A kit for detecting drug-resistant genes of Mycobacterium tuberculosis and its application

A Mycobacterium tuberculosis and gene technology, applied in the direction of microbial determination/testing, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of low sensitivity, long cycle, low capreomycin resistance, etc.

Active Publication Date: 2017-10-27
BOAO BIOLOGICAL CO LTD +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] Capreomycin resistance is closely related to related gene mutations, including 16S rRNA gene and tlyA gene, however, the most important drug resistance gene is 16S rRNA gene, and about 78-80% of capreomycin resistance is related to the mutation of this gene Related, especially the single base substitution of 1401(A→G), the mutation rate is about 72%, but this mutant only causes capreomycin low resistance, 1402(C→T) and 1484(G→T) The mutation rate in serotonin is low, about 5-6%, but they can lead to strains that are highly resistant to capreomycin
[0013] The clinical detection of Mycobacterium tuberculosis has always relied mainly on drug susceptibility testing, but the traditional drug susceptibility testing on solid medium has a long period and low sensitivity, which seriously affects the early diagnosis and treatment of patients.

Method used

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  • A kit for detecting drug-resistant genes of Mycobacterium tuberculosis and its application
  • A kit for detecting drug-resistant genes of Mycobacterium tuberculosis and its application
  • A kit for detecting drug-resistant genes of Mycobacterium tuberculosis and its application

Examples

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Embodiment 1

[0205] Example 1, Primers and Probes for Detecting Mycobacterium tuberculosis drug-resistant gene mutations

[0206] 1. Primer design and synthesis

[0207] The drug resistance genes of Mycobacterium tuberculosis to be detected are: rifampicin resistance (rpoB), isoniazid resistance (katG and / or inhA), streptomycin resistance (rpsL), ethambutol resistance (embB) and Fluoroquinolone (gyrA) gene.

[0208] In order to effectively amplify the target gene in the sample, the genome sequence of Mycobacterium tuberculosis H37RV was retrieved from the NCBI database, and the rpoB gene whose GenBank Accession Number is NC_000962 nucleotide 759807-763325 / GenBank Accession Number is NC_000962 No. 2153889 -2156111 nucleotide katG gene / GenBank Accession Number is NC_000962 inhA gene / GenBank Accession Number is NC_000962 781560-781934 nucleotide rpsL gene / GenBank Accession Number is NC_000962 4246514 -4249810 nucleotide embB gene / GenBankAccession Number is NC_000962 7302-9818 nucleotide gyr...

Embodiment 2

[0221] Example 2, real-time fluorescent quantitative PCR detection of multiple mutation sites in the drug-resistant gene of Mycobacterium tuberculosis

[0222] 1. The template used for PCR amplification is rpoB 511CTG→CCG or rpoB 533CTG→CCG or rpoB516GAC→GTC or rpoB 526CAC→CTC or rpoB 526CAC→CGC or rpoB 526CAC→TAC or rpoB526CAC→GAC or rpoB 531TCG→TGG or katG 315AGC→ AAC or katG 315AGC→ACC or inhA-15C→A or rpsL 43AAG→AGG or rpsL 88AAG→AGG or embB 306ATG→ATA or embB 306ATG→GTG or gyrA90GCG→GTG or gyrA 94GAC→GGC or rrs 1401A→G or rrs 1402C→ T or rrs 1484G→T mutation site genomic DNA (i.e. mutant genome) or H37RV genomic DNA (i.e. wild type genome).

[0223] Both the above-mentioned mutant genome and the wild-type genome were subjected to nucleic acid sequencing, and the sequencing results confirmed that the above-mentioned mutant genome and the wild-type genome used as templates were DNA containing target mutant bases or the wild-type genome.

[0224] 2. Real-time fluorescent qu...

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Abstract

The invention discloses a kit for detecting mycobacterium tuberculosis drug resistance gene and application of the kit. The invention discloses a probe set which can be used for detecting or assisting detection that whether resistance mutation is produced or not in a mycobacterium tuberculosis gene of a sample, wherein the probe set comprises at least one of the following probes any shown in sequences (1)-(20): (1) a sequence shown in SEQ ID No.13; (2) a sequence shown in SEQ ID No.14; (3) SEQ ID No.15; and (4) a sequence shown in SEQ ID No.16. The probe set disclosed by the invention has the advantages that a high-efficiency, sensitive and multiple real-time fluorescent PCR technology, mutation condition of seven drug resistant genes of mycobacterium tuberculosis in a to-be-detected sample can be detected in a short time, result sensitivity is high, specificity is good, and an analysis process is simple and convenient.

Description

technical field [0001] The invention relates to a kit for detecting drug-resistant genes of mycobacterium tuberculosis and an application thereof, belonging to the field of biotechnology. Background technique [0002] Tuberculosis is the infectious disease with the highest mortality rate caused by a single bacterial infection. At present, about 1 / 3 of the people in the world are infected with Mycobacterium tuberculosis, and the number of people who die from tuberculosis every year reaches 3 million. my country is one of the 22 countries with severe tuberculosis epidemics in the world, ranking second in the world. The fifth national tuberculosis epidemiological sampling survey showed that the prevalence rate of tuberculosis among people aged 15 and over was 459 / 100,000, and the multidrug resistance rate of tuberculosis patients was 6.8%. The co-occurrence of tuberculosis and AIDS and other diseases, as well as the phenomenon of multidrug resistance, have made the prevention...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/6858C12Q2561/113C12Q2563/107C12Q2537/143
Inventor 徐洪涛王璨项光新邢婉丽程京梁建琴赵雁林
Owner BOAO BIOLOGICAL CO LTD
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