Primer probes and probe for real-time fluorescent polymerase chain reaction (PCR) detection of Mycobacterium Tuberculosis and using method of primer probes

A Mycobacterium tuberculosis, real-time fluorescence technology, applied in the biological field, can solve the problems of low MTB bacteria count and difficulty in detection in patients

Inactive Publication Date: 2012-07-11
GUANGZHOU SUPBIO BIO TECH & SCI
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Problems solved by technology

At present, smear-negative patients account for about 60% of the total number of TB patients, and t

Method used

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  • Primer probes and probe for real-time fluorescent polymerase chain reaction (PCR) detection of Mycobacterium Tuberculosis and using method of primer probes
  • Primer probes and probe for real-time fluorescent polymerase chain reaction (PCR) detection of Mycobacterium Tuberculosis and using method of primer probes
  • Primer probes and probe for real-time fluorescent polymerase chain reaction (PCR) detection of Mycobacterium Tuberculosis and using method of primer probes

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Embodiment 1: The primer and probe design of MTB real-time fluorescent PCR detection method

[0022] A highly conserved IS6110 gene (Genebank No.NC_000962) with multiple copies was selected from the MTB genomic DNA. For this gene, 28 sets of primers were screened out based on the principle that the annealing temperature of the primers was 55-65°C with various software. After comparing the results by PCR, primers SEQ ID NO.1 and NO.2 were selected, and Blast homology analysis showed that the selected primers were specific sequences of the TB complex and had no homology with other sequences. According to the principle that the primer annealing temperature is 60°C and the probe annealing temperature is about 10°C higher than the primer annealing temperature, the probe SEQ ID NO.3. The length of the amplified product is 70bp.

[0023] The 5' end of the probe is labeled with a FAM luminescent fluorophore, and the 3' end is labeled with a Taqman-MGB quenching fluoropho...

Embodiment 2

[0024] Embodiment 2: MTB real-time fluorescent PCR detection clinical specimen specific operation of the present invention

[0025] 1) Preparation of reaction solution: According to the above-mentioned content of the invention 2, the PCR reaction was prepared in the reagent preparation area, and the concentrations of each substance were as follows: 20mM Tris-HCl (pH8.0), 20mM KCl, 5mM (NH 4 ) SO 4 , 2mM MgCl 2 , 0.2mM dNTP, 0.4mM primer 1, 0.4mM primer 2, 0.2mM probe, 1.5U Hot Start Taq emzyme and 0.1U UNG;

[0026] When preparing the reaction solution, the reaction solution of 2 control tubes should be included, including: negative control and positive control.

[0027] 2) Add template: In the sample area, add 2ul template DNA to each reaction, use sterile saline for negative control, and use standard Mycobacterium tuberculosis strain H for positive control 37 R V Genomic DNA. Cover reaction tubes with caps or 96-well plate membranes.

[0028] 3) PCR dete...

Embodiment 3

[0031] Embodiment 3: Research on the specificity of MTB real-time fluorescent PCR detection method

[0032] The DNA of 15 kinds of non-tuberculous mycobacteria in the national standard product of the MTB fluorescent PCR detection kit was selected as a template, including: Mycobacterium avium, Mycobacterium terrescens, Mycobacterium stutzeri, Mycobacterium kansasii, Mycobacterium asiatica Mycobacterium, Mycobacterium scrofula, Mycobacterium gordonii, Mycobacterium pyogenes, Mycobacterium fortuitously, Mycobacterium phlei, Nocardia brasiliensis, Corynebacterium Beijing, Pneumococcus, Legionella pneumophila, Pertussis Bordetella. MTB standard strain H 37 Rv DNA was used as a positive control in this experiment. RT PCR of the present invention is used for detection, the amount of template is 2ul, and each reaction is repeated three times.

[0033] The results showed that only the MTB standard strain H 37 The Rv DNA test was positive, and the others were all negative, c...

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Abstract

The invention discloses a group of specific primers and a probe, which are designed and screened according to an IS6110 sequence of M. Tuberculosis (MTB), and a method for real-time fluorescent polymerase chain reaction (PCR) detection of DNA of the MTB by using the primers and the probe. A PCR reaction system comprises anti-pollution UNG enzyme, and belongs to the field of biotechnology. The real-time fluorescent PCR detection method established by the primers and the probe has high sensitivity and specificity, and particularly has higher tested positive rate for low MTB carried smear negative sputum specimens and blood samples than other similar detection methods.

Description

technical field [0001] The invention aims at designing and screening a group of specific primers and probes aimed at the IS6110 sequence of M. tuberculosis (MTB) IS6110, which are used for real-time fluorescent PCR detection of tuberculosis mycobacterium, and belong to the field of biotechnology. Background technique [0002] TB is a chronic infectious disease caused by MTB infection that seriously endangers human health. MTB can be latently infected in the human body for several years or even a lifetime. One-third of the global population (about 2 billion) is considered to be infected with latent TB. About 44.6% of the Chinese population has latent TB infection, and more than 10 of them % of people will develop active disease during their lifetime. According to the World Health Organization (WHO) 2010 Global TB Control Report, in 2009, there were about 1.7 million TB deaths worldwide, and about 9.4 million new TB patients. Most of these patients are concentrated in develo...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11G01N21/64
Inventor TUOFUZHU(朱托夫)
Owner GUANGZHOU SUPBIO BIO TECH & SCI
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