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Primers, probes, method and kit for detecting mycobacterium tuberculosis specific gene

A technology of mycobacterium tuberculosis and mycobacteria, applied in the field of molecular biology, can solve the problems of low sensitivity, cumbersome operation, and poor repeatability, and achieve the effects of strong specificity, high sensitivity, and high accuracy

Inactive Publication Date: 2018-03-27
宁波基内生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The invention provides a primer, a probe, a method and a kit for detecting specific genes of Mycobacterium tuberculosis, which solve the problem of using bacterial culture to identify Mycobacterium tuberculosis (MTB) and non-tuberculous mycobacteria (NTM) in the prior art, Not only the technical problems of cumbersome operation, time-consuming and laborious, low sensitivity and poor repeatability

Method used

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  • Primers, probes, method and kit for detecting mycobacterium tuberculosis specific gene
  • Primers, probes, method and kit for detecting mycobacterium tuberculosis specific gene
  • Primers, probes, method and kit for detecting mycobacterium tuberculosis specific gene

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Embodiment 1

[0025] The embodiment of the present invention provides a kind of primer and probe that detects Mycobacterium tuberculosis specific gene, detects the nucleotide sequence of the primer of DNA gyrase B subunit (gyrB) gene of Mycobacterium tuberculosis specific gene such as SEQ ID NO: 2 Shown in ~ 3, the nucleotide sequence of the Taqman probe that detects Mycobacterium tuberculosis-specific gene DNA gyrase B subunit (gyrB) gene is shown in SEQ ID NO: 4; Detect non-tuberculous mycobacterium 16S ribosomal DNA gene The nucleotide sequence of the primer (16S rDNA) is shown in SEQ ID NO: 6-7, and the nucleotide sequence of the Taqman probe for detecting the non-tuberculous mycobacterium 16S ribosomal DNA gene (16S rDNA) is shown in SEQ ID NO: 8;

[0026] Wherein, the sequence of the specific primer designed for the Mycobacterium tuberculosis specific gene DNA gyrase B subunit (gyrB) gene is as follows:

[0027] Upstream primer P-gyrB-F: 5'-GAGATGGCGTTCCTCAACAAG-3'

[0028] Downstrea...

Embodiment 2

[0038] The embodiment of the present invention also provides a method for detecting Mycobacterium tuberculosis specific genes, comprising:

[0039] Step 101, sample nucleic acid preparation to obtain a nucleic acid template;

[0040] Step 102, designing specific primers and fluorescently labeled probes for the Mycobacterium tuberculosis-specific gene DNA gyrase B subunit (gyrB) gene and the non-tuberculous mycobacterium 16S ribosomal DNA gene (16S rDNA);

[0041] Wherein, the nucleotide sequence of the primer for detecting Mycobacterium tuberculosis gyrB is shown in SEQ ID NO: 2-3, and the nucleotide sequence of the Taqman probe for detecting Mycobacterium tuberculosis gyrB is shown in SEQ ID NO: 4; The nucleotide sequence of the primer for bacillus 16S rDNA is shown in SEQ ID NO: 6-7, and the nucleotide sequence of the Taqman probe for detecting non-tuberculous mycobacterium 16S rDNA is shown in SEQ ID NO: 8.

[0042] Step 103, configuring an enzyme reagent, wherein the enzy...

Embodiment 3

[0055] The embodiment of the present invention also provides a kit for detecting specific genes of Mycobacterium tuberculosis, including nucleic acid extraction solution, first primer-probe mixed solution, second primer-probe mixed solution, PCR reaction enzyme system, negative control products, positive control products, and packaging boxes for separating and collectively packaging these reagent bottles or tubes, wherein the first primer-probe mixture is rotated by deoxyribonucleoside triphosphate dN(U)TP, Mycobacterium tuberculosis-specific gene DNA Composed of upstream and downstream primers of the enzyme B subunit (gyrB) gene and a fluorescently labeled probe, the second primer-probe mixture consists of deoxyribonucleoside triphosphate dN(U)TP, non-tuberculous mycobacterium 16S ribosomal DNA Gene (16S rDNA) upstream and downstream primers and a fluorescent labeling probe; wherein, the sequence of the gyrB gene-specific upstream and downstream primers is shown in SEQ ID NO: ...

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Abstract

The invention relates to the technical field of molecular biology and discloses primers, probes, method and kit for detecting a mycobacterium tuberculosis specific gene. Through a Taqman probe real-time fluorescence PCR method, primers and fluorescent labeled probes are designed according to (gyrB) and 16S rDNA nucleic acid conserved regions. An enzyme and a sample nucleic acid are added into a PCR detection mixed solution of the primers and probes, a FAM channel of a fluorescence PCR device is used for amplification and a desired gene is detected through change of a fluorescence signal. The primers, probes, method and kit have the characteristics of high accuracy, strong specificity and high sensitivity and can realize fast and accurate detection of mycobacterium tuberculosis (MTB) and non-tuberculous mycobacteria (NTM).

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a primer, a probe, a method and a kit for detecting specific genes of Mycobacterium tuberculosis. Background technique [0002] Mycobacterium tuberculosis (MTB) is the main pathogenic bacterium that causes human tuberculosis. One-third of the world's population is infected with MTB, which is one of the serious public health threats. In recent years, non-tuberculous mycobacteria (NTM) infection has shown a clear upward trend. There are more than 160 types of NTM, and only a small part of them are pathogenic to humans. They mainly cause chronic lung infections, and NTM lung infections The clinical symptoms are similar to those of MTB infection and other respiratory diseases such as lung cancer, and it is difficult to distinguish them, but the medications are quite different. The commonly used anti-MTB drugs are rifampicin, isoniazid, ethambutol, streptomycin, etc. NTM in...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/04C12N15/11C12R1/32
CPCC12Q1/689C12Q2600/156
Inventor 倪剑锋王伟建刘文黄俊淇翁毅
Owner 宁波基内生物技术有限公司
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