WO2015172734A1 - Combinaison de fragments spécifiques de gène de résistance à un médicament pour quatre types de médicaments de deuxième ligne contre mycobacterium tuberculosis et son utilisation - Google Patents

Combinaison de fragments spécifiques de gène de résistance à un médicament pour quatre types de médicaments de deuxième ligne contre mycobacterium tuberculosis et son utilisation Download PDF

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WO2015172734A1
WO2015172734A1 PCT/CN2015/079022 CN2015079022W WO2015172734A1 WO 2015172734 A1 WO2015172734 A1 WO 2015172734A1 CN 2015079022 W CN2015079022 W CN 2015079022W WO 2015172734 A1 WO2015172734 A1 WO 2015172734A1
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seq
mutation
complement
probe
acid sequence
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PCT/CN2015/079022
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万康林
李桂莲
赵丽丽
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中国疾病预防控制中心传染病预防控制所
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Publication of WO2015172734A1 publication Critical patent/WO2015172734A1/fr

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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • 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
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    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present application generally relates to the field of detection technology for pathogenic bacteria resistance. Specifically, the present application relates to tuberculosis branches of tuberculosis four second-line drugs fluoroquinolones, aminoglycoside kanamycin and amikacin (ampicanacin) and cyclic peptides capreomycin Bacterial resistance test.
  • tuberculosis remains one of the major public health problems in most developing countries, with multi-drug resistant tuberculosis (MDR-TB) and extensive drug resistant tuberculosis (XDR-TB).
  • MDR-TB multi-drug resistant tuberculosis
  • XDR-TB extensive drug resistant tuberculosis
  • the emergence of tuberculosis is even more serious.
  • China is one of the 22 countries with high burden of tuberculosis in the world.
  • the number of new tuberculosis patients ranks second in the world every year, especially the emergence of drug-resistant tuberculosis.
  • Fluoroquinolones, kanamycin, amikacin and capreomycin are four of the second-line anti-tuberculosis drugs recommended by the World Health Organization and play a very important role in the treatment of tuberculosis.
  • the timely detection of drug resistance of M. tuberculosis to these four drugs can guide clinical drug use and avoid delay in treatment, which is of great significance for effectively controlling the generation and spread of drug-resistant tuberculosis.
  • the drug resistance detection methods used in clinical practice include phenotypic detection and genetic detection, and are mainly based on traditional detection. Phenotypic detection methods such as Roche medium ratio method and Roche culture absolute concentration method, however, both methods have obvious limitations, which are affected by the slow growth of Mycobacterium tuberculosis, and take 6-8 weeks or longer to obtain result.
  • the existing genotyping methods are DNA sequencing (1. Wang H, Zhao C, He W, Zhang F, Zhang L, Cao B, et al. High prevalence of fluoroquinolone-resistant group B streptococci among clinical isolates in China And predominance of sequence type 19with serotype III. Antimicrob Agents Chemother 2013, 57:1538-41.), Linear Hybridization (2. Ando H, Mitarai S, Kondo Y, Suetake T, Kato S, Mori T, et al. Evaluation Of a line probe assay for the rapid detection of gyrA mutations associated with fluoroquinolone resistance in multidrug-resistant Mycobacterium tuberculosis.
  • the German HAIN company has a GenoType MTBDRs1 kit for detecting fluoroquinolone resistance-related gene gyrA mutation based on linear hybridization technology, but the kit is limited to the detection of fluoroquinolone resistance in second-line drugs.
  • the present application provides a nucleic acid fragment combination for detection of drug resistance of Mycobacterium tuberculosis, comprising two or all of the following (1) to (3):
  • T mutation at position 271 of SEQ ID NO: 1 is C, which results in the 91st position of the amino acid sequence (SEQ ID NO: 2) encoded by SEQ ID NO: 1 being mutated from Ser to Pro;
  • SEQ ID NO: 1 The A mutation at position 281 of SEQ ID NO: 1 is G, which results in mutation of the amino acid sequence encoded by SEQ ID NO: 1 (SEQ ID NO: 2) from Asp to Gly;
  • SEQ ID NO: 1 The A mutation at position 281 of SEQ ID NO: 1 is C, which results in mutation of the amino acid sequence encoded by SEQ ID NO: 1 (SEQ ID NO: 2) from Asp to Ala;
  • the G mutation at position 280 of SEQ ID NO: 1 is T, which results in mutation of the amino acid sequence of SEQ ID NO: 1 (SEQ ID NO: 2) from Asp to Tyr;
  • the application provides an oligonucleotide probe that specifically binds to the nucleic acid fragment combination of the first aspect.
  • the oligonucleotide probe is selected from at least two or all of the following:
  • SEQ ID NO: 9 or its complement SEQ ID NO: 10 or its complement, SEQ ID NO: 11 or its complement, SEQ ID NO: 12 or its complement, SEQ ID NO: 13 or its complement, SEQ ID NO: 14 or its complement, SEQ ID NO: 15 or its complement, SEQ ID NO: 17 or its complement, SEQ ID NO: 18 or its complement, SEQ ID NO: 20 or its complement, SEQ ID NO: 22 or its complement, SEQ ID NO: 23 or its complement, SEQ ID NO: 24 or its complement.
  • the application provides the use of the oligonucleotide probe of the second aspect in the preparation of a detection tool for M. tuberculosis resistance.
  • the present application provides a M. tuberculosis drug resistance test kit, comprising a solid phase carrier immobilized on the solid phase carrier capable of specifically detecting at least two or more or all of the following mutations Oligonucleotide probes:
  • the oligonucleotide probe is selected from one or more or all of the following:
  • SEQ ID NO: 9 or its complement SEQ ID NO: 10 or its complement, SEQ ID NO: 11 or its complement, SEQ ID NO: 12 or its complement, SEQ ID NO: 13 or its complement, SEQ ID NO: 14 or its complement, SEQ ID NO: 15 or its complement, SEQ ID NO: 17 or its complement, SEQ ID NO: 18 or its complement, SEQ ID NO: 20 or its complement, SEQ ID NO: 22 or its complement, SEQ ID NO: 23 or its complement, SEQ ID NO: 24 or its complement.
  • a wild-type probe corresponding to the mutated partial sequence in the wild-type sequence that is not mutated is specifically immobilized on the solid support, for example, the wild-type probe is selected from one or more of the following One: SEQ ID NOs: 8, 16, 19, 21.
  • the kit further comprises one or more of the following components:
  • a primer pair for amplifying a gene fragment in which the mutation site is located for example, a primer pair selected from one or more of the following: SEQ ID NOs: 25 and 26, 27 and 28, 29 and 30 , 31 and 32;
  • a probe for identifying a Mycobacterium tuberculosis complex for example, the probe sequences are SEQ ID NOs: 6 and 7 or their complementary sequences;
  • a biotin-labeled DNA probe for monitoring the quality of hybridization for example, the sequence of the probe is SEQ ID NO: 5 or its complement.
  • test sample is selected from the group consisting of cultures comprising M. tuberculosis, sputum, pleural effusion, bronchial wash, cerebrospinal fluid, peripheral blood, and pathological tissue.
  • the solid support is a nylon membrane.
  • the present application provides a method for detecting drug resistance of Mycobacterium tuberculosis, comprising contacting a sample with the oligonucleotide probe of the second aspect or the solid phase carrier of the kit of the fourth aspect And detecting and analyzing the hybridization of the sample to an oligonucleotide probe or a solid support.
  • Figure 1 is a diagram showing the use of the exemplary detection membrane strip of the present application for the fluoroquinolone resistance-related genes gyrA, kanamycin, amikacin and capreomycin resistance related genes rrs, eis promoter of Mycobacterium tuberculosis Mutation test results.
  • Figure 2 is a schematic representation of an exemplary solid phase vector of the present application incorporating a probe for detecting mutations in the gyrA gene, the rrs gene, and the eis gene promoter.
  • SEQ ID NO: 1 cDNA/coding sequence of wild type gyrA
  • SEQ ID NO: 2 amino acid sequence of wild type gyrA
  • SEQ ID NO: 3 wild type rrs sequence
  • SEQ ID NO:4 wild type eis promoter (position -139 to -1, wherein the transcription initiation site is the first position);
  • SEQ ID NO: 1-4 are all derived from Mycobacterium tuberculosis, and SEQ ID NOs: 8-30 are based on SEQ ID NO: 1-4;
  • SEQ ID NO: 5 Bio sequence, which is a biotin-labeled DNA probe for monitoring the success of a hybridization experiment
  • SEQ ID NOS: 6 and 7 16S rRNA-1 and 16S rRNA-2 probes for identifying M. tuberculosis complexes
  • SEQ ID NO:8 gyrA 90-94 WT probe for the specific detection of wild-type probes in which the codons of amino acids 90-94 of gyrA are not mutated;
  • SEQ ID NO: 9 gyrA 90GCG-GTG probe for detecting a GCG-GTG mutation mutation detecting probe for specifically detecting the codon 90 of amino acid of gyrA;
  • SEQ ID NO: 10 gyrA 91TCG-CCG probe for specific detection of gyrA 91 a mutation detecting probe for a TCG-CCG mutation at a codon of an amino acid;
  • SEQ ID NO: 11 gyrA 94GAC-GGC probe for mutation detection probe for the specific detection of the codon-generating GAC-GGC mutation of amino acid 94 of gyrA;
  • SEQ ID NO: 12 gyrA 94GAC-GCC probe for mutation detection probe for the specific detection of the codon-generating GAC-GCC mutation of amino acid 94 of gyrA;
  • SEQ ID NO: 13 gyrA 94GAC-TAC probe for mutation detection probe for the specific detection of the codon-generating GAC-TAC mutation of amino acid 94 of gyrA;
  • SEQ ID NO: 14 gyrA 94GAC-AAC probe for mutation detection probe for the specific detection of the codon-generating GAC-AAC mutation of amino acid 94 of gyrA;
  • SEQ ID NO: 15 gyrA 94GAC-CAC probe for mutation detection probe for the specific detection of the codon-generating GAC-CAC mutation of amino acid 94 of gyrA;
  • SEQ ID NO: 16 rrs 1401/1402 WT probe for specific detection of wild type probes at positions 1401 and 1402 of rrs that have not been mutated;
  • SEQ ID NO: 17 rrs 1401A-G probe for specifically detecting a mutation detecting probe at position 1401 where an A-G mutation occurs in rrs;
  • SEQ ID NO:18 rrs 1402C-T probe for specific detection of a mutation detecting probe for the C-T mutation at position 1402 of rrs;
  • SEQ ID NO: 19 rrs 1484 WT probe for specific detection of wild type probe at position 1484 of rrs that has not been mutated;
  • SEQ ID NO: 20 rrs 1484G-T probe for specifically detecting a mutation detecting probe at the 1484th position of rrs in which a G-T mutation occurs;
  • SEQ ID NO: 21 an eis-17 ⁇ +1 WT probe for specifically detecting a wild-type probe that has not been mutated from position -17 to +1 of the eis promoter;
  • SEQ ID NO:22 an eis-14C-T probe for specifically detecting a mutation detecting probe for a C-T mutation at position -14 of the eis promoter;
  • SEQ ID NO:23 an eis-12C-T probe for specifically detecting a mutation detecting probe for a C-T mutation at position -12 of the eis promoter;
  • SEQ ID NO:24 an eis-10G-A probe for specifically detecting a mutation detecting probe at the -10th position of the eis promoter to generate a G-A mutation;
  • SEQ ID NOS: 25 and 26 Forward and reverse amplification primers for the gyrA detection region, amplification of SEQ ID Fragment of the 88th to the 396th position of NO:1;
  • SEQ ID NOs: 27 and 28 forward and reverse amplification primers of the rrs detection region, amplifying a fragment of positions 1223 to 1512 of SEQ ID NO: 3;
  • SEQ ID NOS: 29 and 30 forward and reverse amplification primers for the detection region of the eis promoter, amplifying a fragment from -123 to +205 of the eis promoter region, wherein SEQ ID NO: 4, The transcription start site is the first position;
  • SEQ ID NOS: 31 and 32 Forward and reverse amplification primers for 16S rRNA for amplification of positions 94 to 373 of 16S rRNA.
  • This application is based, at least in part, on the analysis of resistance gene mutation sites in clinical isolates of M. tuberculosis.
  • the inventors of the present application sequenced drug resistance genes related to fluoroquinolones, kanamycin, amikacin, and capreomycin in 252 clinical isolates of Mycobacterium tuberculosis, and were resistant.
  • the genes include the gyrA, rrs, and eis promoters, and the sequencing results were analyzed by examining the domestic and foreign M. tuberculosis resistance-related gene mutations in the CNKI database and the PUBMED database.
  • the present application provides a nucleic acid fragment combination for detection of drug resistance of Mycobacterium tuberculosis, comprising two or all of the following (1) to (3):
  • T mutation at position 271 of SEQ ID NO: 1 is C, which results in the 91st position of the amino acid sequence (SEQ ID NO: 2) encoded by SEQ ID NO: 1 being mutated from Ser to Pro;
  • SEQ ID NO: 1 The A mutation at position 281 of SEQ ID NO: 1 is G, which results in mutation of the amino acid sequence encoded by SEQ ID NO: 1 (SEQ ID NO: 2) from Asp to Gly;
  • SEQ ID NO: 1 The A mutation at position 281 of SEQ ID NO: 1 is C, which results in mutation of the amino acid sequence encoded by SEQ ID NO: 1 (SEQ ID NO: 2) from Asp to Ala;
  • the G mutation at position 280 of SEQ ID NO: 1 is T, which results in mutation of the amino acid sequence of SEQ ID NO: 1 (SEQ ID NO: 2) from Asp to Tyr;
  • (1) above is a mutant nucleic acid fragment directed against the fluoroquinolone resistance gene gyrA
  • (2)-(3) is directed against kanamycin, amikacin, and the capreomycin resistance gene rrs, eis.
  • Mutant nucleic acid fragment Fluoroquinolones are routinely used in the treatment of tuberculosis, including but not limited to, ofloxacin, levofloxacin, ciprofloxacin, and moxifloxacin. According to previous reports, kanamycin, amikacin and capreomycin have cross-resistance, so the detection of drug resistance can be directed to a common drug resistance gene.
  • nucleic acid sequence mutants described above also include complementary sequences thereof.
  • the nucleic acid fragment combination can include all of (1)-(3) to achieve full detection of resistance to the four drugs.
  • the combination of nucleic acid fragments can include all of (1.1)-(1.7), (2.1)-(2.3), (3.1)-(3.3).
  • the combination of nucleic acid fragments can include one or more of (1)-(3) to enable detection of resistance to one or more target drugs.
  • the mutant of the nucleic acid sequence described in (1)-(3) is selected from the full-length coding sequence or a part of the promoter sequence, and such a segment belongs to the "high-risk” segment of the resistance-related mutation. Or called “hypervariable area” or “drug resistance determining area.” Accordingly, the present application also provides variants of any of the above nucleic acid sequence mutants, which may be the nucleic acid sequence A variant obtained by substitution, deletion (eg, truncation at one or both ends) or addition (eg, elongation at one or both ends) of one or more nucleoside segments, as long as the variant is capable of retaining the mutation point. In some embodiments, the variant may also be a fragment of a nucleic acid sequence mutant that retains the mutation site.
  • the application provides an oligonucleotide probe that specifically binds to the nucleic acid fragment combination of the first aspect.
  • the oligonucleotide probe is selected from at least two or all of the following:
  • SEQ ID NO: 9 or its complement SEQ ID NO: 10 or its complement, SEQ ID NO: 11 or its complement, SEQ ID NO: 12 or its complement, SEQ ID NO: 13 or its complement, SEQ ID NO: 14 or its complement, SEQ ID NO: 15 or its complement, SEQ ID NO: 17 or its complement, SEQ ID NO: 18 or its complement, SEQ ID NO: 20 or its complement, SEQ ID NO: 22 or its complement, SEQ ID NO: 23 or its complement, SEQ ID NO: 24 or its complement.
  • the oligonucleotide probe of the mutation of (1.1) in the first aspect is detected as shown in SEQ ID NO: 9 or is a complementary sequence thereof; and the mutation of (1.2) in the first aspect is detected.
  • the oligonucleotide probe is as shown in SEQ ID NO: 10 or is a complementary sequence thereof;
  • the oligonucleotide probe for detecting the mutation in (1.3) of the first aspect is as shown in SEQ ID NO: 11 or is complementary thereto a sequence; detecting the oligonucleotide probe of the mutation in (1.4) of the first aspect as shown in SEQ ID NO: 12 or a complementary sequence thereof; detecting the mutated oligonucleotide of (1.5) in the first aspect
  • the probe is as shown in SEQ ID NO: 13 or is a complementary sequence thereof;
  • the oligonucleotide probe for detecting the mutation in (1.6) of the first aspect is as shown in SEQ ID NO: 14 or is a complementary sequence thereof;
  • the oligonucleotide probe for detecting the mutation of (2.2) in the first aspect is as shown in SEQ ID NO: 18 or is a complementary sequence thereof;
  • the mutated oligonucleotide probe is represented by SEQ ID NO: 20 or is a complementary sequence thereof;
  • the oligonucleotide probe for detecting the mutation of (3.1) in the first aspect is SEQ ID NO: 22 or a complementary sequence thereof;
  • the oligonucleotide probe for detecting the mutation of (3.2) in the first aspect is as shown in SEQ ID NO: 23 or a complementary sequence thereof; and the first aspect (3.3) is detected.
  • the mutated oligonucleotide probe is represented by SEQ ID NO: 24 or is a complementary sequence thereof.
  • G/C content is between 40% and 70%; probe sequence is between 13 and 25 bases; Too many repeats of the same base, especially G should not exceed 4 or more; try to use a probe with C more than G; the mutation site should be located in the center of the probe as much as possible; the Tm value designed using the clone manager should be as much as possible at 60- Between 65 ° C.
  • wild type strain may refer to a drug (eg, a fluoroquinolone, kanamycin, amikacin, and/or capreomycin)-sensitive strain
  • a “mutant strain” may refer to a drug-resistant strain
  • Wild-type sequence may refer to a sequence in which no resistance-related mutation occurs
  • mutant sequence mutant may refer to a sequence in which a drug-resistant mutation described herein occurs
  • wild-type probe may A probe for specifically detecting a sequence in which a drug resistance-related mutation described herein does not occur
  • a “mutation detection probe” may refer to a sequence for specifically detecting a drug resistance-related mutation occurring as described herein. Probe.
  • telomere sequence As used herein, “specifically detect”, “specifically bind” or similar terms refers to the property of being able to detect the presence or absence of a target sequence while not detecting or binding to a non-target sequence.
  • a probe capable of specifically detecting or binding a wild type sequence refers to a probe capable of binding to a wild type sequence or a segment thereof while not binding to a site other than the wild type sequence.
  • a probe capable of specifically detecting or binding a mutant sequence refers to a site capable of binding to a mutant sequence or a segment thereof without binding to a wild type sequence or a wild type sequence and a sequence other than the mutant sequence. Probe.
  • probes that are capable of specifically detecting or binding to a wild-type sequence or a mutant sequence are designed for the sequence of the region in which the mutation site is located. It should also be understood that other techniques can be used to help achieve specific detection or specific binding. For example, by specifically amplifying a gene fragment including a target sequence from a genomic sequence of a sample, the existence of "other sequences" is excluded, and then the designed probe is brought into contact with the amplification product, thereby achieving specific detection or specificity. Combine.
  • the present application provides the oligonucleotide probe of the second aspect in the preparation of a knot Use in detection tools for the protection of nuclear mycobacteria.
  • oligonucleotide probes in the field of molecular biological detection, such as, but not limited to, reverse dot blot hybridization membrane method, reverse dot blot hybridization membrane strip method, linear reverse dot blot hybridization method, biochip method, liquid chip Method, fluid chip method, etc.
  • the detection tool is a membrane or membrane strip that utilizes reverse dot blot hybridization.
  • the present application provides a M. tuberculosis drug resistance test kit, comprising a solid phase carrier immobilized on the solid phase carrier capable of specifically detecting at least two or more or all of the following mutations Oligonucleotide probes:
  • the oligonucleotide probe is selected from one or more or all of the following:
  • SEQ ID NO: 9 or its complement SEQ ID NO: 10 or its complement, SEQ ID NO: 11 or its complement, SEQ ID NO: 12 or its complement, SEQ ID NO: 13 or its complement, SEQ ID NO: 14 or its complement, SEQ ID NO: 15 or its complement, SEQ ID NO: 17 or its complement, SEQ ID NO: 18 or its complement, SEQ ID NO: 20 or its complement, SEQ ID NO: 22 or its complement, SEQ ID NO: 23 or its complement, SEQ ID NO: 24 or its complement.
  • the oligonucleotide probe of the mutation of (a) in the fourth aspect is detected as shown in SEQ ID NO: 9 or is a complementary sequence thereof; and the mutation of (b) in the fourth aspect is detected.
  • the oligonucleotide probe is as shown in SEQ ID NO: 10 or is a complementary sequence thereof;
  • the oligonucleotide probe for detecting the mutation in (c) of the fourth aspect is as shown in SEQ ID NO: 11 or is complementary thereto a sequence; detecting the mutated oligonucleotide probe of (d) in the fourth aspect as shown in SEQ ID NO: 12 or a complementary sequence thereof; detecting the mutated oligonucleotide of (e) in the fourth aspect
  • the probe is as shown in SEQ ID NO: 13 or is a complementary sequence thereof;
  • the oligonucleotide probe for detecting the mutation in (f) of the fourth aspect is as shown in SEQ ID NO: 14 or is a complementary sequence thereof;
  • the wild type vector is further immobilized with a wild type probe that specifically detects a sequence corresponding to the mutant portion of the wild type sequence in which no mutation has occurred, for example, wild type
  • the probe may be selected from one or more of the following: SEQ ID NOs: 8, 16, 19, 21.
  • SEQ ID NOs: 8, 16, 19, 21 The structure and function of the above specific sequences can be found in the "Sequence Description" section of this document. The results of the hybridization obtained from the wild type probe and the mutation detecting probe can be mutually confirmed to further determine the type of mutation.
  • the 5' or 3' end of the probe is modified by amination.
  • the 5' or 3' end of the probe is labeled with biotin.
  • the test sample is selected from the group consisting of cultures comprising M. tuberculosis, sputum, pleural effusion, bronchial wash, cerebrospinal fluid, peripheral blood, and pathological tissue.
  • the sample is subjected to treatment, such as culturing and isolating M. tuberculosis, and isolating and extracting genomic DNA or RNA from M. tuberculosis culture. Therefore, the kit may also include the reagents used in the above steps.
  • the kit can be used for in situ detection, for example, detection at the genomic level.
  • the fragment of the resistance gene is required to be amplified prior to hybridization detection due to the low copy number of the resistance gene in the test sample or M. tuberculosis culture.
  • the amplification method used is a PCR method. Those skilled in the art will appreciate that the amplification product should be specific (i.e., not amplify a fragment other than the target gene) and should contain the location of the mutation site. Those skilled in the art will be able to design suitable primers and amplify the desired gene fragments according to the above requirements.
  • the kit further comprises a primer pair for amplifying the gene fragment in which the mutation site is located, for example, the primer pair may be selected from one or more of the following: SEQ ID NOs: 25 And 26, 27 and 28, 29 and 30, 31 and 32.
  • the kit may also include reagents for the amplification step.
  • the 5' end of the primer is labeled with biotin.
  • the kit further comprises a M. tuberculosis complex 16S rRNA sequence probe for monitoring the PCR system and whether it is a Mycobacterium tuberculosis complex
  • the 16S rRNA sequence probe can be SEQ ID NOs: 6 and 7 or their complementary sequences. If the hybridization results of the two 16S rRNA sequence probes are all positive, the test strain is a Mycobacterium tuberculosis complex strain.
  • the design and use of 16S rRNA sequence probes are well known to those skilled in the art.
  • the kit further comprises a biotin-labeled DNA probe for monitoring whether the hybridization is successful, for example, can be SEQ ID NO: 5 or its complement. If the hybridization result of the probe was positive, it was confirmed that the hybridization was successful.
  • a biotin-labeled DNA probe for monitoring whether the hybridization is successful for example, can be SEQ ID NO: 5 or its complement. If the hybridization result of the probe was positive, it was confirmed that the hybridization was successful.
  • the design and use of labeled biotin DNA probes is well known to those skilled in the art.
  • the sequence used to monitor the hybridization experiment can be any 15-15 base sequence other than the plurality of PCR amplification sequences in the present application. Those skilled in the art will appreciate that other methods of labeling, including but not limited to digoxin labeling, etc., can be used in addition to biotin labeling.
  • the solid support is a membrane strip or membrane. In a specific embodiment, the solid support is a nylon membrane strip or a nylon membrane.
  • Figure 2 shows a schematic diagram of a solid phase support of the present application incorporating a mutation detection probe capable of specifically detecting all of the mutations of (a)-(m) described above, as described above. Wild type probe, 16S rRNA probe and DNA probe labeled with biotin.
  • the present application provides a method for detecting drug resistance of Mycobacterium tuberculosis, comprising contacting a sample with the oligonucleotide probe of the second aspect or the solid phase carrier of the kit of the fourth aspect And detecting and analyzing the hybridization of the sample to an oligonucleotide probe or a solid support.
  • the result of the negative hybridization of the wild type probe indicates that the corresponding gene has a mutation (i.e., the presence of drug resistance), and the positive hybridization result of the mutation detection probe indicates the site where the mutation exists in the corresponding gene.
  • the technical features of the detection method may be according to the technical features disclosed in the oligonucleotide probe of the second aspect or the kit of the fourth aspect.
  • the main instruments or equipment used are as follows:
  • the main prepared solution is as follows:
  • Drug sensitivity tests were performed using the conventional Roche medium ratio method. TB taken grown for 4 weeks at -5 weeks ring Jensen medium 1, bacteria formulated as a grinding McFarland turbidity, and then transferred into saline 10 -2 mg / ml and 10 -4 mg / ml broth Inoculate 10 -3 mg and 10 -5 mg of bacterial liquid onto the medium containing anti-tuberculosis drugs, and incubate at 37 ° C for 4 weeks to observe the results. When the number of growth colonies on the drug-containing medium is greater than or equal to 1% of the number of colonies on the control medium, it is judged that the strain is resistant to the drug.
  • the drug concentration in Roche medium was as follows: fluoroquinolone 3 ⁇ g/ml, kanamycin 30 ⁇ g/ml, capreomycin 40 ⁇ g/ml, and amikacin 30 ⁇ g/ml.
  • the above amplification products were sent to a sequencing company for sequencing.
  • FIG. 1 is a sterile water negative control
  • 2 is a H 37 R V standard sensitive strain, used to test whether the obtained hybridization results are correct
  • 3 7-16 are single quinolone resistant strains
  • 4-6 are quinolone and kanamycin double Drug resistant strains.
  • AB is a 16S rRNA probe (SEQ ID NO: 6-7), and when AB is hybridized positive, the strain is a Mycobacterium tuberculosis complex strain, thereby indicating that the result of CR is reliable.
  • C is a gyrA 90-94 wild type probe (SEQ ID NO: 8), and DJ is a 90th, 91, and 94 amino acid mutant probe of gyrA (SEQ ID NO: 9-15), when wild type C of C
  • DJ is a 90th, 91, and 94 amino acid mutant probe of gyrA (SEQ ID NO: 9-15)
  • K is the rrs 1401/1402 wild type probe (SEQ ID NO: 16).
  • LM is a rrs 1401/1402 mutant probe (SEQ ID NO: 17-18), O is a rrs 1484 wild type probe (SEQ ID NO: 19), and N is a rrs 1484 mutant probe (SEQ ID NO: 20), when the K and O wild type probe positions are deleted, it is judged that there is a mutation of rrs, and if the hybridization signal appears at the corresponding position of the LM and N mutant probes, the specific type of rrs mutation can be known; P is the eis promoter -17 ⁇ +1 wild type probe (SEQ ID NO: 21), QS is an eis promoter mutant probe (SEQ ID NO: 22-24), and is judged as eis when the position of the P wild type probe is deleted.
  • the promoter has a mutation, and the specific type of rrs mutation can be known if a hybridization signal appears at the corresponding position of the QS mutant probe.
  • 3 7-11 is a codon mutation of amino acid sequence of amino acid sequence of gyrA (SEQ ID NO: 2), the mutation type is GCG(Ala)-GTG(Val); 4 is the amino acid sequence of gyrA (SEQ ID The codon of the 90th amino acid of NO: 2), and the base of base 10 of the eis promoter sequence (SEQ ID NO: 4) are mutated, and the mutation types are GCG (Ala)-GTG (Val) and GA, respectively; The codon of the amino acid sequence of the amino acid sequence of gyrA (SEQ ID NO: 2) and the base of the eis promoter sequence (SEQ ID NO: 4) were mutated at base - 14, and the mutation type was TCG (Ser) - CCG (Pro) and CT; 6 is the codon of the amino acid sequence of the amino acid sequence of gyrA (SEQ ID NO: 2), and the 1401 base of the rrs sequence (SEQ ID NO: 3) is mutated
  • GCG(Ala)-GTG(Val) and AG 12 is a codon mutation of amino acid sequence 91 of the amino acid sequence of gyrA (SEQ ID NO: 2), and the mutation type is TCG(Ser)-CCG(Pro); 13-16 is a codon mutation of amino acid sequence 94 of the amino acid sequence of gyrA (SEQ ID NO: 2), and the mutation type is GAC(Asp)-GGC(Gly), respectively.
  • This example tested the effects of fluoroquinolone, kanamycin, amikacin and capreomycin sensitive detection strips prepared according to the present application on a larger scale, and with conventional solids. The results obtained by the medium ratio method (phenotypic detection) and DNA sequencing were cross-compared.
  • the gyrA gene mutation analysis was performed on 214 strains of Mycobacterium tuberculosis clinical isolates, and the results showed 59 strains of fluoroquinolone. Mutations were detected in 40 strains of resistant strains (including mutations at positions 90, 91, and 94 of the gyrA amino acid sequence described above), and 6 of 174 rifampicin-sensitive strains detected mutations; The proportional method is a reference, and the sensitivity and specificity are 67.8% (40/59) and 96.6% (168/174), respectively.

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Abstract

L'invention concerne une combinaison de fragments nucléiques associés à un test de résistance aux médicaments de Mycobacterium tuberculosis de quatre types de médicaments de deuxième ligne, les fluoroquinolones, les aminoglycosides, tels que la kanamycine et l'amikacine (l'amikacine (BBK 8)), et les cyclopeptides, tels que la capréomycine, pour le traitement de la tuberculose et une utilisation correspondante. En particulier, l'invention concerne une combinaison de fragments nucléiques de mutation d'un promoteur de gènes gyrA associés à la résistance aux médicaments de fluoroquinolones, de gènes rrs et eis associés à la résistance aux médicaments d'aminoglycosides, tels que la kanamycine et l'amikacine (amikacine (BBK 8)) et de cyclopeptides, tels que la capréomycine, une sonde oligonucléotidique pour la détection spécifique de mutations, un kit préparé à partir de celle-ci et un procédé de détection pertinent de la résistance aux médicaments.
PCT/CN2015/079022 2014-05-15 2015-05-15 Combinaison de fragments spécifiques de gène de résistance à un médicament pour quatre types de médicaments de deuxième ligne contre mycobacterium tuberculosis et son utilisation WO2015172734A1 (fr)

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CN106480183A (zh) * 2016-09-29 2017-03-08 凯杰（苏州）转化医学研究有限公司 焦磷酸测序检测结核分枝杆菌卡那霉素、阿米卡星、卷曲霉素耐药性的方法
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WO2017095271A1 (fr) * 2015-12-01 2017-06-08 Общество С Ограниченной Ответственностью "Энджентикс" Procédé de détection moléculaire-génétique de la résistance de mycobactéries de la tuberculose à des préparations anti-tuberculose de second rang (fluoroquinolones, aminoglycosides et capréomycine)
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CN114582429A (zh) * 2022-03-03 2022-06-03 四川大学 基于层次注意力神经网络的结核分枝杆菌耐药性预测方法及装置
CN114582429B (zh) * 2022-03-03 2023-06-13 四川大学 基于层次注意力神经网络的结核分枝杆菌耐药性预测方法及装置

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