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Primers, probe, kit, and method used for detecting mycobacterium tuberculosis isoniazid resistance mutation

A technology for Mycobacterium tuberculosis and drug-resistant mutations, applied in biochemical equipment and methods, material stimulation analysis, microbial measurement/inspection, etc., can solve the problems of increased product contamination, high cost, complicated processing steps, etc., and achieve cost-effective Inexpensive, non-pollution, and reliable test results

Inactive Publication Date: 2013-07-31
WUHAN BIOTECH GENE ENG
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] For high-throughput screening technologies such as liquid-phase chip method, MLPA technology, and pyrosequencing, which are applied to tuberculosis drug-resistant mutations, although these methods have a high detection rate, they are also expensive, and the PCR post-processing steps are complicated, which increases product contamination. chances, increasing the requirements for laboratories and personnel

Method used

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  • Primers, probe, kit, and method used for detecting mycobacterium tuberculosis isoniazid resistance mutation
  • Primers, probe, kit, and method used for detecting mycobacterium tuberculosis isoniazid resistance mutation
  • Primers, probe, kit, and method used for detecting mycobacterium tuberculosis isoniazid resistance mutation

Examples

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Embodiment 1

[0057] Example 1. A primer and probe for detecting the isoniazid resistance mutation of Mycobacterium tuberculosis

[0058] Sputum samples were collected from January 2010 to July 2012 in Wuhan Medical Treatment Center. A total of 350 cases were collected. According to the "Tuberculosis Diagnostic Laboratory Inspection Regulations", the acid-fast staining method and bacteria collection method were used for identification, And adopted improved Roche culture medium to isolate and cultivate Mycobacterium tuberculosis, then adopted Bactec960 drug susceptibility identification method to carry out drug susceptibility identification to 285 isolated strains, and obtained 62 strains of isoniazid drug-resistant strains, the embodiment of the present invention is promptly to these 62 strains Isoniazid-resistant strains were tested for drug resistance.

[0059] Primers and fluorescent probes for detection: According to the genome nucleic acid sequence of Mycobacterium tuberculosis H37Rv i...

Embodiment 2

[0070] Example 2. A kit for detecting isoniazid resistance mutations of Mycobacterium tuberculosis

[0071] The kit for detecting the isoniazid resistance mutation of Mycobacterium tuberculosis includes: nucleic acid extraction reaction solution, PCR reaction solution, control reagent, and the above reagents are all placed in the box.

[0072] Specifically, the nucleic acid extraction reaction solution is a TB DNA extraction solution, wherein the TB DNA extraction solution includes 1 mM disodium edetate, 10 mM Tris and 10% Triton-100.

[0073] Specifically, the PCR reaction liquid includes TB enzyme and INH PCR Mix A, wherein, INH PCR Mix A includes 1×PCRBuffer, 3.5mM MgCl 2 , 0.8 mM dNTPs, 0.4 μM dUTP, 0.8 μM forward primer of katG gene, 0.8 μM wild-type forward primer of katG gene, 0.8 μM first mutant forward primer of katG gene, 0.8 μM second mutant type of katG gene Forward primer, 0.8 μM reverse primer of katG gene, 0.2 μM fluorescent probe of katG gene, 0.8 μM forward p...

Embodiment 3

[0078] Embodiment 3. A method for detecting isoniazid resistance mutation of Mycobacterium tuberculosis:

[0079] Mycobacterium tuberculosis clinical sputum DNA extraction (one-step method):

[0080] The source of Mycobacterium tuberculosis used in the embodiment of the present invention is: Wuhan Medical Treatment Center.

[0081] Add 1M NaOH solution 1-3 times the volume of the sputum sample, and liquefy the sputum sample for 15 minutes. The amount of NaOH solution added depends on the viscosity of the sputum sample. For sputum samples with high viscosity, add 2-3 times the volume of the sputum sample, suck out 1mL of the liquefied sputum sample and place it in a 1.5mL centrifuge tube, centrifuge at 10000rpm for 15min to get the filter residue, add 800μL TE washing solution to the filter residue , wherein, the TE washing solution includes 10mM Tris-Cl pH8.5 and 0.5mM EDTA, centrifuges at 13000rpm for 10min, removes the supernatant, and adds 300μL of lysis solution, wherein,...

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Abstract

The invention discloses primers, a probe, a kit and a method used for detecting mycobacterium tuberculosis isoniazid resistance mutation. The invention belongs to the technical field of mycobacterium tuberculosis resistance mutations. The primers and probe used for detecting mycobacterium tuberculosis isoniazid resistance mutation comprises detection-use primers and a fluorescent probe. The kit comprises a nucleic acid extraction reaction solution, a PCR reaction solution, a control reagent, a nucleic acid extraction reaction solution, a PCR reaction solution, and a control reagent. The kit detection method comprises the steps of: (1) reagent preparation; (2) sample collection and processing; (3) sample dosing; (4) PCR amplification; and (5) result analysis and determination. The primers and the probe have high sensitivity and good specificity. The kit has low cost. With the detection method, simultaneous implementation can be carried out without cover opening, pollution is prevented, the result is more reliable, the operation is simple and fast, and quantitation is realized.

Description

technical field [0001] The invention relates to the technical field of detection of drug-resistant mutations of Mycobacterium tuberculosis, in particular to a primer, a probe, a kit and a method for detecting drug-resistant mutations of Mycobacterium tuberculosis isoniazid. Background technique [0002] Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis infection. Mycobacterium tuberculosis can invade various organs of the human body, but mainly invades the lungs and becomes pulmonary tuberculosis. According to the report of the World Health Organization in 2009, one-third of the world's population is currently infected with Mycobacterium tuberculosis, and there are more than 9 million new patients every year, of which 420,000 are multi-drug-resistant tuberculosis and 2 million deaths. my country is the 22nd in the world. One of the countries with a high burden of tuberculosis, the number of patients ranks second in the world after India. my cou...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11G01N21/64
Inventor 唐景峰柳建银程弘夏霍然
Owner WUHAN BIOTECH GENE ENG
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