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Rapid identification method and kit of novel mycobacterium strain

A technology of mycobacteria and kits, which is applied in the field of medical molecular biology diagnosis, and can solve problems such as complicated operation, cross-contamination, expensive spotting system and fluorescent analysis system

Active Publication Date: 2012-08-15
THE 309TH HOSPITAL OF CHINESE PEOPLES LIBERATION ARMY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method is complicated to operate, easy to cause cross-contamination, and the required spotting system and fluorescence analysis system are expensive, so it is difficult to popularize and apply

Method used

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  • Rapid identification method and kit of novel mycobacterium strain
  • Rapid identification method and kit of novel mycobacterium strain
  • Rapid identification method and kit of novel mycobacterium strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1: Design and synthesis of primers and probes

[0054]The 16S rRNA gene is a traditional target gene sequence identified in bacterial taxonomy. The invention analyzes the disclosed 16S rRNA gene sequences of various species of Mycobacterium, and finds Mycobacterium-specific nucleotide sequences and species-specific variable regions. According to the three variable regions of the mycobacterial 16S rRNA gene sequence, three sets of mycobacterium-specific nested primers and mycobacterium species-specific probes were designed, among which the variable region 1 is located in the sequence X55588.1 in the NCBI database The variable region 2 is located between the 55th and 251st bases, the variable region 2 is located between the 1110th and 1314th bases, and the variable region 3 is located between the 366th and 539th bases. Use the software to design primer pairs in the mycobacteria-specific region, and design probes for the species-specific variable regions within th...

Embodiment 2

[0059] Embodiment 2 Mycobacterium strain identification method and establishment of standard curve

[0060] 2.1 Extraction of sample DNA

[0061] Standard strains: Mycobacterium tuberculosis (M.tuberculosis), Mycobacterium kansasii (M.kansasii), Mycobacterium scrofula (M.scrofulaceum), Mycobacterium simiae (M.simiae), Mycobacterium gastrice (M. .gastri), M. marinum, M. ulcerans, M. nonchromogenicum, M. aichiense, M. avium M. avium, M. diernhoferi, M. triviale, M. fortuitum, M. terrae, mycobacterium M.intracellulare, M.gordonae, M.xenopi, M.chelonae subsp.chelonae, M.chelonae M.chelonae subsp.abscessus, M.smegmatis, M.thermoresistibile, M.agri, Mycobacterium vaccae (M.vaccae), Mycobacterium phlei (M.phlei) standard strains were purchased from China Institute of Biological Products.

[0062] The genomic DNA of each of the above standard strains was extracted separately for strain identification and detection, or stored at -20°C for later use.

[0063] 2.2PCR amplification ...

Embodiment 3

[0097] Embodiment 3 clinical identification test

[0098] 3.1 Identification test I

[0099] 187 clinical isolates of Mycobacterium were collected, and the DNA was extracted according to the above method and then dissolved with ddH2O.

[0100] Add 10×PCR buffer (Mg 2+ Free) 2μl, 25mM MgCl 2 1.6 μl, 0.4 μl of 10 mM dNTPs, 0.1 μl each of 10 μM TBleft1, TBleft2 and TBright1, 0.6 μl of 10 μM TBright2, 0.4 μl of each of 10 μM TBProbe1, TBProbe2 and TBProbe3, 0.1 μl of 5 U / μl aTaq DNA polymerase, add 2 μl of the above-mentioned extract to be tested DNA, followed by ddH 2 O Supplement the reaction system to 20 μl. Negative control and blank control were set up in the reaction at the same time, so as to extract from other 14 non-mycobacterium strains (including Acinetobacter baumannii, Stenotrophomonas maltophilia, Enterobacter cloacae, Klebsiella, Staphylococcus aureus, Pseudomonas aeruginosa, Staphylococcus hominis subsp. hominis, Citrobacter freundii, Acinetobacter lophi, Esc...

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Abstract

The invention relates to a double-marking probe detecting and melting curve analyzing method used for identifying mycobacterium strains and a kit using the method to identify various mycobacterium strains at the same time. The kit provided by the invention comprises a primer capable of designing mycobacterium 16S rRNA, a double-marking oligonucleotide probe capable of identifying mycobacterium strains, and a thermostable DNA polymerase without 5' nuclease activity. The detecting method and the kit, which are provided by the invention, can detect and / or identify 24 kinds of common mycobacteria. The method and the kit can judge the results according to melting peaks of different Tm values produced by the sequence hybridization of the probe and the different strains, meanwhile rapidly and accurately detect the mycobacteria, and identify the mycobacteria, non-mycobacteria, mycobacterium tuberculosis compounding groups and nontuberculosis mycobacteria, and the result of identifying the mycobacteria can be reported after 3-4 hours, thus the method and the kid can assist the clinical diagnosis, and guide efficient clinical chemotherapy at the early stage.

Description

technical field [0001] The invention relates to a method and a kit for detecting and / or identifying mycobacterium strains in clinical examination by using nucleic acid amplification and a detection system thereof, and belongs to the technical field of medical molecular biology diagnosis. Background technique [0002] Tuberculosis remains a serious public health problem, especially in developing countries. According to WHO statistics, 1 / 3 of the world's population is infected with Mycobacterium tuberculosis (MTB), which is the largest killer of infectious diseases in the world. my country is one of the 22 countries with a high burden of tuberculosis in the world, and the number of active tuberculosis patients ranks second in the world. At present, the infection rate of Mycobacterium tuberculosis in all age groups in my country is 44.5%, and about 550 million people in the country are infected by Mycobacterium tuberculosis. Simultaneously, non-tuberculous mycobacteria (NTM) ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04G01N21/64
CPCY02A50/30
Inventor 吴雪琼张俊仙富国良孙伟民阳幼荣梁艳
Owner THE 309TH HOSPITAL OF CHINESE PEOPLES LIBERATION ARMY
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