確認された。また、ループプライマー添加による反応効率を確認したところ、初期増幅の時間はループプライマーがない場合は約36分間であるのに対し、有りの場合は約15分間に短縮された。創出したループプライマーが反応速度を加速させるのに適当であることが見いだされた。 2. Results The optimal temperature for effective amplification was identified by incubating the reaction mixture at 60 ° C. to 69 ° C. (FIG. 2). Turbidity measurement confirmed that the optimum temperature for amplification was 68 ° C. When the reaction efficiency by adding the loop primer was confirmed, the initial amplification time was about 36 minutes in the absence of the loop primer, but was shortened to about 15 minutes in the presence of the loop primer. It was found that the created loop primer was suitable for accelerating the reaction rate.
　LAMP法によるKPC β-ラクタマーゼ遺伝子検出の特異性を確認するため、他のタイプのβ-ラクタマーゼを産生する菌株を用いて反応性を査定した。クラスA（CTX-M-2、CTX-M-9、TEM-1およびSHV-1)、クラスB（IMP-1、NDM-1およびVIM-2）、またはクラスD（OXA-48）のβ-ラクタマーゼを産生するE. coli、K. pneumoniae、A. baumanniiまたはP. aeruginosaの11株を対象とし、DNAを抽出して試験した。またネガティブコントロールとして蒸留水を使用した。その結果、いずれの鋳型DNAも増幅されず、KPC β-ラクタマーゼ遺伝子検出におけるLAMP法の特異性が確認された。 In order to confirm the specificity of KPCKβ-lactamase gene detection by the LAMP method, the reactivity was assessed using strains producing other types of β-lactamase. Class A (CTX-M-2, CTX-M-9, TEM-1 and SHV-1), Class B (IMP-1, NDM-1 and VIM-2), or Class D (OXA-48) β -Eleven strains of E. coli, K. pneumoniae, A. baumannii or P. aeruginosa producing lactamase were used for DNA extraction and testing. Distilled water was used as a negative control. As a result, none of the template DNAs were amplified, confirming the specificity of the LAMP method in detecting the KPC β-lactamase gene.
　また、KPC-2 β-ラクタマーゼを産生する4種類のグラム陰性菌（E. coli、S. enterica、P. aeruginosaおよびA. baumannii）を試験した結果、これら全てのDNAはうまく増幅された。さらにこの増幅の特異性は、増幅産物のDNAシークエンス解析により確認した。 In addition, as a result of testing four kinds of gram-negative bacteria (E. coli, S. enterica, P. aeruginosa and A. baumannii) that produce KPC-2 β-lactamase, all these DNAs were successfully amplified. Furthermore, the specificity of this amplification was confirmed by DNA sequence analysis of the amplified product.
　培養したK. pneumoniae ATCC BAA-1705株から抽出したDNAの段階的な10倍希釈液（106-100 CFU per reaction）を用いてLAMP法による増幅を行い、PCRテストの結果と比較することによって、KPC β-ラクタマーゼ遺伝子検出におけるLAMP法の感度を試験した。その結果、LAMP法は100 CFU濃度の細菌の標的遺伝子を検出することが可能であったが（図３、４）、PCR法はその増幅には101 CFU濃度の細菌を必要とした。すなわち、LAMP法はPCRより10倍の感度を有することが確認された。検出限界である100 CFUでさえ、LAMP法ではポジティブコントロール菌株をわずか25分以内で検出することが可能であった（図４）。 To perform amplification by the LAMP method, compared to the results of the PCR test using a staged 10-fold dilutions of the extracted from K. pneumoniae ATCC BAA-1705 strain cultured DNA (10 6 -10 0 CFU per reaction) The sensitivity of the LAMP method in detecting KPC β-lactamase gene was tested. As a result, the LAMP method was able to detect target genes of bacteria having a 10 0 CFU concentration (FIGS. 3 and 4), but the PCR method required bacteria having a 10 1 CFU concentration for amplification. That is, it was confirmed that the LAMP method has a sensitivity 10 times that of PCR. Even the detection limit 10 0 CFU, the LAMP method was able to detect within only 25 minutes to the positive control strain (Figure 4).
　臨床現場での利用を想定し、臨床検体（喀痰、尿、便、血液）にKPC-2産生K. pneumoniae ATCC BAA-1705を107～100 、0 CFU/mlになるように添加したものをサンプルとしてLAMP法を試した。その結果、いずれの検体からもKPC-2産生株を検出することが可能であった（図5）。100 CFU/mlの産生菌であっても、いずれも40分以内に検出可能であった。一方、同じサンプルに対するPCRの感度を測定したところ、検出限界は104　CFU/mlであった。LAMP法による検体からの直接検出では、PCR法と比較して非常に高い感度と迅速性を備わっていることが判った。 Presumed to be used in clinical settings, KPC-2 producing K. pneumoniae ATCC BAA-1705 is added to clinical samples (spider, urine, feces, blood) at 10 7 to 10 0 and 0 CFU / ml The LAMP method was tried using as a sample. As a result, it was possible to detect a KPC-2 producing strain from any sample (FIG. 5). Even 10 0 CFU / ml production bacteria were both detectable within 40 minutes. On the other hand, when the sensitivity of PCR for the same sample was measured, the detection limit was 10 4 CFU / ml. It was found that the direct detection from the specimen by the LAMP method has very high sensitivity and rapidity compared with the PCR method.
実施例２：LAMPプライマーセット（Ｂ）によるKPC β-ラクタマーゼ産生菌の検出
　図７（下段）に示した６個のプライマーセット（Ｂ）を用いたことを除き、実施例１と同様の方法によって菌株から抽出したDNAを対象としてLAMP法の感度を測定した。
　結果は図７（上段）に示したとおりであり、プライマーセット（Ｂ）を用いた場合でも100 CFU濃度の細菌の標的遺伝子を検出することが可能であることが確認された。また、臨床検体（喀痰、尿、便、血液）を対象とした場合も、100CFU濃度のKPC β-ラクタマーゼ産生菌を検出することが可能であった。 Example 2: Detection of KPC β-lactamase-producing bacteria using LAMP primer set (B) According to the same method as in Example 1 except that six primer sets (B) shown in Fig. 7 (lower) were used. The sensitivity of the LAMP method was measured for DNA extracted from the strain.
The results are as shown in FIG. 7 (upper row), and it was confirmed that it was possible to detect a bacterial target gene having a concentration of 10 0 CFU even when the primer set (B) was used. Further, clinical samples (sputum, urine, feces, blood) also intended for, it was possible to detect 10 0 CFU concentration of KPC beta-lactamase producing bacteria.
　本願発明方法は、迅速かつ安価、かつ正確なKPC β-ラクタマーゼ産生菌の検出方法であり、この方法はポイントオブケア（poin-of-care）検査または外来診療場面に適用可能である。このKPC β-ラクタマーゼ産生菌の水平的および垂直的な感染力を考慮すれば、その迅速かつ正確な検出は疫学的な調査の重要な手段であり、適切な抗生物質治療を選択するための手助けであり、これらの耐性決定因子の拡大を最小限に留め、院内感染を制御するための手段となる。 The method of the present invention is a rapid, inexpensive and accurate method for detecting KPC-β-lactamase producing bacteria, and this method can be applied to point-of-care tests or outpatient clinics. Given the horizontal and vertical infectivity of this KPC β-lactamase producing bacterium, its rapid and accurate detection is an important means of epidemiological investigation and helps to select an appropriate antibiotic treatment And minimizes the spread of these resistance determinants and provides a means to control nosocomial infections.

Claims (11)

  1. Klebsiella pneumoniae carbapenemase（KPC） β-ラクタマーゼ産生菌の検出方法であって、検体中に含まれる細菌のDNAを鋳型とするLAMP（loop-mediated isothermal amplification）法によって配列番号１のヌクレオチド配列を増幅し、前記ヌクレオチド配列が増幅された場合に、検体中に含まれる細菌がKPC β-ラクタマーゼ産生菌であることを15分から40分で判定することを特徴とする方法。 A method for detecting Klebsiella pneumoniae carbapenemase (KPC) β-lactamase producing bacteria, wherein the nucleotide sequence of SEQ ID NO: 1 is amplified by a LAMP (loop-mediated isothermal amplification) method using bacterial DNA contained in the sample as a template, A method comprising determining, in 15 to 40 minutes, that a bacterium contained in a specimen is a KPC 検 体 β-lactamase producing bacterium when the nucleotide sequence is amplified.
  2. 検体中の細菌が腸内細菌科を含むグラム陰性桿菌であり、検出されるKPC β-ラクタマーゼ産生菌がKPC-2からKPC-17のいずれかのβ-ラクタマーゼ産生菌である請求項１の方法。 2. The method according to claim 1, wherein the bacterium in the sample is a Gram-negative bacilli containing Enterobacteriaceae, and the detected KPC β-lactamase-producing bacterium is any one of KPC-2 to KPC-17 β-lactamase-producing bacteria. .
  3. LAMP法に使用するプライマーが、配列番号２－５のそれぞれのヌクレオチド配列からなる合成オリゴヌクレオチドである請求項１または２の方法。 The method according to claim 1 or 2, wherein the primer used in the LAMP method is a synthetic oligonucleotide consisting of each nucleotide sequence of SEQ ID NOs: 2-5.
  4. LAMP法に使用するプライマーが、配列番号２－６のそれぞれのヌクレオチド配列からなる合成オリゴヌクレオチドである請求項１または２の方法。 The method according to claim 1 or 2, wherein the primer used in the LAMP method is a synthetic oligonucleotide consisting of each nucleotide sequence of SEQ ID NOs: 2-6.
  5. LAMP法に使用するプライマーが、配列番号７、８、４、５のそれぞれのヌクレオチド配列からなる合成オリゴヌクレオチドである請求項１または２の方法。 The method according to claim 1 or 2, wherein the primer used in the LAMP method is a synthetic oligonucleotide consisting of the nucleotide sequences of SEQ ID NOs: 7, 8, 4, and 5.
  6. LAMP法に使用するプライマーが、配列番号７、８、４および５、または配列番号７、８、４、５と配列番号９または１０、もしくは配列番号７、８、４、５と配列番号９および１０のそれぞれのヌクレオチド配列からなる合成オリゴヌクレオチドである請求項１または２の方法。 Primers used in the LAMP method are SEQ ID NO: 7, 8, 4 and 5, or SEQ ID NO: 7, 8, 4, 5 and SEQ ID NO: 9 or 10, or SEQ ID NO: 7, 8, 4, 5 and SEQ ID NO: 9 and The method according to claim 1 or 2, which is a synthetic oligonucleotide consisting of 10 respective nucleotide sequences.
  7. KPC β-ラクタマーゼ産生菌をLAMP法で検出するためのプライマーセットであって、配列番号２－５のそれぞれのヌクレオチド配列からなる合成オリゴヌクレオチドを含むプライマーセット。 A primer set for detecting a KPC β-lactamase-producing bacterium by the LAMP method, comprising a synthetic oligonucleotide consisting of each nucleotide sequence of SEQ ID NOs: 2-5.
  8. KPC β-ラクタマーゼ産生菌をLAMP法で検出するためのプライマーセットであって、配列番号２－６のそれぞれのヌクレオチド配列からなる合成オリゴヌクレオチドを含むプライマーセット。 A primer set for detecting a KPC β-lactamase producing bacterium by a LAMP method, comprising a synthetic oligonucleotide consisting of each nucleotide sequence of SEQ ID NOs: 2-6.
  9. KPC β-ラクタマーゼ産生菌をLAMP法で検出するためのプライマーセットであって、配列番号７、８、４および５のそれぞれのヌクレオチド配列からなる合成オリゴヌクレオチドを含むプライマーセット。 A primer set for detecting a KPC β-lactamase producing bacterium by a LAMP method, comprising a synthetic oligonucleotide consisting of each nucleotide sequence of SEQ ID NOs: 7, 8, 4 and 5.
  10. KPC β-ラクタマーゼ産生菌をLAMP法で検出するためのプライマーセットであって、配列番号７、８、４、５と配列番号９または１０、もしくは配列番号７、８、４、５、９および１０のそれぞれのヌクレオチド配列からなる合成オリゴヌクレオチドを含むプライマーセット。 A primer set for detecting a KPC β-lactamase producing bacterium by the LAMP method, which is SEQ ID NO: 7, 8, 4, 5 and SEQ ID NO: 9 or 10, or SEQ ID NO: 7, 8, 4, 5, 9 and 10 A primer set comprising a synthetic oligonucleotide consisting of each of the nucleotide sequences.
  11. 請求項７から１０のいずれかに記載のプライマーセット、DNAポリメラーゼおよびLAMP反応混合物を含むKPC β-ラクタマーゼ産生菌の検出キット。 A detection kit for KPC β-lactamase-producing bacteria comprising the primer set according to any one of claims 7 to 10, a DNA polymerase and a LAMP reaction mixture.
PCT/JP2014/061980 2013-05-01 2014-04-30 DETECTION METHOD FOR CARBAPENEM-BASED DRUG-RESISTANT BACTERIUM (KPC β-LACTAMASES PRODUCING BACTERIUM) WO2014178401A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2015514866A JP6358753B2 (en) 2013-05-01 2014-04-30 Method for detecting carbapenem-resistant bacteria (KPCβ-lactamase producing bacteria)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2013-096452 2013-05-01
JP2013096452 2013-05-01

Publications (1)

Publication Number Publication Date
WO2014178401A1 true WO2014178401A1 (en) 2014-11-06

Family

ID=51843525

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2014/061980 WO2014178401A1 (en) 2013-05-01 2014-04-30 DETECTION METHOD FOR CARBAPENEM-BASED DRUG-RESISTANT BACTERIUM (KPC β-LACTAMASES PRODUCING BACTERIUM)

Country Status (2)

Country Link
JP (1) JP6358753B2 (en)
WO (1) WO2014178401A1 (en)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2016189715A (en) * 2015-03-31 2016-11-10 株式会社カネカ Primer for detecting klebsiella pneumoniae, and method
CN106404831A (en) * 2016-12-20 2017-02-15 西北大学 Method for rapidly detecting Beta-lactam antibiotic sensitivity
CN106754906A (en) * 2017-01-11 2017-05-31 博奥生物集团有限公司 LAMP primer composition and its application for detecting seven kinds of drug resistant genes of Carbapenem-resistant class antibiotic
CN107475364A (en) * 2017-06-30 2017-12-15 北京百康芯生物科技有限公司 Drug resistant gene micro-fluidic chip quick detection kit and detection method
CN110129316A (en) * 2018-02-05 2019-08-16 北京智德医学检验所有限公司 It is a kind of for detecting the LAMP primer composition and application of 4 kinds of gramnegative bacteriums in intraocular liquid
WO2020101194A1 (en) * 2018-11-16 2020-05-22 가톨릭대학교산학협력단 Primer set for loop-mediated isothermal amplification reaction for diagnosing carbapenemase-producing enterobacteriaceae, and use thereof
JPWO2021095798A1 (en) * 2019-11-15 2021-11-25 公立大学法人横浜市立大学 Undifferentiated marker gene high-sensitivity detection method
CN114107534A (en) * 2022-01-27 2022-03-01 天津喜诺生物医药有限公司 LAMP primer group for detecting gram-negative bacillus carbapenemase gene and application

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2682299C (en) * 2007-04-06 2015-12-22 Becton, Dickinson And Company Compositions and methods for the identification of a carbapenemase gene
CN104169436A (en) * 2011-12-01 2014-11-26 基因欧姆科技加拿大公司 Molecular assay for the amplification and detection of KPC genes responsible for high-level resistance to carbapenem in gram negative bacteria

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
C. G. GISKE ET AL.: "A sensitive and specific phenotypic assay for detection of metallo-beta- lactamases and KPC in Klebsiella pneumoniae with the use of meropenem disks supplemented with aminophenylboronic acid, dipicolinic acid and cloxacillin", CLINICAL MICROBIOLOGY AND INFECTION, vol. 17, no. 4, 28 June 2010 (2010-06-28), pages 552 - 556, Retrieved from the Internet <URL:http://onlinelibrary.wiley.com/doi/10.1111/j.1469-0691.2010.03294.x/full> *
K. F. ANDERSON ET AL.: "Evaluation of Methods To Identify the Klebsiella pneumoniaeCarbapenemase in Enterobacteriaceae", JOURNAL OF CLINICAL MICROBIOLOGY, vol. 45, no. 8, 20 June 2007 (2007-06-20), pages 2723 - 2725, Retrieved from the Internet <URL:http://jcm.asm.org/content/45/8/2723.full> *
RYUICHI NAKANO ET AL.: "LAMP-ho ni yoru KPC beta- lactamase Sanseikabu Kenshutsuho no Kaihatsu", JOURNAL OF THE JAPANESE ASSOCIATION FOR INFECTIOUS DISEASES, vol. 87, 7 May 2013 (2013-05-07), pages 246 *
SCOTT A. CUNNINGHAM ET AL.: "Rapid and Simultaneous Detection of Genes Encoding Klebsiella pneumoniae Carbapenemase (blaKPC) and New Delhi Metallo-beta-Lactamase (blaNDM) in Gram-Negative Bacilli", JOURNAL OF CLINICAL MICROBIOLOGY, vol. 51, no. 4, 23 January 2013 (2013-01-23), pages 1269 - 1271, Retrieved from the Internet <URL:http://jcm.asm.org/content/51/4/1269.full> *
WEI LIU ET AL.: "Sensitive and Rapid Detection of the New Delhi Metallo-Beta-Lactamase Gene by Loop-Mediated Isothermal Amplification", JOURNAL OF CLINICAL MICROBIOLOGY, vol. 50, no. 5, 22 February 2012 (2012-02-22), pages 1580 - 1585, Retrieved from the Internet <URL:http://jcm.asm.org/content/50/5/1580.full> *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2016189715A (en) * 2015-03-31 2016-11-10 株式会社カネカ Primer for detecting klebsiella pneumoniae, and method
CN106404831A (en) * 2016-12-20 2017-02-15 西北大学 Method for rapidly detecting Beta-lactam antibiotic sensitivity
CN106754906A (en) * 2017-01-11 2017-05-31 博奥生物集团有限公司 LAMP primer composition and its application for detecting seven kinds of drug resistant genes of Carbapenem-resistant class antibiotic
CN107475364A (en) * 2017-06-30 2017-12-15 北京百康芯生物科技有限公司 Drug resistant gene micro-fluidic chip quick detection kit and detection method
CN110129316A (en) * 2018-02-05 2019-08-16 北京智德医学检验所有限公司 It is a kind of for detecting the LAMP primer composition and application of 4 kinds of gramnegative bacteriums in intraocular liquid
WO2020101194A1 (en) * 2018-11-16 2020-05-22 가톨릭대학교산학협력단 Primer set for loop-mediated isothermal amplification reaction for diagnosing carbapenemase-producing enterobacteriaceae, and use thereof
JPWO2021095798A1 (en) * 2019-11-15 2021-11-25 公立大学法人横浜市立大学 Undifferentiated marker gene high-sensitivity detection method
CN114107534A (en) * 2022-01-27 2022-03-01 天津喜诺生物医药有限公司 LAMP primer group for detecting gram-negative bacillus carbapenemase gene and application

Also Published As

Publication number Publication date
JP6358753B2 (en) 2018-07-18
JPWO2014178401A1 (en) 2017-02-23

Similar Documents

Publication Publication Date Title
JP6358753B2 (en) Method for detecting carbapenem-resistant bacteria (KPCβ-lactamase producing bacteria)
JP6258360B2 (en) Methods and kits for detecting antibiotic-resistant bacteria
Nakano et al. Rapid detection of the Klebsiella pneumoniae carbapenemase (KPC) gene by loop-mediated isothermal amplification (LAMP)
CN102899414A (en) Superbacterium gene NDM and KPC duplex fluorescence quantitative polymerase chain reaction (PCR) detection method and kit
JP2014533963A (en) Molecular assay to amplify and detect KPC genes involved in high level resistance to carbapenem in gram-negative bacteria
Soo et al. Rapid and sensitive detection of Acinetobacter baumannii using loop-mediated isothermal amplification
Cheng et al. Rapid detection of bla NDM, bla KPC, bla IMP, and bla VIM carbapenemase genes in bacteria by loop-mediated isothermal amplification
Sakai et al. A novel detection procedure for mutations in the 23S rRNA gene of Mycoplasma pneumoniae with peptide nucleic acid-mediated loop-mediated isothermal amplification assay
Mu et al. Loop-mediated isothermal amplification: Rapid and sensitive detection of the antibiotic resistance gene ISAba1-blaOXA-51-like in Acinetobacter baumannii
Shi et al. WarmStart colorimetric loop-mediated isothermal amplification for the one-tube, contamination-free and visualization detection of Shigella flexneri
EP2753629B1 (en) Methods for detecting lyme disease
US10711316B2 (en) Primers and kits for colony multiplex PCR for the detection of class A, B, C, and D beta-lactamase genes and methods of using thereof
JP5961171B2 (en) Method for detecting toxin-producing Clostridium difficile
WO2012032158A1 (en) Method for detecting the presence of bacterial strains resistant to antibiotics in a biological sample
Dilworth et al. Comparison of polymerase chain reaction for IS6110 and Amplicor in the diagnosis of tuberculosis.
KR101425149B1 (en) Improved method for diagnosing Mycobacterium tuberculosis using one-tube nested real-time PCR
JP2007195421A (en) PRIMER FOR DETECTING METALLO-beta-LACTAMASE GENE, METHOD FOR DETECTING METALLO-beta-LACTAMASE GENE, AND METHOD FOR DETECTING beta-LACTAM MEDICINE-RESISTANT BACTERIUM
Sasano et al. An improved primer design for the loop-mediated isothermal amplification (LAMP) method to detect oxacillinase (OXA)-48 β-lactamase genes in Gram-negative bacteria for clinical applications
EP2300620B1 (en) Lepa / guf1 gene sequences as a diagnostic target for the identification of bacterial species
TIPPARTHI et al. Co-expression of 16S rRNA Methyltransferase and Carbapenemase in Multidrug Resistant Gram Negative Bacteria.
Choi et al. Development of a Two Triplex Real-Time Polymerase Chain Reaction for Rapid Detection of Six Carbapenemase Genes in Enterobacteriaceae
Stewart et al. Using whole-genome sequencing and a pentaplex real-time PCR to characterize third-generation cephalosporin-resistant Enterobacteriaceae from Southeast Queensland, Australia
Armaki et al. Loop-mediated Isothermal Amplification Test for Rapid Identification of Clinical Nocardia Isolates
CN111676303A (en) LAMP primer group and kit for detecting metal beta-lactamase gene blaAFM-1 and application thereof
WILLIAMS 13 PCR and Diagnosis of Tuberculosis

Legal Events

Date Code Title Description
ENP Entry into the national phase

Ref document number: 2015514866

Country of ref document: JP

Kind code of ref document: A

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 14791515

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 14791515

Country of ref document: EP

Kind code of ref document: A1