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Concretion mycobacterium nucleic acid screening method based on loop-mediated isothermal amplification technique

A Mycobacterium tuberculosis, ring-mediated isothermal technology, applied in the field of clinical medicine, can solve the problems of high laboratory safety requirements, poor specificity, and low positive rate.

Inactive Publication Date: 2008-04-09
中华人民共和国徐州出入境检验检疫局
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Problems solved by technology

Although it is the "golden index" for diagnosing pulmonary tuberculosis, its main defect is its low positive rate
(2) Sputum tuberculosis culture, the results are highly reliable, and tuberculosis drug susceptibility test can be done, but it takes 6-8 weeks, and the application is limited. High safety requirements are not conducive to the universal screening of Mycobacterium tuberculosis
(3) Polymerase chain reaction (PCR), which has poor specificity and has the advantage that the sensitivity can reach 98% to 100%. At the same time, it requires PCR amplifiers, gel electrophoresis and other related expensive equipment, which is not conducive to grassroots medical epidemic prevention institutions and on-site rapid determination

Method used

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  • Concretion mycobacterium nucleic acid screening method based on loop-mediated isothermal amplification technique

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Embodiment Construction

[0036] The detection method of the differential diagnosis system is carried out in sequence according to the following steps:

[0037] (1) Nucleic acid extraction: use a part of the patient's sputum for Ziehl-Neelse staining, and add the same amount of 4% NaOH and 2.94% Tri-SodiumCitrate (containing 1% of N-acetyl-L-cysteine) to the remaining sputum , Let it stand at room temperature for 20 minutes to fully liquefy it to clear, then finally prepare the sputum in 20ml phosphate buffer, centrifuge at 4000r / min for 20min, wash twice, and suspend the precipitate in 2ml of the same buffer, take out the processed sputum The liquid sample was centrifuged at 14000r / min for 15min, 100ul of lysate was added to the pellet, incubated at 100°C for 30min, centrifuged at 14000r / min for 3min, and the supernatant was stored at -20°C until use.

[0038](2) LAMP reaction: prepare the above reaction system (10 servings, without Mycobacterium tuberculosis DNA extract), mix the reaction system, and ali...

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Abstract

The invention pertains to the clinical medicine field, disclosing a Mycobacterium tuberculosis DNA screening method which is based on a Loop-mediated isothermal amplification technology. A nucleic acid detecting method which is based on Loop-mediated isothermal amplification technical principle, detects the Mycobacterium tuberculosis in sputum. By adopting specific primers, and utilizing LAMP technology platform to amplify the specific area of target gene, the Mycobacterium tuberculosis in sputum is screened and detected in molecular level with the auxiliary of a series of quality control and internal control detecting system. The invention is characterized by simpleness and convenience, economy, quick speed, sensitivity and specific performance, and has wide application prospect.

Description

Technical field [0001] The invention belongs to the field of clinical medicine and relates to a nucleic acid screening method of Mycobacterium tuberculosis. More specifically, it is a nucleic acid screening method for Mycobacterium tuberculosis based on loop-mediated isothermal amplification technology. Background technique [0002] Tuberculosis is a chronic infectious disease caused by infection with Mycobacterium tuberculosis. In human history, tuberculosis, like smallpox, plague, cholera, and other severe infectious diseases, was once widespread throughout the world. In the 1950s, with the advent of anti-tuberculosis chemotherapeutics, the epidemic of tuberculosis was brought under certain control in developed countries. However, due to various reasons in recent years, the tuberculosis epidemic situation in some countries and regions has risen sharply, seriously endangering human health and becoming a major global public health problem. On April 23, 1993, at the 46th World Hea...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 易海华赵金伟钱志娟房超吴萍兰
Owner 中华人民共和国徐州出入境检验检疫局
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