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Primer probe group for detecting mycobacterium tuberculosis complex and rpoB mutation based on multi-enzyme isothermal rapid amplification technology and application of primer probe group

A technology for mycobacterium tuberculosis and mycobacteria, applied in the field of molecular biology and clinical detection

Active Publication Date: 2020-04-24
ICDC CHINA CDC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there is no method for the identification of Mycobacterium tuberculosis complex strains and the detection of rifampicin resistance of Mycobacterium tuberculosis based on the multi-enzyme constant temperature rapid amplification technology. Therefore, a multi-enzyme constant temperature rapid amplification technology is established. , The detection method used to detect Mycobacterium tuberculosis complex strains and rpoB gene mutation has important practical significance

Method used

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  • Primer probe group for detecting mycobacterium tuberculosis complex and rpoB mutation based on multi-enzyme isothermal rapid amplification technology and application of primer probe group
  • Primer probe group for detecting mycobacterium tuberculosis complex and rpoB mutation based on multi-enzyme isothermal rapid amplification technology and application of primer probe group
  • Primer probe group for detecting mycobacterium tuberculosis complex and rpoB mutation based on multi-enzyme isothermal rapid amplification technology and application of primer probe group

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 Design and synthesis of detection primers and probes suitable for multi-enzyme constant temperature rapid amplification technology

[0056] The present invention has carried out sufficient research and analysis on the genome sequences of Mycobacterium tuberculosis complex strains and non-tuberculous mycobacteria, combined with a large number of practical verifications and found that compared with non-tuberculosis mycobacteria, Mycobacterium tuberculosis complex strain in its rpoB gene ( Rv0667 or the corresponding nucleic acid sequence, the sequence is shown in SEQ ID NO.1), there is a specific base mutation at position 1296, and the site of the Mycobacterium tuberculosis complex strain is A; non-tuberculosis mycobacteria and this site The corresponding site is G. This locus can be used to differentiate between Mycobacterium tuberculosis complex strains and nontuberculous mycobacteria. The results of comparison of the specific sites of Mycobacterium tuberculo...

Embodiment 2

[0127] Example 2 Rapid and visual detection kit based on multi-enzyme constant temperature rapid amplification technology

[0128] This embodiment provides a rapid and visual kit based on the multi-enzyme constant temperature rapid amplification technology for the detection of Mycobacterium tuberculosis complex strains and the detection of rpoB mutations. The kit includes the following components:

[0129] (1) Primers and probes whose sequences are shown in SEQ ID NO.3-7;

[0130] (2) Multi-enzyme constant temperature rapid amplification reaction buffer: including dNTPs, recombinase, constant temperature polymerase, auxiliary protein, exonuclease, polyethylene glycol, Tris-HCl, magnesium acetate and single-chain binding protein;

[0131] (3) Lateral flow test strips and their reaction buffers that can simultaneously detect amplification products of three markers (BIO, DIG, FAM);

[0132] (4) ddH 2 O.

Embodiment 3

[0133] Example 3 Method for detecting Mycobacterium tuberculosis complex strains and rpoB gene mutations based on multi-enzyme constant temperature amplification technology

[0134] 1. Preparation of samples to be tested:

[0135] (1) Add 800µl of ddH to a 1.5ml screw centrifuge tube 2 O, use an inoculation loop to scrape a ring of bacteria to be tested from the Roche solid medium and place it in a centrifuge tube;

[0136] (2) Place the centrifuge tube at 80°C, heat for 30 minutes to inactivate, and centrifuge at 12000rmp for 5 minutes;

[0137] (3) Discard the supernatant and add ddH to the tube 2 O 1ml, mix well;

[0138] (4) Centrifuge at 12000rmp for 5min, discard the supernatant, add 150µl ddH 2 O, slightly shake and mix;

[0139] (5) Place in an environment of 100°C and heat for 20 minutes;

[0140] (6) Centrifuge at 12000rmp for 10min;

[0141] (7) Aspirate the supernatant as the sample to be tested;

[0142] The preparation process of the above samples to be t...

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Abstract

The invention relates to the technical field of molecular biology and clinical detection, in particular to a primer probe group for detecting a mycobacterium tuberculosis complex and rpoB mutations based on a multi-enzyme isothermal rapid amplification technology and application of the primer probe group. A 1296th-site base mutation of an rpoB gene is used as a molecular marker for identifying a mycobacterium tuberculosis complex strain, and a specific primer probe group is designed aiming at amino acid mutations of the 1296th-site and a 516th site, a 526th site and a 531st site of the rpoB; amethod for detecting the mycobacterium tuberculosis complex strain and the rpoB mutations based on a multi-enzyme isothermal rapid amplification technology is developed. The primer probe group disclosed by the invention is high in detection sensitivity and strong in specificity; the method has the advantages of low dependence on equipment, short detection time, realization of rapid and accurate detection, realization of visual detection in combination with a lateral flow test strip, and important application values for the infection detection of the Mycobacterium tuberculosis complex strain and the drug resistance detection of Mycobacterium tuberculosis.

Description

technical field [0001] The invention relates to the technical fields of molecular biology and clinical detection, in particular to a primer probe set and application thereof for detecting Mycobacterium tuberculosis complex and rpoB mutation based on a multi-enzyme constant temperature rapid amplification technology. Background technique [0002] Tuberculosis is mainly caused by Mycobacterium tuberculosis Complex (MTBC), a chronic infectious disease that seriously endangers human health, and the cases caused by Mycobacterium tuberculosis (MTB) infection account for the vast majority most. The Mycobacterium tuberculosis complex mainly includes Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum and Mycobacterium microti, etc.; among them, Mycobacterium microti has been clearly shown to be non-toxic to humans. In addition to pathogenicity, most Mycobacterium tuberculosis complex strains are pathogenic in humans; and produce approximately the same clinical ...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/04C12N15/11C12R1/32
CPCC12Q1/6844C12Q1/689C12Q2600/156C12Q2565/625
Inventor 赵丽丽万康林李马超刘海灿
Owner ICDC CHINA CDC
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