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Method for detecting isoniazid drug resistance of mycobacterium tuberculosis by using pyrosequencing technology

A technology for Mycobacterium tuberculosis and pyrosequencing, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problems of inability to quickly obtain drug resistance, increase in detection time, and many times of reactions, etc. To achieve the effect of reducing the number of reactions, the amount of detection reagents, and shortening the detection time

Active Publication Date: 2013-04-17
SHANGHAI PULMONARY HOSPITAL
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Problems solved by technology

[0006] In the existing conventional methods for detecting drug resistance of Mycobacterium tuberculosis, it is unavoidable to isolate and cultivate Mycobacterium tuberculosis strains in advance from patient sputum specimens, which increases the detection time and the number of reactions that need to be performed during the detection. More, so that the results of drug resistance cannot be quickly drawn

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  • Method for detecting isoniazid drug resistance of mycobacterium tuberculosis by using pyrosequencing technology
  • Method for detecting isoniazid drug resistance of mycobacterium tuberculosis by using pyrosequencing technology
  • Method for detecting isoniazid drug resistance of mycobacterium tuberculosis by using pyrosequencing technology

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Embodiment Construction

[0024] Since the mutation of the KatG gene is mainly concentrated at the 315 site, the pyrosequencing detection method is mainly performed on the 315 site of the isoniazid KatG drug-resistant gene in the present invention. A method for detecting the drug resistance of Mycobacterium tuberculosis to isoniazid provided by the present invention comprises first performing PCR amplification on the KatG gene, and then performing pyrosequencing on the PCR amplification product to determine whether the KatG gene has a mutation site; Among them, pyrosequencing adopts SNP mode, and the nucleotide loading sequence is GTCACAGTCTG.

[0025] Pyrosequencing technology

[0026] Pyrosequencing (Pyrosequencing) technology is a brand-new DNA sequence analysis technology, which can quickly and accurately determine a short target fragment. This technique does not require electrophoresis, and the DNA fragments do not require fluorescent labeling, and the operation is extremely simple.

[0027] P...

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Abstract

The invention provides a method for detecting isoniazid drug resistance of mycobacterium tuberculosis by using a pyrosequencing technology. The method comprises: step 1, performing a PCR amplification for a KatG gene, step 2, performing the pyrosequencing of the PCR amplification products and determining whether or not the KatG gene has mutation points, wherein the pyrosequencing uses a SNP mode, and a nucleotide sampling sequence is GTCACAGTCTG. The method for detecting the isoniazid drug resistance of the mycobacterium tuberculosis by using the pyrosequencing technology can directly detect drug resistance from phlegm samples of patients, do not need to isolate and culture M. tuberculosis strains, and thus, compared with present detection methods, can shorten detection time, and reduce reaction times and amounts of detecting reagents required by the pyrosequencing.

Description

technical field [0001] The invention relates to a method for detecting the drug resistance of Mycobacterium tuberculosis, in particular to a primer and a detection method for directly detecting the drug resistance mutation of Mycobacterium tuberculosis from a sputum sample by using a pyrosequencing method. Background technique [0002] Isoniazid (NH), as an efficient and specific anti-tuberculosis drug, can be converted into an active form mainly through the action of hydrogen peroxide-peroxidase (encoded by the KatG gene) in Mycobacterium tuberculosis to act on the reduction of enoyl-ACP Enzyme, inhibits bacilic acid and cell wall synthesis. Since KatG protein is the only enzyme that can activate isoniazid, the decline or loss of its function will inevitably lead to isoniazid resistance of tuberculosis. Further studies have found that the main cause of clinical isoniazid resistance is not only KatG Complete deletions of genes, point mutations, insertions and deletions of b...

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/02
Inventor 郑瑞娟胡忠义周燕王洁秦莲花
Owner SHANGHAI PULMONARY HOSPITAL
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