WO2004063368A1

WO2004063368A1 - Method of detecting drug-resistance gene contained in tubercle bacillus, primer pair set for pcr, primer set for determining base sequence and reagent kit for diagnnosing drug-resistant tuberculosis

Info

Publication number: WO2004063368A1
Application number: PCT/JP2003/016941
Authority: WO
Inventors: Teruo Kirikae; Junichiro Sekiguchi; Takashi Ohtsuki
Original assignee: Japan Science And Technology Agency
Priority date: 2003-01-14
Filing date: 2003-12-26
Publication date: 2004-07-29
Also published as: JP2004215542A

Abstract

It is possible to provide a method of detecting a drug-resistance gene contained in tubercle bacillus which comprises simultaneously PCR-amplifying 2 or more specific regions carried by 2 or more genes selected from a group of specific genes contained in tubercle bacillus with the use of a specific primer pair for PCR and then directly determining the base sequences of 2 or more target regions contained in the 2 or more specific regions thus amplified; a specific primer set for PCR; a primer set for determining a specific base sequence; a method of quickly and conveniently detecting a drug-resistance gene contained in tubercle bacillus by using a diagnostic reagent kit for tuberculosis resistant to a specific drug; and a primer pair set for PCR, a primer set for determining a base sequence and a diagnostic reagent kit which are appropriately usable in the above method.

Description

Description ^: A method for detecting a drug resistance gene contained in Mycobacterium tuberculosis,

Primer pair set for PCR, primer set for nucleotide sequence determination, and reagent kit for diagnosis of drug-resistant tuberculosis

The present invention relates to a method for detecting a drug-resistant gene contained in Mycobacterium tuberculosis, a primer pair set for PCR, a primer set for nucleotide sequence determination, and a reagent kit for diagnosing drug-resistant tuberculosis. Background art

At present, about 3000 people die from tuberculosis annually in Japan and 3 million people worldwide die from tuberculosis annually. Many types of drugs are known for the treatment of tuberculosis, but it is known that when a drug is administered to a patient, a mutation causes, at a certain frequency, a resistant bacterium that is resistant to the administered drug. I'm sorry. The frequency of occurrence of such resistant bacteria varies depending on the drug. For example, isoniazid (INH) is on the order of 10-6, rifampicin (RFP) is on the order of 1 CI- ⁷ , and streptomycin ( O Ichida one SM) is ^10-6, the order one Etanputoru (EB) 10 4. Pyrazinamide (PZA), kanamycin (KM), piomycin (VM), amikacin (AK), and fluoroquinolone (FQ), which were not approved in Japan as antituberculosis drugs, were used in 1996. There is no data on the frequency of appearance. With regard to resistant bacteria that have resistance to these drugs, there have been reports on resistant bacteria caused by mutations in specific genes.o For resistant strains resistant to RFP, it has been shown that point mutations not found in wild-type M. tuberculosis are present in a narrow region on the rpoB gene encoding the RNA polymerase /? / Subunit. I have. As for the resistant bacteria having resistance to INH, the kat G gene, mab A—inhA, which is a gene coding for cod-peroxidase—-t (Cat al ase—perox i das e) Mutations in the gene have been shown to be involved in resistance. With respect to resistant bacteria having resistance to EB, mutations in the embB gene encoding arabinosyltransferase (Arab inosyltransferase) have been shown to be involved in resistance. With regard to resistant bacteria having resistance to PZA, it has been clarified that a mutation in the pncA gene encoding pyrazinamidase is involved in resistance. With respect to resistant bacteria having resistance to SM, mutations in the rpsL gene encoding ribosomal protein S12 (rib0s0ma1protein in S12), and 16S liposome; RNA (16Sribo Mutations in the cluster A region and the class B region on the rrs gene encoding Soma l RNA) have been shown to be involved in resistance. For resistant strains with resistance to KM, VM or AK, mutations in the class C region of the rrs gene encoding 16S ribosomal RNA (16S ribosomal RNA) are involved in resistance It has been revealed. With respect to resistant bacteria having resistance to FQ, it has been clarified that a mutation in the gyr A gene encoding the DNA gyrase 6 A subunit is involved in the resistance. Figure 2 shows the locations of these resistance-related genes on the M. tuberculosis gene. In the show _(in tuberculosis cavity, usually 1 0 ⁸ Ru is the order number of M. tuberculosis is present. If relative tuberculosis patients carrying 10 ⁹ M. tuberculosis When were administered I NH monotherapy , on average, they survive as INH-resistant bacteria 10 ³ or more bacteria resistant to I NH. the tuberculosis patients. administration of INH, but seems to have been once recovered, is surviving I NH resistant bacteria If RFP is used subsequently in the treatment of the affected patient, the INH-resistant bacterium can be re-equipped by a similar mechanism: This repetition will result in the emergence of refractory M. tuberculosis resistant to many drugs, which will delay patient treatment. Not only hospital-acquired infections In order to prevent the emergence of resistant bacteria, tuberculosis treatment requires two or three drugs to prevent the emergence of resistant bacteria. The mainstream of tuberculosis treatment is two-drug therapy or three-drug therapy to be administered When tuberculosis patients receive two or three drugs from the initial treatment stage, resistant frequency is the administration of drops. I NH and RFP to levels may be largely ignored, the frequency of appearance of resistant ^{^{bacteria, 10_ 6 xl 0- 7 = l}} 0- 13 of the order one, in normal TB patients However, it can be considered that almost no resistant bacteria appear, but the tuberculosis bacterium possessed by a tuberculosis patient is already resistant to one or more of the two or three drugs to be administered. If you have two drugs or three May develop resistant bacteria even if the drug is administered o For this reason, if there is information on the resistance of M. tuberculosis bacteria possessed by M. tuberculosis patients to drugs, it is possible to select and administer two or three drugs that M. tuberculosis patients possess without resistance to M. tuberculosis. So that the emergence of new resistant bacteria is almost eliminated. Therefore, drug susceptibility tests for M. tuberculosis have been conducted to obtain information on the resistance of M. tuberculosis bacteria to drugs possessed by M. tuberculosis patients. The drug susceptibility test is generally a method of examining the presence or absence of bacterial growth in a drug-sensitive medium containing a predetermined drug to determine the presence or absence of resistance to the predetermined drug. Examples of the drug susceptibility medium in the drug susceptibility test include Mycobacteria Growth Indicator tube “MGITj (Nippon Becton, manufactured by Dickinson Co., Ltd.), BACTEC 460TB (Nippon Becton, manufactured by Dickinson Co., Ltd.), and tuberculosis susceptible PZA liquid medium ( Far East Pharmaceutical Co., Ltd.), Bit Sector SR (Far East Pharmaceutical Co., Ltd.), Brosmic MTB-I (Far East Pharmaceutical Co., Ltd.), Elpac Media P (Japan BCG Corporation), Elpac Media III, II (Manufactured by Nippon BCG Co., Ltd.), resistant medium (manufactured by Nissui Pharmaceutical Co., Ltd.), and susceptible medium (manufactured by Nissui Pharmaceutical Co., Ltd.) However, Mycobacterium tuberculosis grows slowly and does not grow or not. It takes several weeks to determine the presence or absence of resistance by checking DNA.In addition, recently, a diagnostic method using DNA hybridization technology has been developed. Japanese Patent Application Laid-Open Publication No. 2001-0103981 (Patent Document 1) discloses that wild-type Mycobacterium tuberculosis susceptible to drugs used for the treatment of tuberculosis and any drug are disclosed. Of the resistant and resistant mutant M. tuberculosis strains on the M. tuberculosis genome involved in resistance to drugs used in the treatment of tuberculosis An oligonucleotide synthesized based on the nucleotide sequence of a gene is a tuberculosis diagnostic kit including a substrate immobilized by covalent bonds, and is a tuberculosis bacterium that hybridizes with a nucleic acid derived from a test tubercle bacillus. the drug resistance paragon or by diagnostic kit DNA Haipuridaize one Chillon technology using _c the diagnostic kit which is disclosed for performing determination of M. tuberculosis infection, by DNA Chidzupu, viewed mutation drug resistance gene Although it is about to be released, it requires a large amount of DNA, the test time is 7 hours, but the time for culture is several weeks, the fact that it can only detect mutations in specific gene sequences, Products (DNA microarray kit “01 igo Array”: manufactured by Nisshinbo Industries, Inc.) involve genes involved in resistance to five drugs: INH, RFP, SM, KM, and EB Different only there is a problem such as that can not be detected. In both tuberculosis drug susceptibility tests, the bacteria to be tested need to be cultured for weeks or months to isolate them. ^) Ο Furthermore, PCR is performed by extracting genes from tuberculosis bacteria obtained from sputum, etc. There is a method for detecting genes involved in drug resistance by directly sequencing the amplified product. According to this method, it is not necessary to culture M. tuberculosis in several weeks. However, for each gene involved in resistance: Experiments on PCR amplification and direct nucleotide sequencing are performed and require a great deal of effort, and are not suitable for daily use in laboratories. Accordingly, the present invention provides a method for quickly and easily detecting a drug resistance gene contained in M. tuberculosis, a primer set for PCR, a primer set for nucleotide sequence determination, and a drug which can be suitably used in the method. An object of the present invention is to provide a reagent kit for diagnosing drug-resistant tuberculosis. Disclosure of the invention

Under such circumstances, the present inventors have conducted intensive studies and have found that the genes contained in M. tuberculosis, rP0B, katG, mabA-inhA, embB, pncA, rpsL, rrs, and Two or more specific regions of two or more genes selected from the gene group consisting of gyr A are combined into one unit using two or more pairs of PCR primers corresponding to the two or more specific regions. Amplification of PCR at once by a PCR amplification device, and then direct sequencing of two or more amplified specific regions enables rapid and easy detection of drug-resistant genes contained in M. tuberculosis. And completed the present invention. That is, the present invention (1) comprises a DNA derived from Mycobacterium tuberculosis and rp oB which is a gene contained in the Mycobacterium tuberculosis; kat G, mabA-inhA, embB, pncA, rps L _s rrs, and gyr A Using two or more types of PCR primer pairs corresponding to two or more specific regions of two or more genes selected from a gene group, the two or more specific regions are used to amplify one PCR The PCR is amplified at once by an instrument, and then the two or more target regions are amplified using two or more base sequence determination primers corresponding to two or more target regions included in the two or more specific regions amplified. It is intended to provide a method for detecting a drug resistance gene contained in M. tuberculosis by direct sequencing. By adopting such a configuration, two or more genes selected from the gene group consisting of rpoB ヽ katG, mabA-inhA, embB, pncA, rpsL, rrs, and gyrA, which are genes contained in M. tuberculosis, PCR amplification of two or more specific regions of all genes can be performed easily and quickly, so that drug resistance genes contained in M. tuberculosis can be detected quickly and easily. Out method. Further, the present invention (2) is a DNA derived from Mycobacterium tuberculosis, rp oB is a gene contained in said binding Kakukin, kat G, mabA- inhA, embB , from p ncA, rps L _s rrs, and gyr A Using two or more PCR primer pairs corresponding to two or more specific regions of two or more genes selected from the gene group, the two or more specific regions can be used as one PCR amplification device PCR amplification at a time, and then using the two or more base sequence determination primers corresponding to the two or more target regions included in the amplified two or more specific regions, the two or more target regions The present invention provides a method for detecting drug-resistant genes contained in M. tuberculosis by performing a sequence reaction and determining the nucleotide sequence at a time by using a single sequencing device using the obtained sequence reaction product. Things. By adopting such a configuration, rpoB, katG, mabA-inhA, embB, pncA, which are genes contained in M. tuberculosis: two or more selected from the gene group consisting of rpsL, rrs, and gyrA Since PCR amplification and direct nucleotide sequencing of two or more specific regions of this gene can be performed simply and quickly, a quick and simple method for detecting a drug resistance gene contained in M. tuberculosis can be provided. Further, the present invention (3) provides a method according to the present invention, wherein the two or more PCR primer pairs are a primer pair comprising a primer having the nucleotide sequence of SEQ ID NO: 1 and a primer pair comprising a primer having the nucleotide sequence of SEQ ID NO: 2; A primer pair consisting of a primer containing the base sequence of SEQ ID NO: 34 and a primer pair consisting of a primer containing the base sequence of SEQ ID NO: 3 and a primer containing the base sequence of SEQ ID NO: 4 , A primer pair containing the nucleotide sequence of SEQ ID NO: 5 and a primer pair containing the nucleotide sequence of SEQ ID NO: 6, a primer pair containing the nucleotide sequence of SEQ ID NO: 7, and a primer containing the nucleotide sequence of SEQ ID NO: 8 A primer pair comprising a primer comprising the nucleotide sequence of SEQ ID NO: 9 and a primer pair comprising a primer comprising the nucleotide sequence of SEQ ID NO: 10, a primer comprising the nucleotide sequence of SEQ ID NO: 11 and a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 12 A primer pair containing the nucleotide sequence of SEQ ID NO: 13 and a primer pair consisting of a primer containing the nucleotide sequence of SEQ ID NO: 14 and a primer containing the nucleotide sequence of SEQ ID NO: 15, and SEQ ID NO: 16 A primer pair consisting of a primer pair with a primer containing the base sequence of It is intended to provide a method for detecting a drug resistance gene contained in M. tuberculosis according to the invention (1) or (2), which is at least two types of primer pairs selected from the group A. By adopting such a configuration, in addition to the effects of the invention (1) or (2), rpoB, katG, mabA-inhAsembB, pncA, rpsLrr, which are genes contained in M. tuberculosis, Two or more specific regions possessed by two or more genes selected from the gene group consisting of s and gyrA are sequenced by a conventional direct nucleotide sequence determination method in a gene selected from the aforementioned gene group of M. tuberculosis. This has the effect of being able to amplify as a broader region than the region that is. Further, the present invention (4) provides an oligonucleotide pair comprising the oligonucleotide primer of SEQ ID NO: 1 and the oligonucleotide nucleotide of SEQ ID NO: 2; And an oligonucleotide primer consisting of the oligonucleotide of SEQ ID NO: 34, the oligonucleotide of SEQ ID NO: 3 and the oligonucleotide of SEQ ID NO: 4 An oligonucleotide pair consisting of an oligonucleotide pair consisting of an oligonucleotide of SEQ ID NO: 5 and an oligonucleotide pair consisting of an oligonucleotide of SEQ ID NO: 6; an oligonucleotide pair consisting of an oligonucleotide of SEQ ID NO: Ί and an oligonucleotide of SEQ ID NO: 8; SEQ ID NO: An oligonucleotide pair consisting of the oligonucleotide of SEQ ID NO: 9 and the oligonucleotide of SEQ ID NO: 10; an oligonucleotide pair consisting of the oligonucleotide of SEQ ID NO: 11 and the oligonucleotide of SEQ ID NO: 12; the oligonucleotide pair of SEQ ID NO: 13 and Oligonucleotide pairs consisting of the oligonucleotides of SEQ ID NO: 14 and oligonucleotide pairs consisting of the oligonucleotides of SEQ ID NO: 15 and the oligonucleotides of SEQ ID NO: 16 Is there is provided a method of detecting a drug-resistant gene contained in the M. tuberculosis described in a more Origonukureochidope § the invention (1) or (2). By adopting such a configuration, the same effects as those of the invention (3) can be obtained. Also, the present invention (5) provides a method for detecting a drug resistance gene contained in M. tuberculosis according to any one of the inventions (1) to (4), wherein the PCR amplification is a shuttle PCR amplification. To provide a way to: By adopting such a configuration, in addition to exhibiting the same effects as any of the inventions (1) to (4), in addition to the above, the PCR amplification with PCR primers, and Since the extension of the complementary strand by DNA polymerase can be carried out under the same temperature conditions, the genes contained in M. tuberculosis, rp0B, katG, mabA—inhA, embB, pncA, rpsL, rrs, and gyr An effect is obtained that PCR can be more easily performed on two or more specific regions of two or more genes selected from the gene group consisting of A. In addition, the present invention (6) is characterized in that the two or more types of primers for determining a base sequence include 16 primers including a primer having a base sequence of SEQ ID NO: 17 to a primer having a base sequence of SEQ ID NO: 32 The present invention provides a method for detecting a drug resistance gene contained in M. tuberculosis according to any one of the above inventions (1) to (5), which is at least two types of primers selected from a group consisting of the following primers: By adopting such a configuration, in addition to the effects achieved by any one of the inventions (1) to (5), the genes contained in M. tuberculosis rpB, katG, mabA-iniiA, embB, Two or more specific regions of two or more genes selected from the gene group consisting of pnc A rps L rrs and gyr A are ligated by a conventional direct nucleotide sequencing method to a gene selected from the aforementioned gene group of M. tuberculosis. It can be amplified as a wider region than the region to be sequenced, and has the effect that it is possible to find a novel mutation that was overlooked in the conventional direct base sequencing. In addition, the present invention (7) provides the method according to (2), wherein the two or more primers for determining a base sequence are selected from an oligonucleotide group consisting of 16 oligonucleotides of oligonucleotides of SEQ ID NO: 17 to SEQ ID NO: 32 The present invention also provides a method for detecting a drug resistance gene contained in M. tuberculosis according to any one of the inventions (1) to (5), which is the above oligonucleotide. By adopting such a configuration, the same effect as the invention (6) can be obtained. Further, the present invention (8) provides the Mycobacterium tuberculosis according to any one of the inventions (1) to (7), wherein the direct nucleotide sequencing is performed by the Daiichi-Mine-Ichi-Otsu method. And a method for detecting a drug resistance gene contained in the gene. By adopting such a configuration, in addition to the effects of the invention of any one of the inventions (1) to (7), the sequence of one target region in one tube can be directly determined for nucleotide sequencing. The effect is that the reaction can be performed. Further, the present invention (9) provides an oligonucleotide pair consisting of the oligonucleotide of SEQ ID NO: 1 and the oligonucleotide of SEQ ID NO: 2, an oligonucleotide pair consisting of the oligonucleotide of SEQ ID NO: 33 and the oligonucleotide of SEQ ID NO: 34, An oligonucleotide pair consisting of the oligonucleotide of SEQ ID NO: 3 and the oligonucleotide of SEQ ID NO: 4, an oligonucleotide pair consisting of the oligonucleotide of SEQ ID NO: 5 and the oligonucleotide of SEQ ID NO: 6, the oligonucleotide and the sequence of SEQ ID NO: 7 The oligonucleotide pair consisting of the oligonucleotide No. 8 and the oligonucleotide No. 9 and the oligonucleotide No. 10 and the oligonucleotide No. 11 and the oligonucleotide No. 12 From an oligonucleotide pair consisting of an oligonucleotide of SEQ ID NO: 13, an oligonucleotide pair consisting of the oligonucleotide of SEQ ID NO: 13 and the oligonucleotide of SEQ ID NO: 14, and an oligonucleotide pair consisting of the oligonucleotide of SEQ ID NO: 15 and the oligonucleotide of SEQ ID NO: 16 And a primer pair set for PCR, which is one or more oligonucleotide pairs selected from the group consisting of: Also, the present invention (10) provides a primer comprising the nucleotide sequence of SEQ ID NO: 1 and a primer comprising a primer comprising the nucleotide sequence of SEQ ID NO: 2. A primer pair comprising a primer having the nucleotide sequence of SEQ ID NO: 33 and a primer having the nucleotide sequence of SEQ ID NO: 34, a primer having the nucleotide sequence of SEQ ID NO: 3 and the nucleotide sequence of SEQ ID NO: 4 A primer pair consisting of a primer, a primer containing the nucleotide sequence of SEQ ID NO: 5 and a primer pair consisting of a primer containing the nucleotide sequence of SEQ ID NO: 6, a primer containing the nucleotide sequence of SEQ ID NO: 7, and SEQ ID NO: A primer pair containing a base sequence of SEQ ID NO: 9 and a primer pair consisting of a primer containing a base sequence of SEQ ID NO: 10 and a primer pair consisting of a primer containing a base sequence of SEQ ID NO: 10, and the base sequence of SEQ ID NO: 11 Primer pair consisting of edible primer and primer containing nucleotide sequence of SEQ ID NO: 12, base of SEQ ID NO: 13 A primer pair comprising a sequence-containing primer and a primer comprising the nucleotide sequence of SEQ ID NO: 14, and a primer pair comprising a primer comprising the nucleotide sequence of SEQ ID NO: 15 and a primer comprising the nucleotide sequence of SEQ ID NO: 16 An object of the present invention is to provide a primer set for PCR, which is one or more primer pairs selected from a primer pair group consisting of pairs. Further, the present invention (11) comprises an oligonucleotide of SEQ ID NO: 1 to an oligonucleotide of SEQ ID NO: 16, an oligonucleotide of SEQ ID NO: 33 and an oligonucleotide of 18 of SEQ ID NO: 34 An object of the present invention is to provide a primer pair set for PCR having as a primer one or more kinds of oligonucleotides selected from an oligonucleotide group. Further, the present invention (12) provides a primer comprising the nucleotide sequence of SEQ ID NO: 1 to 16 primers comprising the nucleotide sequence of SEQ ID NO: 16 And a primer pair set for PCR having at least one primer selected from the group consisting of a primer comprising the nucleotide sequence of SEQ ID NO: 33 and a primer comprising the nucleotide sequence of SEQ ID NO: 34 is there. Further, the present invention (13) provides a primer for determining a base sequence, which is one or more oligonucleotides selected from the oligonucleotide group consisting of 16 oligonucleotides of the oligonucleotides of SEQ ID NO: 17 to SEQ ID NO: 32. A set is provided. Also, the present invention (14) provides one or more primers selected from a primer group consisting of 16 primers comprising a primer having the nucleotide sequence of SEQ ID NO: 17 and a primer having the nucleotide sequence of SEQ ID NO: 32 The present invention provides a set of primers for base sequence determination. Further, the present invention (15) provides a primer pair set for PCR described in any one of the above-mentioned inventions (9) to (12) and a nucleotide sequence set forth in the above-mentioned invention (13) or (14). It is intended to provide a reagent kit for diagnosing drug-resistant tuberculosis, comprising a primer set. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows eight PCR products obtained as a result of agarose gel electrophoresis. FIG. 2 is a diagram showing the locations of genes involved in drug resistance on M. tuberculosis genes. BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, embodiments of the present invention will be described, but the present invention is not limited to these embodiments.

Hereinafter, a first method for detecting a drug resistance gene contained in the Mycobacterium tuberculosis of the present invention will be described. Preparation of DNA derived from Mycobacterium tuberculosis can be obtained by extracting DNA from Mycobacterium tuberculosis from a tissue extract such as sputum, cerebrospinal fluid, gastric juice, feces, or lymph nodes. For example, a conventional method can be used to extract DNA from Mycobacterium tuberculosis in sputum, but the method of J. BEIGE et al. (J. Clin. Micro obio l., 33: 90- 95. 1995) or the following extraction method is preferable. That is, 500 μl of sputum was pretreated by the N-acetyl-L-cysteine (NALC) -NaOH method, centrifuged at 12,000 rpm for 15 minutes, Eliminate Sesame. Next, add 10% Chelix 100 resin (Nippon Bio-Rad Laboratories) dissolved in sterile distilled water to the sediment, add Ο Ο ΟμΙ, and vortex, then heat at 45 ° C for 45 minutes. After heating at 100 ° C for 10 minutes, centrifuge again at 12,000 rpm for 15 minutes, and use the supernatant as a sample. <Selection of two or more genes and two or more specific regions>

Also, two or more genes are selected from the gene group consisting of rp0B, katG, mabA-inhAsembBspncA, rpsL, rrs, and gyrA, which are genes included in M. tuberculosis. Then, for each of the two or more selected genes, a specific region for PCR amplification is selected, and two or more specific regions are selected. The entire genomic sequence of Mycroberacterium tuberculosis was reported by Cole, S. T et al. (Nature 393 (6685), p537-544 (1998)), and GeneBank (Accessi on No .: NC-000962). The rpo B gene is equivalent to 759805 base pairs to 765323 base pairs on the genome sequence, the kat G gene is equivalent to 2153887 base pairs to 2 156109 base pairs on the genome sequence, and the mabA — inhA gene Corresponds to 1673438 base pairs to 1675009 base pairs on the genome sequence, the embB gene corresponds to 42465 11 base pairs to 4249807 base pairs on the genome sequence, and the pncA gene corresponds to 2288679 base pairs to 22 89239 base pairs of the rps L gene, which corresponds to 751558 base pairs to 781932 base pairs of the genomic sequence, and the rrs gene corresponds to 1471844 base pairs of the genome sequence. This corresponds to base pair 1473380, and the gyrA gene corresponds to base pairs 7302 to 9818 on the genome sequence. The specific region selected for PCR amplification differs for each gene. The base pair numbers in the specific region of each gene described below are determined by defining the first base pair of the start codon of the sense codon as the first base pair. Regarding the rPoB gene, the specific region is preferably a region containing the 1.276 base pairs to 1356 base pairs, and is a region containing the 1199 base pairs to 1903 base pairs or 1256 base pairs. Most preferably, it is a region containing base pairs to the 1570th base pair. For the kat G gene, the specific region is preferably a region containing the 1st to 2205 base pairs, and most preferably a region consisting of the 1st to 2223 base pairs. For the mabA-inhA gene, the specific region is the primary 81 salt It is preferably a region containing a base pair to the 104th base pair, and most preferably a region consisting of the -217th base pair to the 114th base pair. For the emb B gene, the specific region is preferably a region containing 855 base pairs to 372 base pairs, and a region consisting of 640 base pairs to 3387 base pairs. Is most preferable. For the pncA gene, the specific region is preferably a region containing the 12th to 546th base pairs, more preferably a region consisting of the 80th to 590th base pairs. Most preferred. For the rps L gene, the specific region is preferably a region containing 1115 base pairs to 273 base pairs, and a region consisting of 4 base pairs to 5775 base pairs. Most preferred. For the rrs gene, the specific region is preferably a region containing the 512th to 142nd base pairs, and the region consisting of the 428th to 176th base pairs. Is most preferable. As for the gyrA gene, the specific region is preferably a region containing the 262nd base pair to the 285th base pair, and is preferably a region consisting of the -1st base pair to the 397th base pair. Most preferred. When selecting genes and specific regions corresponding to genes, it is preferable to select three or more genes and three or more specific regions, and more preferably four or more genes and four or more specific regions. More preferably, the above genes and 5 or more specific regions are selected, more preferably, 6 or more genes and 6 or more specific regions are selected, and more preferably, 7 or more genes and 7 or more specific regions are selected. Most preferably, all eight genes and eight specific regions are selected. In previous studies, the mechanism by which M. tuberculosis acquired drug resistance in the eight genes described above was based on relatively restricted codons within the genome gene. It has been clarified that this mechanism is based on the generation of resistance genes to base substitutions (mutations). So far, base substitutions at relatively limited codons in the genomic gene on these generated resistance genes have been demonstrated. Research that focuses on mutations. For example, in the rp0B gene, PCR amplification and direct nucleotide sequencing have been performed on a limited region around codon CAA at position 513 and a limited region around codon TCG at position 513. In kat G, PCR amplification and direct nucleotide sequencing have been performed on a limited region around the 315th codon AGC. In mab A-inhA, PCR amplification and direct nucleotide sequencing have been performed on a limited region around the C base 15 bases upstream of the G base of the start codon. In embB, PCR amplification and direct nucleotide sequencing have been performed on a limited region around the codon CAG at position 497. In pncA, PCR amplification and direct nucleotide sequencing have been performed on a limited region around the 51st codon CAC. In rps L, PCR amplification and direct sequencing have been performed on a restricted region around codon AAG at position 43 and a restricted region around codon AAG at position 88. In rrs, the 5th base C: a limited region around the 516th base C (the region in C 1 usterA for resistance to streptomycin), the 903th base Τ the 906th base A restricted region around base Α (the region in C 1 uster B for resistance to streptomycin) and a restricted region around base 1399 base C to base 1402 base G (kanamycin, piomycin, And the region in C1 uster C for resistance to amikacin) have been subjected to PCR amplification and direct sequencing. In gyrA, PCR amplification and direct amplification of a restricted region around the 95th codon AGC Nucleotide sequencing has been performed. The present invention also focuses on the above-mentioned 8 genes, and performs PCR amplification as a specific region covering the above-described codons or bases in two or more genes selected from these 8 genes. Not only limited codons such as the conventional PCR amplification and direct nucleotide sequencing described above, but also a wide-ranging region containing a region where mutations related to resistance may occur, considering the mechanism of resistance. Area. As a result, it was possible to find new mutations by devising the conditions for direct sequencing. Therefore, it was possible to identify mutations that might have been overlooked.

Next, for each of the two or more selected genes, a PCR primer pair capable of amplifying the selected specific region is determined, and two types corresponding to the two or more specific regions of the two or more genes are determined. Prepare the above primer pair for PCR. The two or more PCR primer pairs are preferably designed so as to be able to amplify two or more specific regions and to perform PCR amplification under the same temperature conditions. For the rp0B gene, the primer pair for PCR was a primer pair containing the nucleotide sequence of SEQ ID NO: 1 and a primer pair consisting of a primer containing the nucleotide sequence of SEQ ID NO: 2 or a primer containing the nucleotide sequence of SEQ ID NO: 33 It is particularly preferable that the primer pair is a primer pair comprising the base sequence of SEQ ID NO: 34 and SEQ ID NO: 34, and the primer pair for PCR is an oligonucleotide pair consisting of the oligonucleotide of SEQ ID NO: 1 and the oligonucleotide of SEQ ID NO: 2, or Arrangement? ] The oligonucleotide pair consisting of the oligonucleotide of No. 33 and the oligonucleotide of SEQ ID No. 34 is most preferable. It is most preferable because it can amplify the preferable region, that is, the 1st to 156th base pairs, and is suitable for shuttle PCR amplification. For the kat G gene, it is particularly preferable that the PCR primer pair is a primer pair comprising a primer containing the nucleotide sequence of SEQ ID NO: 3 and a primer containing the nucleotide sequence of SEQ ID NO: 4, and the primer for PCR is preferably used. Is an oligonucleotide pair consisting of the oligonucleotide of SEQ ID NO: 3 and the oligonucleotide of SEQ ID NO: 4, it is possible to amplify the most preferred region, the first base pair to the second base pair, It is most preferable because it is suitable for shuttle PCR amplification. For the mab A-inh A gene, the primer pair for PCR is particularly preferably a primer pair comprising a primer having the nucleotide sequence of SEQ ID NO: 5 and a primer having the nucleotide sequence of SEQ ID NO: 6, and the primer for p When one pair is an oligonucleotide pair consisting of the oligonucleotide of SEQ ID NO: 5 and the oligonucleotide of SEQ ID NO: 6, the most preferred region, the first 217 base pair to the first 145 base pair, is widened. This is most preferable because it can be performed and is suitable for shuttle PCR amplification. With regard to the emb gene, it is particularly preferable that the PCR primer pair is a primer pair comprising a primer having the nucleotide sequence of SEQ ID NO: 7 and a primer having the nucleotide sequence of SEQ ID NO: 8, and the PCR primer pair is preferably a SEQ ID NO: 7 Oligonucleotide pair consisting of the oligonucleotide of SEQ ID NO: 8 and the oligonucleotide of SEQ ID NO: 8 can amplify the most preferable region, the 640th base pair to the 338th base pair, and the shuttle PCR amplification Most preferred because it is suitable for With regard to the pncA gene, it is particularly preferable that the PCR primer pair is a primer pair comprising a primer having the nucleotide sequence of SEQ ID NO: 9 and a primer having the nucleotide sequence of SEQ ID NO: 10. Is the oligonucleotide of SEQ ID NO: 9 and An oligonucleotide pair consisting of the oligonucleotides of SEQ ID NO: 10 can amplify the most preferred region, the first 80 to 590 base pairs, and is suitable for shuttle PCR amplification. Most preferred. With respect to the rps L gene, it is particularly preferable that the PCR primer pair is a primer pair comprising the nucleotide sequence of SEQ ID NO: 11 and a primer pair comprising a primer having the nucleotide sequence of SEQ ID NO: 12, When one pair is an oligonucleotide pair consisting of the oligonucleotide of SEQ ID NO: 11 and the oligonucleotide of SEQ ID NO: 12, it is possible to amplify the fourth to fifth base pairs, which are the most preferable regions. And most suitable for shuttle PCR amplification. With regard to the rrs gene, it is particularly preferable that the PCR primer pair is a primer pair comprising a primer having the nucleotide sequence of SEQ ID NO: 13 and a primer having the nucleotide sequence of SEQ ID NO: 14; Is the oligonucleotide pair consisting of the oligonucleotide of SEQ ID NO: 13 and the oligonucleotide of SEQ ID NO: 14, it is possible to amplify the most preferred region, 428 base pairs to 175 base pairs. It is most preferable because it is possible and suitable for shuttle PCR amplification. For the gyrA gene, it is particularly preferable that the primer pair for PCR is a primer pair comprising a primer having the nucleotide sequence of SEQ ID NO: 15 and a primer pair comprising a primer having the nucleotide sequence of SEQ ID NO: 16, If the pCR primer pair is an oligonucleotide pair consisting of the oligonucleotide of SEQ ID NO: 15 and the oligonucleotide of SEQ ID NO: 16, the most preferable region, the first base pair to the 397th base pair, It is most preferred because it can be amplified and is suitable for shuttle PCR amplification. In addition, the oligonucleotide of SEQ ID NO: 1 corresponds to the 11th of the rpoB gene. Sense base primer corresponding to 999 base pairs to 1226 base pairs, the oligonucleotide of SEQ ID NO: 2 is an antisense primer corresponding to 1903 base pairs to 1880 base pairs of the rp0B gene, SEQ ID NO: Thirty-three oligonucleotides are the sense primers corresponding to 1256-base pair of the rpoB gene to the 127th base pair, and the oligonucleotide of SEQ ID NO: 34 is a base pair of 155 base pairs of the rp0B gene. It is an antisense primer corresponding to the first 570 base pairs. The oligonucleotide of SEQ ID NO: 3 is a sense primer corresponding to the 1st to 28th base pairs of the kat G gene, and the oligonucleotide of SEQ ID NO: 4 is the 2223 to 2220 base pairs of the kat G gene. An antisense primer corresponding to a base pair. The oligonucleotide of SEQ ID NO: 5 is a sense primer corresponding to the first 217 base pairs to the first 198 base pairs of the mab A—inhA gene, and the oligonucleotide of SEQ ID NO: 6 is a mab A— It is an antisense primer corresponding to the 1145th to the 126th base pairs of the inhA gene. The oligonucleotide of SEQ ID NO: 7 is a sense primer corresponding to nucleotides 640 to 665 of the embB gene, and the oligonucleotide of SEQ ID NO: 8 is nucleotides 387 to 3363 of the embB gene. Antisense primer corresponding to the pair. The oligonucleotide of SEQ ID NO: 9 is a sense primer corresponding to the -80th base pair to the 61st base pair of the pncA gene, and the oligonucleotide of SEQ ID NO: 10 is the 59th base pair of the pncA gene. It is an antisense primer corresponding to base pairs to the 572th base pair. The oligonucleotide of SEQ ID NO: 11 is a sense primer corresponding to the 4th to 23rd base pairs of the rpsL gene, and the oligonucleotide of SEQ ID NO: 12 is 575th of the rpsL gene. It is an antisense primer corresponding to base pairs to the 556th base pair. The oligonucleotide of SEQ ID NO: 13 is It is a sense primer corresponding to 428 base pairs to 447 base pairs, and the oligonucleotide of SEQ ID NO: 14 is an antisense primer corresponding to 1st to 56 base pairs to 1737 base pairs of the rrs gene. The oligonucleotide of SEQ ID NO: 15 is a sense primer corresponding to the 1st to 19th base pairs of the gyrA gene, and the oligonucleotide of SEQ ID NO: 16 is the 397th to 19th base pairs of the gyrA gene. It is an antisense primer corresponding to 379 base pairs. Here, the base pair number is such that the start base of the start codon of the type I chain is defined as the first base. <PCR amplification of two or more specific regions>

Next, using two pieces of PCR primer pairs corresponding to two or more specific regions with DNA extracted from M. tuberculosis, two or more specific regions CR amplify. In PCR amplification, the region between the primers is amplified based on the type II DNA by the catalytic action of a thermostable polymerase. PCR amplification is performed by converting type I DNA into a single strand (denaturation), binding this single strand to a primer (annealing), and finally extending the complementary strand by the catalytic action of a thermostable polymerase (extension). Reaction) or normal PCR amplification consisting of three steps, or shuttle PCR amplification. Usually, in two or more primer pairs, the optimal annealing temperature differs for each primer, and the extension reaction time varies in proportion to the length of the product to be amplified. In performing normal PCR amplification in the present invention, all the annealing temperatures of the primers constituting two or more PCR primer pairs corresponding to two or more specific regions must be constant. In the case of the present invention, two or more specific areas, also P In order to amplify regions with different lengths of CR products at one time using a single PCR device, shuttle PCR amplification is preferable because it is more practical. Shuttle PCR amplification is preferable because annealing with a PCR primer and extension of a complementary strand by DNA polymerase in PCR amplification can be performed under the same temperature conditions. Shuttle PCR amplification is a known method, and kits for performing shuttle PCR amplification include P1 atinum Rtaq DNA polymerase (made by Invitrogen Corporation) and TakaRa Z—taq ^TM ( Pyr obest R DNA Polymerase (manufactured by Yukala Bio Inc.) is commercially available, and the equipment for PCR is Gen e Amp PGR SYSTEM 9700 t hermo cy c 1 er (Applied Biosystems), i—Cyc 1er (Nippon Bio's Laboratories), and TakaRa PCR Thermal Cycler GP (Yukara Bio Inc.) are commercially available. ing. In addition, at the time of shuttle PCR amplification, two or more PCR primer pairs were selected from the oligonucleotide pairs consisting of the nine oligonucleotide pairs described above, or two or more oligonucleotide pairs selected from the above-mentioned oligonucleotide pairs. When two or more primer pairs are selected from a primer pair consisting of different types of primer pairs, shuttle PCR amplification can be suitably performed under the same temperature conditions using a single PCR amplification device. The reason why when two or more oligo nucleotide pairs or the two or more primer pairs are used as two or more PCR primer pairs, shuttle PCR can be suitably amplified under the same temperature conditions is as follows. The following I think. Since a strict annealing temperature is not required in shuttle PCR, the optimal temperature for each oligonucleotide nucleotide or primer of the two or more oligonucleotide pairs or the two or more primer pairs is set. There is no need to set an annealing temperature, and amplification can be performed at once under the same temperature condition in which annealing and extension reaction are performed simultaneously. Conversely, in the shuttle PCR method, there is a concern that non-specific reactions are likely to occur, and the lengths of the products differ. However, for the two or more oligonucleotide pairs or the two or more primer pairs, the same extension reaction time should be used in shuttle PCR amplification, and conditions for good reproducibility and non-specific reaction should not occur. Settings have been made. In addition, the two or more oligonucleotide pairs or the two or more primer pairs satisfy the temperature condition of a polymerase (Z-taq; TAKARA, manufactured by Yukara Bio Inc.) suitable for shuttle PCR. I have. Furthermore, the above-mentioned two or more kinds of oligonucleotide pairs or the above-mentioned two or more kinds of primer pairs are designed so that the bonding between primers (primer-dimer formation) is unlikely to occur as in the case of ordinary PCR primers. . In addition, since the two or more oligonucleotide pairs or the two or more primer pairs are designed so that the site binding to the two or more specific regions during annealing is not a high mutation site, the strain There is no difference in the binding site at the time of annealing depending on the presence or absence of the mutation, so that there is no difference in the degree of growth depending on the strain. A pair of two or more primers for PCR is composed of three or more oligonucleotide pairs selected from the above-mentioned nine oligonucleotide pairs or More preferably, it is a pair of three or more primers selected from the nine primer pairs described above. The two or more PCR primer pairs are four or more oligonucleotide nucleotide pairs selected from the nine types of oligonucleotide pairs or four or more primer pairs selected from the nine types of primer pairs. More preferred. Two or more: Five or more primer pairs whose PCR primers are selected from the nine oligonucleotide pair groups or five or more primer pairs selected from the nine primer pair groups More preferably, it is a pair. The two or more PCR primer pairs are six or more oligonucleotide pairs selected from the nine oligonucleotide pairs or the six or more primer pairs selected from the nine primer pairs. And more preferably. More preferably, the two or more PCR primer pairs are 7 or more oligonucleotide pairs selected from the nine types of oligonucleotide pairs or seven or more primer pairs selected from the eight types of primer pairs. Preferred. The two or more PCR primer pairs are eight or more oligonucleotide pairs selected from the nine oligonucleotide pairs or eight or more primer pairs selected from the nine primer pairs. And more preferably. If two or more PCR primer pairs are eight oligonucleotide pairs selected from the above nine types or eight primer pairs selected from the above nine types, the number of samples to be amplified by PCR becomes eight. Therefore, it is most preferable. The reason is described below. The PCR equipment currently on the market has a structure of 8 x 12 we 11 and has a structure in which eight types of samples can be amplified simultaneously by PCR. Ube is also commercially available. Therefore, if there are fewer sambles than 8 species, If the sample is more than 9 types, it is necessary to perform PCR amplification twice, and the reaction time will be doubled, resulting in a loss of time and cost. Furthermore, using eight types of samples enables PCR amplification of twelve samples at a time, which also helps prevent sample mix-up.

The PCR-amplified products in two or more specific regions are subjected to removal and purification of excess primer using Micro Spin ^™ Columns (manufactured by Amersham Biosciences) or the like. Appropriate selection of two or more target areas included in two or more specific areas. As described above, the nucleotide sequence of each gene was reported by Cole, S. T et al. (Natur e 393 (6685), p537-544 (1998)), and Gene Bank (Accession No .: NC-0) ◦ 0962) disclosed above. The preferred region of the target region selected for direct base sequencing will vary from gene to gene. The base pair numbers in the target region of each gene described below are such that the start base of the start codon of the type I chain of each gene is the first base. Regarding the rpoB gene, the target region is preferably a region containing a base pair located at the 5′-end of the 1529th base pair of the complementary chain to the 鎵 -type chain of the specific region of the rpoB gene described above, Most preferably, it is a region containing 1529 to 1199 base pairs of the complementary strand. With respect to the kat G gene, the target region is a region containing base pairs located at the 5th and 5th ends of the 鎖 -type strand in the specific region of the above-described specific region of the kat G gene, 574 base pairs of the type chain ~ a region containing the base pair located at the 3 'end of the type I chain in the specific region of the kat G gene as described above, 1162 base pairs of the type chain ~ the above-mentioned kat G gene A region containing a base pair located at the 3 ′ end of the type I strand in the specific region of It is preferably at least one type of region consisting of a region containing a base pair located at the 3 ′ end of the type II strand of the specific region of the above-mentioned kat G gene. The target region includes a region containing the 689th base pair to the 1st base pair of the complementary strand, a region containing the 574th base pair to the 1324th base pair of the 錶 type strand, One species selected from the group consisting of a region containing 62 base pairs to 192 base pairs and a region containing 172nd base pairs to 222 bp of type I chain It is more preferable that the above-mentioned region be used. Regarding the kat G gene, it is more preferable that the target region is two or more regions selected from the above-mentioned region group, and the target region is three or more regions selected from the above-mentioned region group. More preferably, the target region is most preferably all four regions included in the region group. As for the mab A-inh A gene, the target region is located at the 2′-117 base pair of the 鐯 -chain to the base pair located at the 3 ′ end of the 錡 -chain of the specific region of the mab A-inh A gene described above. And most preferably a region containing the 2nd to 17th base pairs to the 90th base pair of the type I chain. Regarding the emb B gene, the target region is a region containing the base pair located at the 3 ′ end of the 錶 type strand in the specific region of the emb emb B gene, from the 646th base pair of the 錶 type chain to the 錶 type chain. The first 462 base pair of the strand to the region containing the base pair located at the 3 'end of the type I strand in the specific region of the emb B gene described above, the first 596 base pair of the complementary strand to the emb described above region containing a base pair located at the 5 ⁵ end of錶型chain of a specific region of the B gene, ³⁵ of the second 0 0 7 base pairs to錄型strand of a specific region of emb B genes described above in鎵型chain A region containing a base pair located at the end, and a region containing the base pair located at the 3 ′ end of the type I strand of the above-mentioned specific region of the type embB gene- It is preferably one or more regions selected from a region group consisting of regions. The region in which the target region contains base chain 646 to base pair 136 of base type I A region containing the 1462th base pair to the 2221st base pair of the type I strand, a region containing the 1596th base pair to the 846th base pair of the complementary chain, A region comprising the 207th base pair to 257th base pair of the type chain, and a region comprising the 258th base pair to the 3331 base pair of the type strand More preferably, it is at least one region selected from the group. For the embB gene, it is more preferable that the target region is two or more types of regions selected from the above-mentioned region group, and that the target regions is three or more types of regions selected from the above-mentioned region group. More preferably, the target region is more preferably four or more regions included in the region group, and most preferably, the target region is all five regions included in the region group. For pnc A gene, the target region, first of錡型strand - is an area containing 8 0 base pairs to base pairs located at the 3 ⁵ end of錶型chain of a specific region of the pnc A gene described above Most preferably, it is a region containing the −80th base pair to the 590th base pair of the 錶 type chain. rps L gene One to information, the target region, preferably as a base pair located 3 ⁵ end of錡型chain of a specific region of the fourth base pairs ~ rps L gene as described above in銪型chain,錶Most preferably, it is a region containing the fourth to fifth base pairs of the template strand. For the rrs gene, the target region is a region containing the 979th base pair of the complementary strand to the base pair located at the 5 ′ end of the 鎵 type strand of the above-described specific region of the rss gene; preferable to be one or more regions selected from 2 9 1 base pairs to a region group ing from a region containing a base pair located at the 5 ⁵ end of鍀型chain in a specified area of rrs genes described above, the complementary strand A region composed of two regions, that is, a region containing the 979th base pair to the 428th base pair and a region containing the 1st 921st base pair to the 1756th base pair of the type I chain More preferably, it is one or more regions selected from the group. Further, it is more preferable that the target region is the two regions described above. About gyr A gene Is preferably a base pair located at the 3 ′ end of the type I strand from the first base pair of the type I strand to the specific region of the gyrA gene described above. Most preferably, it is a region consisting of 7 base pairs. In this paragraph, type II strand refers to a strand that is directly used as type II when a gene is transcribed into RNA, and a complementary strand refers to a strand that forms a duplex with type II strand. Is shown. When selecting target regions, it is preferable to select all target regions corresponding to three or more genes, more preferably to select all target regions corresponding to four or more genes, and to select targets corresponding to five or more genes. More preferably, all the regions are selected, more preferably, all target regions corresponding to 6 or more genes are selected, and further preferably, all target regions corresponding to 7 or more genes are selected, and all 8 genes are selected. It is most preferable to select 16 target regions corresponding to In addition, all the target regions corresponding to the gene refer to, for example, all the above-mentioned five types of target regions in the embB gene.

The two or more target regions are directly sequenced using two or more base sequence determination primers corresponding to the selected two or more target regions. Known primers can be used as two or more base sequence determination primers. Preferred primers for the primers for nucleotide sequencing differ for each gene. For the gene rpoB gene, a primer containing the oligonucleotide of SEQ ID NO: 17 or the nucleotide sequence of SEQ ID NO: 17 is particularly preferred. For the kat G gene, the four oligos of SEQ ID NOS: 18-21 One or more oligonucleotides in the oligonucleotide group consisting of nucleotides, or one of the primers consisting of four primers, one from the primer containing the nucleotide sequence of SEQ ID NO: 18 to the primer containing the nucleotide sequence of SEQ ID NO: 18 Particularly preferred are at least two kinds of primers, and more preferably two or more, more preferably three or more, and most preferably all four of the above-mentioned oligonucleotide group or primer group of primers for nucleotide sequence determination. For the mab A-inh A gene, a primer containing the oligonucleotide of SEQ ID NO: 22 or the nucleotide sequence of SEQ ID NO: 22 is particularly preferred. Regarding the emb B gene, one or more oligonucleotides selected from the oligonucleotide group consisting of the five oligonucleotides of SEQ ID NOS: 23 to 27, or primers 1 to SEQ ID NO: including the nucleotide sequence of SEQ ID NO: 23 One or more primers selected from a primer group consisting of five primers of primers containing 27 nucleotide sequences are particularly preferable, and the primer for nucleotide sequence determination is preferably at least two kinds of the above oligonucleotide groups or one primer group. Are more preferable, three or more are more preferable, four or more are more preferable, and all five are most preferable. For the pncA gene, an oligonucleotide of SEQ ID NO: 28 or a primer containing the nucleotide sequence of SEQ ID NO: 28 is particularly preferred _{(for the} rps L gene, the oligonucleotide of SEQ ID NO: 29 or the primer of SEQ ID NO: 29) Particularly preferred is a primer containing a base sequence: For the rrs gene, one or more oligonucleotides selected from the oligonucleotide group consisting of two oligonucleotides of SEQ ID NOs: 30 and 31, or the bases of SEQ ID NO: 30 One or more primers selected from a primer group consisting of a primer containing a sequence and a primer containing the nucleotide sequence of SEQ ID NO: 31 are particularly preferred. The primer for nucleotide sequencing is selected from the group consisting of the oligonucleotide group or the primer group 2 Seeds are the best Is also preferred. For the gyrA gene, a primer containing the oligonucleotide of SEQ ID NO: 32 and a primer containing the nucleotide sequence of SEQ ID NO: 32 are particularly preferred. The oligonucleotide of SEQ ID NO: 17 is a primer for determining a nucleotide sequence corresponding to the 1529th base pair to the 1512th base pair of the complementary strand of the rpoB gene. The oligonucleotide of SEQ ID NO: 18 is a primer for nucleotide sequence determination corresponding to the 689th base pair to 670th base pair of the complementary strand of the kat G gene, and the oligonucleotide of SEQ ID NO: 19 is A primer for determining the nucleotide sequence corresponding to the 5th 74th base pair to the 5'th 93rd base pair of the 鎖 chain, the oligonucleotide of SEQ ID NO: 20 is the 1st 162 base of the kat G gene 鎖 chain Pairing to a base sequence determination primer corresponding to the 1st base pair, and the oligonucleotide of SEQ ID NO: 21 is the 1st to 29th base pair to the 1st This is a primer for nucleotide sequence determination corresponding to 1748 base pairs, and the oligonucleotide of SEQ ID NO: 22 corresponds to the -217 base pairs to the first 198 base pairs of the type I chain of the mab A-inh A gene. Antisense primer. The oligonucleotide of SEQ ID NO: 23 is a primer for base sequence determination corresponding to the 646th base pair to 665th base pair of the 錶 type chain of the embB gene, and the oligonucleotide of SEQ ID NO. 24 is the 錶 type of the embB gene. This is a primer for nucleotide sequence determination corresponding to the 1462nd base pair to the 1481th base pair of the chain. The oligonucleotide of SEQ ID NO: 25 is a primer for nucleotide sequence determination corresponding to the 1596th base pair to the 1577th base pair of the complementary strand of the embB gene, and the oligonucleotide of SEQ ID NO: 26 is A primer for determining a base sequence corresponding to base No. 007 to base pair 2026 of the 錶 type strand, and the oligonucleotide of SEQ ID NO: 27 is embB This is a sense primer corresponding to the 2581st base pair to the 2601st base pair of the type I chain of the gene. The oligonucleotide of SEQ ID NO: 28 is an antisense primer corresponding to the -80th base pair to the 61st base pair of the type I chain of the pncA gene. The oligonucleotide of SEQ ID NO: 29 is a primer for nucleotide sequence determination corresponding to the 4th to 23rd base pairs of the type I chain of the rps L gene. The oligonucleotide of SEQ ID NO: 30 is a primer for nucleotide sequence determination corresponding to the 979th to 959th base pairs of the complementary strand of the rrs gene, and the oligonucleotide of SEQ ID NO: 31 Is a primer for nucleotide sequence determination corresponding to the 1st to 129th base pairs of the type III strand of the rrs gene. The oligonucleotide of SEQ ID NO: 32 is a primer for nucleotide sequence determination corresponding to the first to 19th base pairs of the type I chain of the gyrA gene. Here, the base pair number is such that the start base of the start codon of type I chain is defined as the first base. <Direct nucleotide sequencing>

As a method of direct nucleotide sequencing, two or more target regions are subjected to a sequence reaction at one time with one sequence reaction device using two or more base sequence determination primers, and then the sequence reaction obtained by the sequence reaction is performed. A preferred method is to run the single-sequence reaction product at a time using one base sequence determination device (auto sequencer) and analyze and edit the base sequence by software. . A sequence reaction method in which two or more types of base sequence determination primers are used to perform a sequence reaction in two or more stages with one sequence reaction device, and one base sequence determination device (automatic sequencer) The present invention also encompasses a method in which electrophoresis is performed two or more times according to 1) and the base sequence is analyzed and edited by software. Sequence for performing a sequence reaction As the reaction equipment (thermal cycler), GeneAmp PCR SYSTEM 9700 thermocycle (manufactured by Applied Biosystems), i Cycler thermal cycler (manufactured by Nippon Bio-Rad Laboratories) And TakaRa PCR Thermal C cler GP (manufactured by Yukara Bio Co., Ltd.). As a base sequence determination device for directly migrating a sequence reaction product during base sequence determination, 16 capillaries are used. Formula AB I PRI SM 3100 Genetic Ana lyzer, 48 capillaries Applied B ios ystems 3730 DNA Analyzer, 96 capillaries Complete set AB I PR I SM 3700 DNA Ana 1 yzer Instruments that can be used to analyze and edit base sequences include Sequencing Analyzes is S of tware (Applied Bios ystems), Genetyx (Genetics) ), DNAS ISP ro (Hitachi Software Co., Ltd.) Manufactured). Hereinafter, a second method for detecting a drug resistance gene contained in the Mycobacterium tuberculosis of the present invention will be described. <Preparation of DNA derived from Mycobacterium tuberculosis>, <Selection of two or more genes and two or more specific regions>, <Determination of two or more PCR primer pairs>, <PCR of two or more specific regions Amplification> and <Separation of PCR product and selection of target region> are performed in the same manner as in the first method. The sequence reaction kit, electrophoresis equipment, and base sequence determination and editing software used for direct base sequence determination are the same as those described above. The second method differs from the first method in that two or more target regions described above are directly sequenced at a time by one base sequencer. You. Hereinafter, this point will be described in detail. Usually, in a sequence reaction using two or more primers for sequencing, the optimum annealing temperature differs for each primer, and the extension reaction time is proportional to the length of the product to be subjected to the sequence reaction. different. In the second method, in order for two or more target regions to be subjected to a sequence reaction at one time by one sequence reaction device, the sequence reaction should be performed at an optimum annealing temperature separately for each primer. The annealing temperatures of the primers constituting the two or more primers for determining the base sequence corresponding to the above target regions must all be kept constant. In the case of the present invention, in order for two or more target regions, which are also regions having different product lengths due to the sequence reaction, to undergo a sequence reaction at a time with one sequence reaction device, the annealing temperature is required during the reaction. It is preferable to use a method in which everything is constant because it is practical. The primer for nucleotide sequence determination is a group of oligonucleotides selected from the above 16 oligonucleotides or a group of primers selected from the above 16 primers, which can sequence all target regions corresponding to three or more types of genes. Is preferred. More preferably, it is a group of oligonucleotides selected from the above 16 oligonucleotides or a group of primers selected from the above 16 primers, which can sequence all target regions corresponding to four or more genes. More preferably, it is a group of oligonucleotides selected from the above-mentioned 16 oligonucleotides or a group of primers selected from the above 16 primers, which can sequence all target regions corresponding to 5 or more genes. All target regions corresponding to 6 or more genes can be sequenced. More preferably, it is an oligonucleotide group selected from the 16 oligonucleotides or a primer group selected from the 16 primers described above. More preferably, it is a group of oligonucleotides selected from the above 16 oligonucleotides or a group of primers selected from the above 16 primers, which can sequence all target regions corresponding to 7 or more genes. The oligonucleotide group selected from the above 16 oligonucleotides or the primer group selected from the above 16 primers, which can sequence all target regions corresponding to the three genes, are, for example, A primer group consisting of the above-mentioned six primers or the above-mentioned six oligonucleotides capable of sequencing all the above-mentioned six target regions of the rpoB gene, katG gene, and mabA-inhA gene is shown.

16 target regions corresponding to all 8 genes can be sequenced. If the above 16 oligonucleotides or 16 primers are used, 16 samples will be used as the sequence reaction product. The most preferred. The reason is described below. Instruments for direct sequencing, such as the ABI 310 Sequencer, use 16 samples as the basis for a single reaction. Therefore, if the number of samples is less than 16, the cost is lost, and if the number of samples is more than 17, it is necessary to perform the sequence reaction in two steps, so the reaction time is doubled, and the time and cost are reduced. Loss. As described above, the above-mentioned 16 oligonucleotides or 16 primers are designed so that the most preferable target region can be subjected to a sequence reaction at one time to directly determine the nucleotide sequence. Next, the primer pair set for PCR of the present invention will be described. The present invention The primer pair set for PCR of the present invention is described in the above-mentioned inventions (9) to (12). The primers constituting these primer pair sets are obtained by using a fully automatic oligonucleotide synthesizer ( It can be easily synthesized and prepared using Applied Biosystem 380B). The PCR primer pair set of the present invention is used for PCR amplification in the first or second method for detecting a drug resistance gene contained in the above-described tuberculosis bacterium of the present invention. Using a pair of PCR primers, the two or more specific regions can be suitably used when shuttle PCR amplification is performed at once by one PCR amplification device. Next, the primer set for determining a nucleotide sequence of the present invention will be described. The primer set for determining a nucleotide sequence according to the present invention is described in the inventions (13) to (14) described above. The primers constituting these primer sets are fully automated oligos. It can be easily synthesized and prepared using a nucleotide synthesizer. The primer set for nucleotide sequence determination of the present invention can be used for direct nucleotide sequencing in the first or second method for detecting the drug resistance gene contained in the above-described tuberculosis bacterium of the present invention, particularly in the case of a reaction. Through a sequence reaction using two or more primers for sequencing at a constant annealing temperature, the two or more target regions are directly sequenced at once by a single sequencing device. In this case, it can be suitably used. Next, the reagent kit for diagnosing drug-resistant tuberculosis of the present invention will be described. The reagent kit for diagnosing drug-resistant tuberculosis according to the present invention is described in the above-mentioned invention (15). Easily synthesized with an oligonucleotide synthesizer Can be prepared. The reagent kit for diagnosing drug-resistant tuberculosis of the present invention can be suitably used in the above-mentioned first method or second method for detecting a drug-resistant gene contained in the tuberculosis bacterium of the present invention. (Example)

Hereinafter, the present invention will be described more specifically with reference to examples. However, this is merely an example and does not limit the present invention. Example 1

From 35 patients, 35 sputum specimens were collected. Pretreatment and DNA extraction of 35 sputum samples were performed by the following methods. That is, 500 μΐ of sputum is pretreated by the N-acetyl-L-cystein (NALC) -NaOH method, centrifuged at 12,000 r.p.m for 15 minutes, and the supernatant is removed. Next, add 10% Chelix 100 resin (Nippon Bio's Ladder Laboratories) dissolved in sterile distilled water to the sediment, add ポ Ο Ο μΙ, and stir with Portex, then heat at 45 ° C for 45 minutes. After heating at 100 ° C for 10 minutes, centrifuge again at 12,000 rpm for 15 minutes, and use the supernatant as a sample.

According to the method described above, eight genes of rp0B, katG, mabA-inhA, embB, pncA, rpsL, rrs, and gyrA were selected from DNA extracted for each strain, and one specific region was selected for each gene. A total of eight specific areas were selected. In all strains, the specific region is defined as a 705 base pair from the 1199th base pair to the 1903 base pair as the rpoB gene. Or a region consisting of 315 base pairs from 1256 base pairs to 1570 base pairs. As the kat G gene, in all strains, the specific region was defined as a region consisting of 2223 base pairs from 1st base pair to 2223 base pairs. As the mabA-inhA gene, in all strains, the specific region was a region consisting of 1362 base pairs from 217 base pairs to 1145 base pairs. As the embB gene, in all strains, the specific region was a region consisting of 2748 base pairs from 640 base pairs to 3387 base pairs. As the pncA gene, in all strains, the specific region was a region consisting of 670 base pairs from the -80th base pair to the 590th base pair. As the rps L gene, in all strains, the specific region was defined as a region consisting of 572 base pairs from the fourth base pair to the 575 base pair. As the rrs gene, in all strains, the specific region was a region consisting of 1329 base pairs from 428 base pairs to 1756 base pairs. As the gyrA gene, in all the strains, the specific region was a region consisting of 398 base pairs from the 1st base pair to the 397 base pair.

<8 types of primer pairs for PCR>

One primer pair was prepared for each of the eight specific regions corresponding to the eight genes described above. Oligonucleotide pairs of SEQ ID NOs: 1 and 2 or oligonucleotide pairs of SEQ ID NOs: 33 and 34 were used as primer pairs for the specific region corresponding to the rpoB gene. Oligonucleotide pairs of SEQ ID NOS: 3 and 4 were used as a primer pair for the specific region corresponding to the kat G gene. Oligonucleotide pairs of SEQ ID NOS: 5 and 6 were used as a primer pair for the specific region corresponding to the mabA-inhA gene. For the specific region corresponding to the emb B gene, Nucleotide pairs were used as primer pairs. Oligonucleotide pairs of SEQ ID NOS: 9 and 10 were used as primer pairs for the specific region corresponding to the pncA gene. For the specific region corresponding to the rps L gene, the oligonucleotide pairs of SEQ ID NOS: 11 and 12 were used as a primer pair. Oligonucleotide pairs of SEQ ID NOS: 13 and 14 were used as a primer pair for the specific region corresponding to the rrs gene. Oligonucleotide pairs of SEQ ID NOS: 15 and 16 were used as primer pairs for the specific region corresponding to the gyrA gene.

The DNA extracted by the above method is designated as type 、, and the above-mentioned eight specific regions are charged with a primer pair selected from the above-mentioned eight primer pairs, one for each PCR tube. Using one PCR tube, shuttle PCR amplification was performed once for each strain using one PCR amplification device. The procedure for shuttle PCR amplification is shown below.

The composition of each PCR tube was determined as follows: type III DNA 5.0 zl, Z-Taq polymerase 1.25 U (Yukarabaio), dNTP20 Ojl (Yukara Bio Inc.), One primer pair selected from the above eight primer pairs is kept at 200 nM, and 10 XZtaq Buf fer 5/1 (manufactured by Yukala Bio Inc.) is added. Was set to 50〃1. Used: PCR tubes are eight tubes numbered 1-8. The No. 1 PCR tube contains the oligonucleotide pairs of SEQ ID Nos. 1 and 2 or the oligonucleotide pairs of SEQ ID Nos. 33 and 34, and the No. 2 PCR tube contains the oligonucleotide pairs of SEQ ID Nos. 3 and 4. Nucleotide pairs, the No. 3 PCR tube with the oligonucleotide pairs of SEQ ID Nos. 5 and 6, the No. 4 PCR tube with the oligonucleotide pairs of SEQ ID Nos. 7 and 8, and the No. 5 PCR tube. Indicates the oligonucleotide pairs of SEQ ID NOS: 9 and 10, the PCR tube of No. 6 contains the oligonucleotide pairs of SEQ ID NOS: 11 and 12, and the PCR tube of No. 7 shows the oligonucleotide pairs of SEQ ID NOs: 13 and 14. The oligonucleotide pair of SEQ ID NOS: 15 and 16 was introduced into the PCR primer No. 8 as a primer pair for PCR. The above-mentioned eight specific regions contained in the eight PCR tubes prepared as described above were used as a PCR amplification device by using a Gene Amp PCR SYSTEM 9700 thermocycle (manufactured by Applied Biosystems). All were amplified by shuttle PCR with the same temperature profile. The temperature profile was denaturation at 95 ° C for 1 second and annealing and extension reaction 68 for 30 seconds. The temperature profile was repeated for 30-35 cycles. Thereafter, each of the eight PCR tubes was purified by agarose-gel electrophoresis using Micro Spin TMCo 1 umns (manufactured by Amersham Biosciences), and purified as described above. Eight PCR products were extracted from each of the eight specific regions. Fig. 1 shows the results of agarose gel electrophoresis. In FIG. 1, No. 1 shows the band of the PCR product of the above specific region for the rpoB gene, No. 2 shows the band of the above specific region of the kat G gene which shows the PCR product, and No. 3 shows the ma b A— The band of the PCR product of the specific region for the inhA gene, No. 4 is the band of the PCR product of the specific region for the em bB gene, and No. 5 is the band for the pncA gene. The band indicated by the PCR product of the specific region above, No. 6 is the band indicated by the PCR product of the above specific region for the rps L gene, and No. 7 is the PCR product of the above specific region for the rrs gene: The band No. 8 shows the band indicated by the PCR product of the specific region of the gyrA gene.

For the rpoB gene, the target region is defined as a region consisting of the first base pair to the first base pair of the complementary strand, or a region consisting of the first base pair to the first base pair. And For the kat G gene, the first target region is the region consisting of base pairs 689 to 1 of the complementary strand, and the second target region is the base chain 574 to base 1 of the type I strand. A region consisting of 324 base pairs, a third target region being a region consisting of base pairs 162 to 192, and a fourth target region consisting of base 1領域 A region consisting of 29 base pairs to 2nd 223 base pairs. For the mabA-inhA gene, the target region was defined as a region consisting of the -217 base pair to the 903-base pair of the type I chain. For the emb B gene, the first target region is a region consisting of base pair 646 to pair 136 base pairs of the type I chain, and the second target region is region 146 base pair 1 of base type II of the type II chain. The third target region is a region consisting of the 1st 966th base pair to the 846th base pair of the complementary strand; the fourth target region is the 200th base pair of the 錡 type strand. The region consisting of 7 base pairs to 2757 base pairs, and the fifth target region was the region consisting of 2581 base pairs to 3331 base pairs of the type I chain. For the pncA gene, the target region was a region consisting of the −80th base pair to the 590th base pair of the type I chain. For the rps L gene, the target region was a region containing the fourth base pair to the fifth base pair of the type I chain. For the rrs gene, the first target region is a region consisting of the 979th base pair to the 4288th base pair of the complementary strand, and the second target region is The region was composed of 6 base pairs. For the gyrA gene, the target region is defined as the region consisting of the first base pair to the third base pair of the 錡 -type chain. Was

One primer was prepared for each of the 16 target regions corresponding to the eight genes described above. The oligonucleotide of SEQ ID NO: 17 was used as a primer for the target region corresponding to the rpoB gene. The oligonucleotide of SEQ ID NO: 18 was used as a primer for the first target region of the katG gene. For the second target region of the kat G gene, the oligonucleotide of SEQ ID NO: 19 was used as a primer. The oligonucleotide of SEQ ID NO: 20 was used as a primer for the third target region of the katG gene. The oligonucleotide of SEQ ID NO: 21 was used as a primer for the fourth target region of the katG gene. The oligonucleotide of SEQ ID NO: 22 was used as a primer for the target region of the mab A-inhA gene. The oligonucleotide of SEQ ID NO: 23 was used as a primer for the first target region of the embB gene. For the second target region of the embB gene, the oligonucleotide of SEQ ID NO: 24 was used as a primer. The oligonucleotide of SEQ ID NO: 25 was used as a primer for the third target region of the embB gene. For the fourth specific region of the embB gene, the oligonucleotide of SEQ ID NO: 26 was used as a primer. The oligonucleotide of SEQ ID NO: 27 was used as a primer for the fifth specific region of the embB gene. For the target region corresponding to the pncA gene, the oligonucleotide of SEQ ID NO: 28 was used as a primer. For the target region corresponding to the rpsL gene, the oligonucleotide pair of SEQ ID NO: 29 was used as a primer pair. Using the oligonucleotide of SEQ ID NO: 30 as a primer for the first target region of the rrs gene Was. The oligonucleotide of SEQ ID NO: 31 was used as a primer for the second target region of the rrs gene. For the target region corresponding to the gyrA gene, the oligonucleotide of SEQ ID NO: 32 was used as a primer.

Sequencing: The sequencing reaction (Sequencing reaction) is performed using a dye using a BigDye terminat orcclesequencing FS Read Reac tion kit V3,0 (Applied Biosystems, Inc.). I went by the one-one-one-one-one method. The composition of the sequence reaction tube was as follows. Premix (Sequence buffer; 5 x Sequen cng Buf fer 4/1, Deoxyribonucleotide triphosphate; dNTP mix 1/1, Dideoxy Yuichi Minei Yoichi; Dye D eoxy Terminat ors 0.5 1, DNA polymerase Amp 1 iT aq, FS 4〃 1; total 8 1 (Ap 1 ied Biosystems, Inc.)), one P product 1 in one tube 1. A primer suitable for determining the nucleotide sequence of the above target region of the resistance gene, 3.2 pmol, was added, and the total amount was made 201 with sterile distilled water. The sequence reaction tubes used were 16 tubes of Nos. 1 to 16 _(The No. 1 sequence reaction tube contains the PCR product obtained from the No. 1 PCR tube and the SEQ ID No. 17). The oligonucleotide and the PCR product obtained from the PCR tube No. 2 and the oligonucleotide of SEQ ID NO: 18 were placed in the No. 2 sequence reaction tube. The PCR product obtained from the No. 2 PCI tube and the oligonucleotide of SEQ ID NO: 19 were placed in the No. 3 sequence reaction tube, and the No. 2 PCR product was placed in the No. 4 sequence reaction tube. The PCR product obtained by the PCR tube and the oligonucleotide of SEQ ID NO: 20 were combined with the PCR product obtained by the PCR tube of No. 2 and the oligonucleotide of SEQ ID NO: 20. The oligonucleotide and the PCR product obtained from the PCR reaction tube No. 6 and the oligonucleotide of SEQ ID NO: 22 were placed in the tube for the sequencing reaction No. 6 in the tube for the sequencing reaction No. 7 The PCR product obtained from the No. 4 PCR tube and the oligonucleotide of SEQ ID NO: 23 were used for the PCR product obtained using the No. 4 PCR tube, and the PCR product obtained using the No. 4 PCR tube was used for the Sequence No. Products and sequences In the No. 9 sequence reaction tube, the PCR product obtained from the No. 4 PCR tube and the oligonucleotide of SEQ ID NO: 25 were placed in the No. 9 sequence reaction tube. In Ube, the PCR product obtained with the No. 4 P tube and the oligonucleotide of SEQ ID NO: 26 were used. In the No. 11 sequence reaction tube, the No. 4 PCR tube was used. The PCR product obtained with the oligonucleotide of SEQ ID NO: 27, the PCR product obtained by the PCR tube of No. 5 and the oligonucleotide of SEQ ID NO: 28 were placed in the tube for sequence reaction No. 12, The PCR reaction tube No. 13 contains the PCR product obtained from the PCR tube No. 6 and the oligonucleotide of SEQ ID NO: 29, and the PCR reaction tube No. 14 contains the PCR product No. 7. For PCR The PCR product obtained in the tube and the oligonucleotide of SEQ ID NO: 30 were placed in the tube for sequence reaction No. 15, and the PCR product obtained in the tube for PCR in No. 7 and the oligonucleotide of SEQ ID NO: 31. And the No. 16 sequence reaction tube contains 8 The PCR product obtained by the No. PCR tube and the oligonucleotide of SEQ ID NO: 32 were introduced. The temperature profile of the sequence reaction is as follows. 25 cycles of denaturation at 96 ° C for 10 seconds, annealing at 50 ° C for 5 seconds, and extension reaction at 60 ° C for 4 minutes were repeated. The sequence reaction was performed using the 16 tubes described above and the Gene Amp PCR System 9700 thermo cycler (manufactured by Applied Bios ystems) described above as a sequence reaction device. I went every time. After the amplification reaction, unreacted dye and excess primer are removed using Centri-sep s in columns (manufactured by Princeton Separateions) as a column, and the sequence reaction product is purified. did. Next, the column eluate was centrifuged at 1700 rpm for 15 minutes at 50 ° C. using a concentrating centrifuge (manufactured by Tomiichi Kogyo Co., Ltd.), and dried. Aplied B iosyst ems) (15〃1) was added, and the mixture was rapidly cooled after heating at 95 ° C for 2 minutes. In addition, by placing the sequence reaction from one tube per well, 16 tubes of sequence reaction can be placed in a 96-well plate (2 lanes in 2 lanes of 8-well XI). Then, the gel was electrophoresed without waste by using a set of 16 capillaries ABI 3100 autosequencer. Electrophoresis was performed for each strain.

Software for analyzing and editing nucleotide sequences includes Sequencing Analysys is S of twere and Applied Bi. osystems) was used to compare the nucleotide sequence of these known drug-sensitive M. tuberculosi sH37Rv strains, and the presence or absence of the mutation was analyzed for each strain. For each gene, the location of the mutation found, the mutation, and the number of strains in which the mutation was found are described in detail below.

Sense codon numbers shown below are numbered with the start codon of the sense codon as the first codon. In addition, the base pair numbers are given as base pair numbers with the first base pair of the start codon of the sense codon as the first base pair. Regarding the rpo B gene, one out of 35 strains has the 513th sense codon CAA (translated into glutamine and Gin) mutated to codon CTA (translated into oral isin and Leu), and 35 Six of the strains have the 531st sense codon TCG (translated into serine and Ser) mutated to codon TTG (translated into leucine and Leu), and one out of 35 strains The 526th sense codon CAC (translated to histidine and His) is mutated to ACC (translated to tyrosine and Thr), and the mutation was confirmed in a total of 8 strains out of 35 strains. Was a strain. Among the mutations in the gene, the mutation at the 526th sense codon is a novel discovery. Regarding the kat G gene, one out of 35 strains has the 295th sense codon CAG (translated into glutamine, G1n) mutated to CCG (translated into proline, Pro), One out of 35 strains has the 297th sense codon GGC (translated into glycine and Gly) mutated to GTC (translated into valine and Va1), and 4 out of 35 strains have 31 The fifth sense codon AGC (translated to serine and Ser) is mutated to AC C (translated to threonine and Thr), and two out of 35 strains have the 324nd sense codon ACC ( Threonine and Thr) were mutated to CCC (proline and Pro), and mutations were confirmed in a total of 8 strains out of 35 strains. Among the gene mutations, mutations at the 295th sense codon, the 297th sense codon, and the 324nd sense codon are new discoveries. Regarding the mab A-inh A gene, 4 out of 35 strains had the C base at the 15th base pair mutated to a T base, and the mutation was confirmed in 35 out of 35 strains in total. There were four strains. Regarding the embB gene, one of 35 strains had the 497th sense codon CAG (translated into glutamine and Gin) mutated to CGG (translated into arginine and Arg), and 35 Three of the strains have the 306th sense codon ATG (translated into methionine and met) mutated to ATA (translated into isoleucine and lie), and 35 strains have been confirmed to be mutated Among them, the total was 4 shares. Regarding the pncA gene, one out of 35 strains had the fourth sense codon GCG (translated into alanine and Ala) mutated to GAG (translated into glutamine and G1u). One of the strains has the 10th senscodon CAG (translated to glutamine and Gin) mutated to CCG (translated to proline and Pro), and one out of 35 strains has The third sense codon, GAC (translated to Asparagine, Asp), is mutated to GCC (Translated to Alanine, Ala), and one out of 35 strains However, the 5th first sense codon CAC (translated to histidine and His) is mutated to CAG (translated to glutamine and Gin), and one out of 35 strains is transformed into the 148th strain. Sense codon CGC (translated to arginine and Arg) was mutated to AGC (translated to serine and Ser), and the mutation was confirmed in 5 of 34 strains in total. Among the gene mutations, the fourth mutation in the sense codon and the 148th mutation in the senscodon are new findings. Regarding the rps L gene, one of 35 strains was found to have the 43rd sense codon AAG (translated to lysine and Lys) mutated to AGG (translated to arginine and Arg), and the mutation was confirmed. The total number of strains was 35 out of 35. Regarding the rrs gene, 1 out of 35 strains had the A base at the 1400th base pair mutated to a T base, and the mutation was confirmed in 1 out of 35 strains in total. The 1400th base pair mutation is a novel discovery. Regarding the gyr A gene, one out of 35 strains has the 90th sense codon GCG (translated to Alanine, Ala) mutated to GTG (valine, Va1), One of the 35 strains was confirmed. Summarizing the above, the time required from the extraction of M. tuberculosis DNA to the completion of the nucleotide sequence analysis was about 6.5 hours, which was longer than the conventional drug susceptibility test for more than two weeks. And conventional PCR amplification and direct salt The time required to detect a strain having a resistance gene could be significantly reduced compared to the time required to determine the base sequences of the above eight genes by base sequencing. In addition, it was found that this method can be used as epidemiological information because it can identify gene mutations. In addition, 1 out of 8 strains having a mutation in the rpoB gene, 4 out of 8 strains having a mutation in the kat G gene, 2 out of 4 strains having a mutation in the pncA gene, and a mutation in the rrs gene A strain with a novel mutation, which has not been reported so far, was found for one strain having the same. Example 2

The conventional drug susceptibility test was compared with Example 1 <Results of nucleotide sequence analysis> as follows.

A conventional drug sensitivity test was performed. <Sputum pretreatment and DNA extraction of Mycobacterium tuberculosis> 34 strains of Mycobacterium tuberculosis from 34 patients were analyzed by the method of SAB IN et al. gy 37 (1), p45-48, 1999). A drug susceptibility test was performed for each strain using the grown strains. For INH, RFP and EMB, drug susceptibility tests were performed according to the method of SABINE et al. (Journal of Clinical Microblog 1 ogy 37 (1), p45-48, 1999). For PZ A, a drug susceptibility test was performed according to the method of APD AV IES et al. (JournalofClinicMicromicology, 38 (10), p3686-3688, 2000) . As a result, out of 35 clinical isolates examined, Nineteen strains were sensitive to all drugs. Ten out of 35 strains were found to be resistant to RFP. 15 out of 35 strains were found to be resistant to INH. Four out of 35 strains were found to be resistant to EMB. It was found that 5 out of 35 strains had resistance to PZA.

Regarding the 19 strains that were sensitive to all drugs in the drug sensitivity test, no mutation in the resistance gene was observed in any of the 19 strains in the results of the nucleotide sequence analysis in Example 1. In the drug susceptibility test, among the 10 strains having resistance to RFP, in the results of the nucleotide sequence analysis of Example 1, 8 of the 10 strains showed mutations related to RFP resistance, oB gene mutation was detected. In the drug susceptibility test, 15 of the 15 strains having resistance to INH were analyzed by the nucleotide sequence of Example 1, and as a result of the analysis of the kat G gene, which is a mutation related to the resistance to INH, in 9 of the 15 strains, Mutations were detected, and in 4 of the 15 strains, a mutation in the mabA-inhA gene, which is a mutation involved in resistance to INH, was detected. In 13 out of 15 strains having resistance to INH, mutations in genes involved in resistance were confirmed. In the drug susceptibility test, four of the four strains having resistance to EMB in the results of the analysis of the base sequence in Example 1 showed that in four of the four strains, the embB gene, which is a mutation relating to the resistance to EMB, was analyzed. Mutation was detected. In the drug sensitivity test, five of the five strains having resistance to PZA showed the pncA gene mutation, which is a mutation involved in resistance to PZA, in five of the five strains in the results of the nucleotide sequence analysis of Example 1. Mutation was detected. Summarizing the above results, the results of the nucleotide sequence analysis in Example 1 show that 8 out of 10 RFP-resistant strains (80%) by drug sensitivity test and 13 out of 15 INH-resistant strains by drug sensitivity test (80%) Mutations in genes involved in resistance were detected in 4 out of 4 EMB resistant strains (100%) and 5 out of 5 PZA resistant strains (100%) by drug susceptibility tests. As a result, resistant bacteria in the drug susceptibility test could be detected. Since the method for detecting a drug resistance gene of this example is performed by PCR amplification, relatively small amounts of DNA and bacteria are sufficient. Therefore, it is possible to perform a direct test from a patient sputum sample. Also, since the base sequence is directly decoded, there is no oversight due to sequence differences. In addition, it can be used as epidemiological information between strains (it is relatively accurate to determine whether they are the same strain). In addition, a simple kit can be used for the eight primers for PCR and the sixteen primers for base sequence determination used in Example 1 if they have a sequencer (AB13100) and a PCR amplifier. . Industrial applicability

According to the present invention, a method for quickly and easily detecting a drug resistance gene contained in M. tuberculosis, a primer pair set for PCR, a primer set for nucleotide sequence determination, and a drug which can be suitably used in the method A kit for diagnosis of resistant tuberculosis can be provided. The method for detecting a drug resistance gene of the present invention can be easily used as a rapid drug susceptibility test for tuberculosis in a bacterial testing department of a medical facility, a health center, a local health research institute, a bacterial testing center, and the like.

Claims

The scope of the claims

1. the DNA from M. tuberculosis, rp 0 B is a gene contained in said binding Kakukin, kat G _N mabA- inhA 2 which, embB, pnc A, selected from the gene group consisting of rps L _s rrs _s and gyr A Using two or more types of PCR primer pairs corresponding to two or more specific regions of two or more genes, the two or more specific regions can be simultaneously treated by one PCR amplification device. PCR amplification, and then directly base-sequencing the two or more target regions using two or more kinds of base-sequence determination primers corresponding to two or more target regions contained in the two or more amplified specific regions A method for detecting a drug resistance gene contained in M. tuberculosis.

2. DNA selected from the group consisting of DNA from Mycobacterium tuberculosis and the genes contained in the Mycobacterium tuberculosis, rpoB, katG, mabA-inhA, embB, pncA, rpsL, rrs, and gyrA 2 Using two or more types of PCR primer pairs corresponding to two or more specific regions of two or more genes, the two or more specific regions are once treated by one PCR amplification device. To: PCR amplification, followed by a sequence reaction of the two or more target regions using two or more primers for sequencing that correspond to two or more target regions included in the two or more specific regions. A method for detecting a drug resistance gene contained in Mycobacterium tuberculosis by determining a nucleotide sequence at a time using a single nucleotide sequencing device using the obtained sequence reaction product.

3. The two or more types of PCR primer pairs contain the nucleotide sequence of SEQ ID NO: 1. The primer pair comprises a primer and a primer pair comprising the nucleotide sequence of SEQ ID NO: 2, and the nucleotide sequence of SEQ ID NO: 33 Primers and primers containing primers SEQ ID NO: 34 A primer pair comprising a primer having the nucleotide sequence of SEQ ID NO: 3 and a primer having the nucleotide sequence of SEQ ID NO: 4, a primer having the nucleotide sequence of SEQ ID NO: 5 and a primer having the nucleotide sequence of SEQ ID NO: 6 A primer pair containing the nucleotide sequence of SEQ ID NO: 7 and a primer pair consisting of a primer containing the nucleotide sequence of SEQ ID NO: 8, a primer containing the nucleotide sequence of SEQ ID NO: 9, and the nucleotide sequence of SEQ ID NO: 10 A primer pair containing the base sequence of SEQ ID NO: 11 and a primer pair consisting of a primer containing the base sequence of SEQ ID NO: 12 and a primer containing the base sequence of SEQ ID NO: 13 Primer and a pair of primers containing the nucleotide sequence of SEQ ID NO: 14, and SEQ ID NO: 15 A primer pair comprising two or more primer pairs selected from a primer pair consisting of a primer pair comprising a primer comprising a nucleotide sequence and a primer comprising the nucleotide sequence of SEQ ID NO: 16.3. 2. The method for detecting a drug-resistant gene contained in M. tuberculosis according to item 1.

4. The two or more PCR primer pairs comprise an oligonucleotide pair comprising the oligonucleotide of SEQ ID NO: 1 and an oligonucleotide of SEQ ID NO: 2, an oligonucleotide pair of SEQ ID NO: 33 and an oligonucleotide of SEQ ID NO: 34 An oligonucleotide pair consisting of the oligonucleotide pair of SEQ ID NO: 3 and the oligonucleotide of SEQ ID NO: 4; an oligonucleotide pair consisting of the oligonucleotide of SEQ ID NO: 5 and the oligonucleotide of SEQ ID NO: 6; An oligonucleotide pair consisting of an oligonucleotide and an oligonucleotide of SEQ ID NO: 8; an oligonucleotide pair consisting of an oligonucleotide of SEQ ID NO: 9 and an oligonucleotide of SEQ ID NO: 10; an oligonucleotide pair of SEQ ID NO: 11 and SEQ ID NO: 1 Consisting of two oligonucleotides From the oligonucleotide pair consisting of the oligonucleotides of SEQ ID NO: 13 and the oligonucleotide of SEQ ID NO: 14 and the oligonucleotide pair of the oligonucleotides of SEQ ID NO: 15 and the oligonucleotide of SEQ ID NO: 16 3. The method for detecting a drug resistance gene contained in M. tuberculosis according to claim 1 or 2, wherein the method is a combination of two or more oligonucleotides selected from the group consisting of:

5. The method for detecting a drug resistance gene contained in Mycobacterium tuberculosis according to any one of claims 1 to 4, wherein the PCR amplification is a shuttle PCR amplification.

6. The two or more types of primers for determining a base sequence are selected from the group consisting of primers consisting of 16 primers of a primer containing a base sequence of SEQ ID NO: 17 to a primer containing a base sequence of SEQ ID NO: 32 The method for detecting a drug resistance gene contained in Mycobacterium tuberculosis according to any one of claims 1 to 5, wherein the two or more primers are selected.

7.The two or more nucleotide sequencing primers are two or more oligonucleotides selected from the oligonucleotide group consisting of the oligonucleotides of SEQ ID NO: 17 to the oligonucleotides of SEQ ID NO: 32 to 16 The method for detecting a drug resistance gene contained in Mycobacterium tuberculosis according to any one of claims 1 to 5, wherein the method comprises the steps of:

8. The method for detecting a drug-resistant gene contained in Mycobacterium tuberculosis according to any one of claims 1 to 7, wherein the direct nucleotide sequencing is performed by a dye terminator method.

9. An oligonucleotide pair consisting of the oligonucleotide of SEQ ID NO: 1 and the oligonucleotide of SEQ ID NO: 2, an oligonucleotide consisting of the oligonucleotide of SEQ ID NO: 33 and the oligonucleotide of SEQ ID NO: 34 Oligonucleotide pair consisting of the oligonucleotide of SEQ ID NO: 3 and the oligonucleotide of SEQ ID NO: 4, oligonucleotide pair consisting of the oligonucleotide of SEQ ID NO: 5 and the oligonucleotide of SEQ ID NO: 6, the oligonucleotide pair of SEQ ID NO: 7 and the SEQ ID NO: 8 An oligonucleotide pair consisting of the oligonucleotide of SEQ ID NO: 9 and an oligonucleotide pair consisting of the oligonucleotide of SEQ ID NO: 10; an oligonucleotide consisting of the oligonucleotide of SEQ ID NO: 11 and the oligonucleotide of SEQ ID NO: 12 An oligonucleotide pair consisting of a nucleotide pair, an oligonucleotide of SEQ ID NO: 13 and an oligonucleotide of SEQ ID NO: 14, and an oligonucleotide of SEQ ID NO: 15 and an oligonucleotide of SEQ ID NO: 16 PCR primer pairs cell Uz bets, characterized in that the one or more oligonucleotide Dopea selected from oligonucleotide pair group ing from Li Cheng oligonucleotide pair.

10. A primer pair comprising a primer containing the nucleotide sequence of SEQ ID NO: 1 and a primer containing the nucleotide sequence of SEQ ID NO: 2, a primer containing the nucleotide sequence of SEQ ID NO: 33 and the nucleotide sequence of SEQ ID NO: 34 A primer pair comprising a primer comprising the nucleotide sequence of SEQ ID NO: 3 and a primer pair comprising a primer comprising the nucleotide sequence of SEQ ID NO: 4, and a primer comprising the nucleotide sequence of SEQ ID NO: 5 A primer pair containing the base sequence of SEQ ID NO: 6; a primer pair containing the base sequence of SEQ ID NO: 7 and a primer pair consisting of a primer containing the base sequence of SEQ ID NO: 8; containing the base sequence of SEQ ID NO: 9 A primer pair consisting of a primer and a primer containing the nucleotide sequence of SEQ ID NO: 10, the base sequence of SEQ ID NO: 11 Primer one pair with primers containing a base sequence of the primer first and SEQ ID NO: 1 2 containing, containing the nucleotide sequence of the primers and SEQ ID NO: 1 4 containing the nucleotide sequence of SEQ ID NO: 1 3 One or more primer pairs selected from a primer pair consisting of a primer pair consisting of a primer pair and a primer pair comprising a primer containing the nucleotide sequence of SEQ ID NO: 15 and a primer containing the nucleotide sequence of SEQ ID NO: 16 A primer pair set for PCR, characterized in that:

11. Oligonucleotides selected from the oligonucleotides of SEQ ID NO: 1 to the oligonucleotides of SEQ ID NO: 16 and the oligonucleotides consisting of the oligonucleotides of SEQ ID NO: 33 and the 18 oligonucleotides of the oligonucleotides of SEQ ID NO: 34 ′ A primer pair set for PCR, characterized by having one or more oligonucleotides as primers.

1 2. A primer containing the base sequence of SEQ ID NO: 1 ^ ^ a primer containing a base sequence of SEQ ID NO: 16, a primer containing a base sequence of SEQ ID NO: 33 and a primer containing a base sequence of SEQ ID NO: 33 and SEQ ID NO: 3 A primer pair set for PCR, comprising one or more primers selected from a group of primers comprising primers having the following nucleotide sequences:

13. A base sequence determination characterized by being at least one oligonucleotide selected from the group consisting of oligonucleotides consisting of the oligonucleotides of SEQ ID NO: 17 to oligonucleotides of SEQ ID NO: 32 to SEQ ID NO: 32 Primer set.

14. A primer comprising the nucleotide sequence of SEQ ID NO: 17 to a primer comprising the nucleotide sequence of SEQ ID NO: 32 to 16 or more primers selected from the group consisting of 16 primers. Primer set for determining base sequence.

15. A PCR primer pair set according to any one of claims 9 to 12 and a base sequence determination primer set according to claim 13 or 14. Diagnostic kit for drug-resistant tuberculosis.