+

WO2004063368A1 - Procede de detection d'un gene resistant aux medicaments contenu dans le bacille de tuberculose, ensemble de paire d'amorce pour pcr, ensemble d'amorce pour determiner la sequence de base et kit de reactifs pour diagnostiquer la tuberculose resistant aux medicaments - Google Patents

Procede de detection d'un gene resistant aux medicaments contenu dans le bacille de tuberculose, ensemble de paire d'amorce pour pcr, ensemble d'amorce pour determiner la sequence de base et kit de reactifs pour diagnostiquer la tuberculose resistant aux medicaments Download PDF

Info

Publication number
WO2004063368A1
WO2004063368A1 PCT/JP2003/016941 JP0316941W WO2004063368A1 WO 2004063368 A1 WO2004063368 A1 WO 2004063368A1 JP 0316941 W JP0316941 W JP 0316941W WO 2004063368 A1 WO2004063368 A1 WO 2004063368A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
primer
oligonucleotide
pair
nucleotide sequence
Prior art date
Application number
PCT/JP2003/016941
Other languages
English (en)
Japanese (ja)
Inventor
Teruo Kirikae
Junichiro Sekiguchi
Takashi Ohtsuki
Original Assignee
Japan Science And Technology Agency
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Japan Science And Technology Agency filed Critical Japan Science And Technology Agency
Publication of WO2004063368A1 publication Critical patent/WO2004063368A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • 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.
  • 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.
  • mutations in the embB gene encoding arabinosyltransferase (Arab inosyltransferase) have been shown to be involved in resistance.
  • resistant bacteria having resistance to PZA it has been clarified that a mutation in the pncA gene encoding pyrazinamidase is involved in resistance.
  • RNA 16Sribo Mutations in the cluster A region and the class B region on the rrs gene encoding Soma l RNA
  • KM rib0s0ma1protein in S12
  • 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.
  • FIG. 2 shows the locations of these resistance-related genes on the M. tuberculosis gene.
  • 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.
  • 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.
  • 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.)
  • Mycobacterium tuberculosis grows slowly and does not grow or not.
  • 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.
  • INH, RFP, SM, KM, and EB Different only there is a problem such as that can not be detected.
  • the bacteria to be tested need to be cultured for weeks or months to isolate them.
  • PCR is performed by extracting genes from tuberculosis bacteria obtained from sputum, etc.
  • An object of the present invention is to provide a reagent kit for diagnosing drug-resistant tuberculosis. Disclosure of the invention
  • 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.
  • 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
  • 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.
  • two or more genes selected from the gene group consisting of rpoB ⁇ 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.
  • 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
  • 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.
  • 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.
  • 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
  • tuberculosis according to the invention (1) or (2), which is at least two types of primer pairs selected from the group A.
  • 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.
  • 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
  • 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.
  • 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:
  • 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.
  • 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.
  • 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.
  • 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.
  • the present invention 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 present invention 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • embodiments of the present invention will be described, but the present invention is not limited to these embodiments.
  • 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.
  • a tissue extract such as sputum, cerebrospinal fluid, gastric juice, feces, or lymph nodes.
  • 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.
  • 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
  • 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
  • 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
  • the rrs gene corresponds to 1471844 base pairs of the genome sequence.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • embB PCR amplification and direct nucleotide sequencing have been performed on a limited region around the codon CAG at position 497.
  • pncA PCR amplification and direct nucleotide sequencing have been performed on a limited region around the 51st codon CAC.
  • 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.
  • 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 ⁇ the 906th base A restricted region around base ⁇ (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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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,
  • 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.
  • 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.
  • 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,
  • 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.
  • 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
  • the oligonucleotide of SEQ ID NO: 34 is a base pair of 155 base pairs of the rp0B gene.
  • the oligonucleotide of SEQ ID NO: 3 is a sense primer corresponding to the 1st to 28th base pairs of the kat G gene
  • the oligonucleotide of SEQ ID NO: 4 is the 2223 to 2220 base pairs of the kat G gene.
  • 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
  • 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
  • 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.
  • the oligonucleotide of SEQ ID NO: 13 is It is a sense primer corresponding to 428 base pairs to 447 base pairs
  • 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
  • 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.
  • 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 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.
  • 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.
  • all the annealing temperatures of the primers constituting two or more PCR primer pairs corresponding to two or more specific regions must be constant.
  • two or more specific areas also P
  • 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.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • 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. .
  • 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.
  • 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.
  • PCR-amplified products in two or more specific regions are subjected to removal and purification of excess primer using Micro Spin TM Columns (manufactured by Amersham Biosciences) or the like. Appropriate selection of two or more target areas included in two or more specific areas.
  • 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 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 ⁇ -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.
  • the target region is a region containing base pairs located at the 5th and 5th ends of the ⁇ -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
  • 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 ⁇ 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.
  • the target region is located at the 2′-117 base pair of the ⁇ -chain to the base pair located at the 3 ′ end of the ⁇ -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.
  • the target region is a region containing the base pair located at the 3 ′ end of the ⁇ type strand in the specific region of the emb emb B gene, from the 646th base pair of the ⁇ type chain to the ⁇ type chain.
  • 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.
  • 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.
  • 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.
  • the target region, first of ⁇ strand - is an area containing 8 0 base pairs to base pairs located at the 3 5 end of ⁇ 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 ⁇ type chain.
  • the target region preferably as a base pair located 3 5 end of ⁇ chain of a specific region of the fourth base pairs ⁇ rps L gene as described above in ⁇ chain, ⁇ Most preferably, it is a region containing the fourth to fifth base pairs of the template strand.
  • 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 ⁇ 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 ⁇ 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.
  • 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.
  • type II strand refers to a strand that is directly used as type II when a gene is transcribed into RNA
  • a complementary strand refers to a strand that forms a duplex with type II strand. Is shown.
  • all target regions corresponding to three or more genes 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.
  • a primer containing the oligonucleotide of SEQ ID NO: 17 or the nucleotide sequence of SEQ ID NO: 17 is particularly preferred.
  • 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.
  • a primer containing the oligonucleotide of SEQ ID NO: 22 or the nucleotide sequence of SEQ ID NO: 22 is particularly preferred.
  • 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.
  • 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:
  • a primer containing a base sequence is particularly preferred.
  • 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.
  • 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
  • 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 ⁇ chain
  • the oligonucleotide of SEQ ID NO: 20 is the 1st 162 base of the kat G gene ⁇ chain Pairing to a base sequence determination primer corresponding to the 1st base pair
  • 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
  • the oligonucleotide of SEQ ID NO: 22 corresponds to the -217 base pairs to the first 198 base pairs
  • 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 ⁇ type chain of the embB gene
  • the oligonucleotide of SEQ ID NO. 24 is the ⁇ 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
  • 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 ⁇ type strand
  • 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
  • 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.
  • the base pair number is such that the start base of the start codon of type I chain is defined as the first base.
  • 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.).
  • thermocycle manufactured by Applied Biosystems
  • i Cycler thermal cycler manufactured by Nippon Bio-Rad Laboratories
  • TakaRa PCR Thermal C cler GP manufactured by Yukara Bio Co., Ltd.
  • ⁇ 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.
  • 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.
  • the sequence reaction 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the primer pair set for PCR of the present invention will be described.
  • 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.
  • the two or more specific regions can be suitably used when shuttle PCR amplification is performed at once by one PCR amplification device.
  • 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.
  • 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.
  • 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).
  • 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)
  • Example 1
  • Pretreatment and DNA extraction of 35 sputum samples were performed by the following methods. That is, 500 ⁇ 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 ⁇ ⁇ ⁇ ⁇ , 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.
  • NALC N-acetyl-L-cystein
  • 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.
  • the specific region was defined as a region consisting of 2223 base pairs from 1st base pair to 2223 base pairs.
  • the specific region was a region consisting of 1362 base pairs from 217 base pairs to 1145 base pairs.
  • the specific region was a region consisting of 2748 base pairs from 640 base pairs to 3387 base pairs.
  • the specific region was a region consisting of 670 base pairs from the -80th base pair to the 590th base pair.
  • 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.
  • the specific region was a region consisting of 1329 base pairs from 428 base pairs to 1756 base pairs.
  • the specific region was a region consisting of 398 base pairs from the 1st base pair to the 397 base pair.
  • 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.
  • 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.
  • a primer pair selected from the above-mentioned eight primer pairs one for each 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.
  • 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.
  • 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.
  • 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.
  • 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
  • No. 3 shows the ma b A—
  • 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.
  • the first target region is the region consisting of base pairs 689 to 1 of the complementary strand
  • the second target region is the base chain 574 to base 1 of the type I strand.
  • the target region was defined as a region consisting of the -217 base pair to the 903-base pair of the type I chain.
  • the first target region is a region consisting of base pair 646 to pair 136 base pairs of the type I chain
  • 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 ⁇ 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.
  • the target region was a region consisting of the ⁇ 80th base pair to the 590th base pair of the type I chain.
  • the target region was a region containing the fourth base pair to the fifth base pair of the type I chain.
  • 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.
  • the target region is defined as the region consisting of the first base pair to the third base pair of the ⁇ -type chain.
  • 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.
  • 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.
  • 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.
  • 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.
  • the oligonucleotide of SEQ ID NO: 28 was used as a primer.
  • the oligonucleotide pair of SEQ ID NO: 29 was used as a primer pair.
  • the oligonucleotide of SEQ ID NO: 31 was used as a primer for the second target region of the rrs gene.
  • the oligonucleotide of SEQ ID NO: 32 was used as a primer.
  • Sequencing reaction 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.
  • 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.
  • 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.
  • the PCR reaction tube No. 14 contains the PCR product No. 7.
  • 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.
  • 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.
  • sequence reaction 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.
  • Sequencing Analysys is S of twere and Applied Bi. osystems
  • Sense codon numbers shown below are numbered with the start codon of the sense codon as the first codon.
  • 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.
  • 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 is mutated to ACC (translated to tyrosine and Thr), and the mutation was confirmed in a total of 8 strains out of 35 strains.
  • the mutation at the 526th sense codon is a novel discovery.
  • 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)
  • 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.
  • 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.
  • 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
  • GAC transcription to Asparagine, Asp
  • CAC codon to His
  • CAG translated to glutamine and Gin
  • one out of 35 strains is transformed into the 148th strain.
  • Sense codon CGC was mutated to AGC (translated to serine and Ser), and the mutation was confirmed in 5 of 34 strains in total.
  • the fourth mutation in the sense codon and the 148th mutation in the senscodon are new findings.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • this method can be used as epidemiological information because it can identify gene mutations.
  • 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.
  • Example 1 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.
  • 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
  • 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.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention propose un procédé pour détecter un gène résistant aux médicaments, contenu dans le bacille de tuberculose, qui consiste à amplifier simultanément par PCR deux ou plusieurs régions spécifiques transportées par deux ou plusieurs gènes sélectionnés dans un groupe de gènes spécifiques, contenus dans le bacille de tuberculose, avec l'utilisation d'une paire d'amorces spécifiques pour le PCR puis à déterminer directement les séquences de base de deux ou plusieurs régions cibles, contenues dans une ou plusieurs régions spécifiques amplifiées de cette manière. L'invention concerne également un ensemble de paire d'amorce pour PCR, un ensemble d'amorce destiné à déterminer une séquence de base spécifique, un procédé pour détecter de façon rapide et aisée un gène de résistance aux médicaments contenu dans le bacille de tuberculose grâce à l'utilisation d'un kit de réactifs diagnostiques destiné à détecter la tuberculose résistant à un médicament spécifique, et un ensemble de paire d'amorces pour PCR, un ensemble d'amorces pour déterminer une séquence de base et un ensemble de réactifs de diagnostique, qui peut s'utiliser de façon appropriée dans le procédé ci-décrit.
PCT/JP2003/016941 2003-01-14 2003-12-26 Procede de detection d'un gene resistant aux medicaments contenu dans le bacille de tuberculose, ensemble de paire d'amorce pour pcr, ensemble d'amorce pour determiner la sequence de base et kit de reactifs pour diagnostiquer la tuberculose resistant aux medicaments WO2004063368A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2003-5370 2003-01-14
JP2003005370A JP2004215542A (ja) 2003-01-14 2003-01-14 結核菌に含まれる薬剤耐性遺伝子を検出する方法、ｐｃｒ用プライマーペアセット、塩基配列決定用プライマーセット、及び薬剤耐性結核の診断用試薬キット

Publications (1)

Publication Number Publication Date
WO2004063368A1 true WO2004063368A1 (fr) 2004-07-29

Family

ID=32709016

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2003/016941 WO2004063368A1 (fr) 2003-01-14 2003-12-26 Procede de detection d'un gene resistant aux medicaments contenu dans le bacille de tuberculose, ensemble de paire d'amorce pour pcr, ensemble d'amorce pour determiner la sequence de base et kit de reactifs pour diagnostiquer la tuberculose resistant aux medicaments

Country Status (2)

Country Link
JP (1) JP2004215542A (fr)
WO (1) WO2004063368A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1866023B (zh) * 2006-05-25 2010-11-03 上海市肺科医院 一种同步检测多种结核杆菌特异性分泌抗原的方法
CN102229988A (zh) * 2011-05-25 2011-11-02 厦门大学 一种结核分枝杆菌利福平耐药突变的检测方法及试剂盒
CN101419237B (zh) * 2008-09-03 2012-10-24 深圳市东湖医院 结核菌感染的酶联免疫斑点诊断试剂盒及特异性抗原制取方法
CN104450877A (zh) * 2014-07-03 2015-03-25 北京圣谷同创科技发展有限公司 利福平、异烟肼、氟喹诺酮类药物四个结核耐药基因检测
CN106399539A (zh) * 2016-10-25 2017-02-15 石河子大学 一种用于结核杆菌链霉素耐药快速检测引物及其试剂盒

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP5904432B2 (ja) * 2008-07-02 2016-04-13 ニプロ株式会社 結核菌におけるイソニアジド感受性を検出するための方法および試験片
CN102105596A (zh) * 2008-07-02 2011-06-22 尼普洛株式会社 用于检测结核杆菌中的异烟肼敏感性的方法和试验片
KR102030005B1 (ko) * 2018-05-24 2019-11-08 연세대학교 원주산학협력단 개선된 퀀타매트릭스 어세이 플랫폼 기반 결핵균 검출 및 결핵균의 리팜핀, 아이소니아지드, 에탐부톨, 퀴놀론, 아미카신, 카나마이신, 카프레오마이신와 같은 이차 주사제 및 스트렙토마이신 약제에 대한 내성여부를 동시 확인할 수 있는 진단법 및 그 키트

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1076099A2 (fr) * 1999-08-03 2001-02-14 Nisshinbo Industries, Inc. Trousse pour la diagnose des bacilles de tubercule

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1076099A2 (fr) * 1999-08-03 2001-02-14 Nisshinbo Industries, Inc. Trousse pour la diagnose des bacilles de tubercule

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BOCKSTAHLER L E: "PEPTIDE NUCLEIC ACID PROBE DETECTION OF MUTATIONS IN MYCOBACTERIUM TUBERCULOSIS GENES ASSOCIATED WITH DRUG RESISTANCE", BIOTECHNIQUES, vol. 32, no. 3, March 2002 (2002-03-01), pages 508 - 514, XP001194469 *
KIM B J, ET AL: "DETECTION OF RIFAMPIN-RESISTANT MYCOBACTERIUM TUBERCULOSIS IN SPUTA BY NESTED PCR-LINKED SINGLE-STRAND CONFORMATION POLYMORPHISM AND DNA SEQUENCING", J CLIN MICROBIOL, vol. 39, no. 7, July 2001 (2001-07-01), pages 2610 - 2617, XP000294870 *
TILLIB S V, ET AL: "INTEGRATION OF MULTIPLE PCR AMPLIFICATIONS AND DNA MUTATION ANALYSES BY USING OLIGONUCLEOTIDE MICROCHIP", ANAL BIOCHEM., vol. 292, no. 1, 1 May 2001 (2001-05-01), pages 155 - 160, XP002974871 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1866023B (zh) * 2006-05-25 2010-11-03 上海市肺科医院 一种同步检测多种结核杆菌特异性分泌抗原的方法
CN101419237B (zh) * 2008-09-03 2012-10-24 深圳市东湖医院 结核菌感染的酶联免疫斑点诊断试剂盒及特异性抗原制取方法
CN102229988A (zh) * 2011-05-25 2011-11-02 厦门大学 一种结核分枝杆菌利福平耐药突变的检测方法及试剂盒
CN104450877A (zh) * 2014-07-03 2015-03-25 北京圣谷同创科技发展有限公司 利福平、异烟肼、氟喹诺酮类药物四个结核耐药基因检测
CN106399539A (zh) * 2016-10-25 2017-02-15 石河子大学 一种用于结核杆菌链霉素耐药快速检测引物及其试剂盒

Also Published As

Publication number Publication date
JP2004215542A (ja) 2004-08-05

Similar Documents

Publication Publication Date Title
US11479819B2 (en) Non-invasive method for monitoring transplanted organ status in organ-transplant recipients
CN110719957B (zh) 用于核酸靶向富集的方法和试剂盒
EP3957753A1 (fr) Amorces de réaction en chaîne de polymérase et sondes pour le bacille de koch
WO2001075171B1 (fr) Methodes, compositions et necessaires correspondants servant au depistage et a la surveillance du cancer du sein
CN109312410B (zh) 用于从福尔马林固定的石蜡包埋的生物样品中表征抗药性细菌的方法和组合物
EP3225696A1 (fr) Ensemble d'amorces et procédé d'amplification d'exons du gène pkd1 et du gène pkd2
KR20130044217A (ko) 녹농균 혈청형 분석법 및 키트 및 상기 방법 및 키트에 유용한 올리고뉴클레오타이드 서열들
US7381547B2 (en) Methods and compositions to detect bacteria using multiplex PCR
KR101979202B1 (ko) 퀀타매트릭스 어세이 플랫폼 기반 헬리코박터 파이로리균 및 그 균의 항생제 내성에 관련된 유전자의 검출을 확인할 수 있는 진단법 및 그 응용
US20110287965A1 (en) Methods and compositions to detect clostridium difficile
JP2023512559A (ja) 体液においてｔｂを検出するためのｃｒｉｓｐｒに基づくアッセイ
WO2004063368A1 (fr) Procede de detection d'un gene resistant aux medicaments contenu dans le bacille de tuberculose, ensemble de paire d'amorce pour pcr, ensemble d'amorce pour determiner la sequence de base et kit de reactifs pour diagnostiquer la tuberculose resistant aux medicaments
KR100950495B1 (ko) 특정 프라이머 세트를 포함하는 결핵균을 검출하기 위한등온증폭반응용 프라이머 조성물, 키트 및 상기 프라이머세트를 이용하여 결핵균을 검출하는 방법
CN110656188A (zh) 检测引发血流感染的杆菌的引物和/或探针组合物及其应用
KR102555330B1 (ko) SARS-CoV-2 진단용 조성물, 키트 및 이를 이용한 SARS-CoV-2의 진단방법
WO2002022872A1 (fr) Procede et kit de pcr multiplexe et oligonucleotides destines a la detection et l'identification de mycobacterium au moyen dudit procede
JPH10500011A (ja) 直列変異反復配列オリゴタイピングによる結核菌群細菌の検出および鑑別
KR20160009397A (ko) 약제 내성 결핵균을 검출하는 방법
AU2004271214B2 (en) Primers for use in detecting beta-lactamases
KR101765677B1 (ko) 결핵 및 비결핵 항산균 검출용 프라이머 세트 및 이를 이용한 검출 방법
CA2520512A1 (fr) Methodes, compositions et trousses pour la detection et pour la surveillance d'un cancer du poumon
US20050239067A1 (en) Method of detecting and quantifying hemolysin-producing bacteria by overwhelmingly detecting and quantifying thermostable hemolysin-related genes (tdh-related hemolysin genes) of food poisoning bacteria
JP2005204582A (ja) オリゴヌクレオチド及びそれを用いた非定型抗酸菌群の検出方法
US20090253129A1 (en) Identification of usa300 community-associated methicillin-resistant staphylococcus aureus
KR20100012319A (ko) 패혈증­유발 미생물의 분류 및 동정 방법

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): CN KR US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PT RO SE SI SK TR

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载