Method of extracting mycobacterium tuberculosis DNA and kit for detecting multiple drug resistance of mycobacterium tuberculosis

A technology of Mycobacterium tuberculosis and kits, applied in the field of biomedical detection, can solve the problems of DNA extraction difficulties, time-consuming and labor-intensive problems

Inactive Publication Date: 2014-07-09
南京爱必梦生物材料有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0012] The invention provides a method for extracting Mycobacterium tuberculosis DNA to solve the problems of difficult DNA extraction, time-consuming and labor-intensive problems in the prior art

Method used

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  • Method of extracting mycobacterium tuberculosis DNA and kit for detecting multiple drug resistance of mycobacterium tuberculosis
  • Method of extracting mycobacterium tuberculosis DNA and kit for detecting multiple drug resistance of mycobacterium tuberculosis
  • Method of extracting mycobacterium tuberculosis DNA and kit for detecting multiple drug resistance of mycobacterium tuberculosis

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Experimental program
Comparison scheme
Effect test

preparation example Construction

[0343] Kit preparation:

[0344] 1) Prepare sputum sample liquefaction agent: Dilute 100% NaOH to a final concentration of 4%.

[0345] 2) Prepare DNA extraction solution: prepare 500ml DNA extraction solution, please refer to the following for specific methods

[0346] Preparation of DNA extraction solution (500ml)

[0347] component name

high concentration stock solution

Amount added

Final concentration

Guanidine isothiocyanate (GITC)

177.5 grams

3M

Dithiothreitol (DTT)

1M

500μl

1mM

Sodium Citrate

1.47 grams

10mM

Sodium dodecyl sulfonate (SDS)

10%

25ml

0.5%

Antifoaming agent (Antifoam)

10%

1ml

0.002%

Tris(Tris)

1M

50ml

100mM

Ethylenediaminetetraacetic acid (EDTA)

0.5M

100μl

0.1mM

Ethanol

100%

100ml

20%

nuclease-free water

Make up to 500ml

[0348] 3) The primer sequences ...

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Abstract

The invention discloses a method of extracting mycobacterium tuberculosis DNA and a kit for rapidly and accurately detecting multiple drug resistance (MDR) of mycobacterium tuberculosis. The method includes: extracting DNA to be detected from a clinical sample, determining mycobacterium tuberculosis infection by utilization of fluorescence real-time quantification PCR, indentifying whether the mycobacterium tuberculosis is drug-resistant or not and identifying the drug-resistant bacterial strain causing infection, and finally determining the pathogen infection quantity. The method can be used for detecting the whole drug-resistant genes with 29 sites that are determined to cause tubercle bacillus resistance to drugs (comprising rifampicin, isoniazide and ethambutol), and is the most comprehensive rapid molecular diagnosis system comprising tubercle bacillus drug-resistant genes so far. Through unique primers, probes and selection of fluorochrome marking designs, all the drug-resistant gene sites are detected by application of multiple qPCR. The application is simple, convenient and rapid. The results are accurate and reliable. The method and the kit have great practical value and wide application prospects for rapidly diagnosing tubercle bacillus infection and determining drug resistance, thus performing targeted treatment.

Description

technical field [0001] The invention relates to the technical field of biomedical detection, in particular to a method for extracting Mycobacterium tuberculosis DNA and a kit for rapidly and accurately detecting multiple drug resistance of Mycobacterium tuberculosis. Background technique [0002] Tuberculosis remains a global problem. At present, about 2 billion people are infected in the world, and about 8 million to 10 million new tuberculosis patients appear every year, and about 2 million to 3 million deaths due to tuberculosis every year. Tuberculosis has become one of the diseases that seriously endanger human health in the 21st century. China is also one of the countries where the incidence of tuberculosis is relatively serious. Nearly half of the population has been infected with tuberculosis bacteria. There are currently 5 million tuberculosis patients, and 1.5 million new patients are added every year. [0003] Although tuberculosis is no longer the kind of plague...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/68
Inventor 李沛祥
Owner 南京爱必梦生物材料有限公司
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