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Loop-mediated isothermal amplification (LAMP) detection method for mycobacterium tuberculosis, and special primer and kit thereof

A detection kit and technology for Mycobacterium tuberculosis, applied in the biological field, can solve the problems of the LAMP-specific primers and kits for Mycobacterium tuberculosis, and achieve the effect of being suitable for large-scale promotion and application, broad market prospects, and simple operation

Active Publication Date: 2013-07-24
INST OF PLA FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far, there are no LAMP-specific primers and kits for detecting Mycobacterium tuberculosis on the market.

Method used

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  • Loop-mediated isothermal amplification (LAMP) detection method for mycobacterium tuberculosis, and special primer and kit thereof
  • Loop-mediated isothermal amplification (LAMP) detection method for mycobacterium tuberculosis, and special primer and kit thereof
  • Loop-mediated isothermal amplification (LAMP) detection method for mycobacterium tuberculosis, and special primer and kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1. Primer Design for LAMP Detection of Mycobacterium tuberculosis

[0036] The Mycobacterium tuberculosis sequence (GenBank No.: CP002992.1) was retrieved from the American Gene Database, and homology analysis was performed by BLAST software to obtain the specific conserved target sequence IS6110 of Mycobacterium tuberculosis (sequence 1 in the sequence table), and then according to the conserved target DNA sequence, with software Primer design V4 design is used for the primer that LAMP detects to Mycobacterium tuberculosis, designs 5 sets of primers (JH164, JH118, JH99, JH75, JH54), through experimental comparison (referring to embodiment 2), finally selected The primer combination JH164 consisting of 6 primers (the 6 primers can be combined in any ratio, a specific optimized combination is given as an example in the reaction system of Example 2), and its primer sequences are shown in Table 1.

[0037] Table 1 is used for the primer JH164 that LAMP detects to m...

Embodiment 2

[0039] Embodiment 2, the establishment of the LAMP detection method of Mycobacterium tuberculosis of the present invention

[0040] The primer combination JH164 (containing six primers) listed in Table 1 for LAMP detection of Mycobacterium tuberculosis was used for LAMP detection of Mycobacterium enteritidis (from the Center for Infectious Disease Control, Institute of Disease Control and Prevention, Chinese People's Liberation Army) to obtain the best response System and reaction conditions, while using other 4 sets of primers (JH118, JH99, JH75, JH54) as a control, the specific method is as follows:

[0041] 1. Determination of the best response system

[0042] 1) Use the genomic DNA containing Mycobacterium tuberculosis (use Promega's Wizard Genomic DNA purification Kit commercial DNA extraction kit to extract the nucleic acid in the sample to be tested) as a template, and carry out LAMP amplification under the guidance of the primer combination JH164 (including six primers...

Embodiment 3

[0051] Embodiment 3, the specificity and sensitivity of the LAMP detection method of Mycobacterium tuberculosis of the present invention

[0052] One, the specific detection of the LAMP detection method of Mycobacterium tuberculosis of the present invention

[0053] Mycobacterium tuberculosis (the strains were from PLA 309 Hospital); Neisseria meningitidis, Streptococcus pneumoniae, Legionella, Haemophilus influenzae, Klebsiella pneumoniae (the above strains were all from the National CDC); Vibrio parahaemolyticus, Salmonella enteritidis, Salmonella paratyphi A, Shigella flexneri, Shigella sonnei, enteroinvasive Escherichia coli, pathogenic Escherichia coli, enterotoxigenic Escherichia coli (the above strains were obtained from Chinese People’s Liberation Army Disease Prevention Genomic DNA from the Control Center for Infectious Diseases) was used as a template. Double-distilled water was used as a negative control to test the specificity of the best LAMP detection method for...

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Abstract

The invention discloses a loop-mediated isothermal amplification (LAMP) detection method for mycobacterium tuberculosis, and a special primer and a kit thereof. The special primer contains a primer combination consisting of six primers, namely a forward inner primer (FIP), a backward inner primer (BIP), a left forward (LF) primer, a left backward (LB) primer, an F3 primer and a B3 primer. The LAMP detection method can be used for quickly, conveniently and efficiently detecting the mycobacterium tuberculosis with high specificity and high sensitivity under the isothermal condition, does not require complex instruments, and can be used for detecting pure bacteria samples, samples of clinical sputum and cerebrospinal fluid and the like. The method provides a new technical platform for detecting the mycobacterium tuberculosis, and can be used for screening and detecting the mycobacterium tuberculosis in basic-level medical health units and disease prevention and control centers.

Description

technical field [0001] The invention belongs to the molecular biology detection method of bacteria in the field of biotechnology, in particular to the LAMP detection method of Mycobacterium tuberculosis and its special primers and kit. Background technique [0002] Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis infection. Mycobacterium tuberculosis may invade various organs of the human body, but mainly invades the lungs, which is called pulmonary tuberculosis. Tuberculosis, also known as tuberculosis and "white plague", is an ancient infectious disease, and tuberculosis has existed since human beings existed. So far, tuberculosis is still a serious infectious disease. According to statistics, 1 / 3 of the world's population is infected with Mycobacterium tuberculosis. According to the WHO, about 8 million new cases occur each year and at least 3 million people die from the disease. my country is one of the 22 countries with a high burden...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
Inventor 袁静黄留玉刘威尹志涛邹大阳熊鸿燕
Owner INST OF PLA FOR DISEASE CONTROL & PREVENTION
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