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Method for detecting mycobacterium tuberculosis and special-purpose primers therefor

A technology of Mycobacterium tuberculosis and primer pair, applied in the biological field, can solve problems such as poor specificity, and achieve the effects of high accuracy, high sensitivity and short time consumption

Active Publication Date: 2012-11-21
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] 3 Polymerase chain reaction (PCR), the sensitivity is improved compared with conventional culture methods and acid-fast staining methods, but the specificity is poor
At present, there is no report on the detection of Mycobacterium tuberculosis by MDA-PCR two-step method at home and abroad

Method used

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  • Method for detecting mycobacterium tuberculosis and special-purpose primers therefor
  • Method for detecting mycobacterium tuberculosis and special-purpose primers therefor
  • Method for detecting mycobacterium tuberculosis and special-purpose primers therefor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Embodiment 1, the acquisition of Mycobacterium tuberculosis specific primer

[0052] IS6110 is a unique insertion sequence of Mycobacterium tuberculosis complex, and the number of copies of this insertion sequence in the genome is 1-20. Selecting the insertion sequence as the detection target sequence of Mycobacterium tuberculosis can obtain higher sensitivity and specificity .

[0053]Using Primer V5.0 to design primers with IS6110 as the target sequence, the sequence is as follows:

[0054]

Embodiment 2

[0055] Embodiment 2, detect Mycobacterium tuberculosis

[0056] 1. Template preparation

[0057] Extraction of Mycobacterium tuberculosis H37Rv (Zhang Yuanyuan, Zhang Xuanmin, Zhao Xiuqin, Zeng Lingcheng, Liu Zhiguang, Wang Xilin, Wang Yan, Du Xinling, Han Yueling, Wan Kanglin. Establishment and evaluation of a PCR technique for direct detection of tuberculosis sputum specimens. Chinese Communist Party of Animals Disease Journal, 2008, 24 (11): 1017-1021. The public can obtain from the Institute of Microbiology, Chinese Academy of Sciences.) DNA as a template, carry out gradient dilution, get concentration respectively and be 300fg / μl and 30fg / μl and carry out following as template reaction.

[0058] 2. MDA-PCR two-step reaction procedure

[0059] 1) MDA reaction

[0060] The nucleotide sequence of the MDA random primer is 5'-NNNNNN-3', and a sulfur-phosphorus bond is formed between the 1st and 2nd nucleotides at the 3' end, and a sulfur-phosphorus bond is formed between th...

Embodiment 3

[0073] Embodiment 3, the sensitivity analysis of detection method

[0074] 1. Preparation of DNA

[0075] ①Take 500 μl of clinical sample sputum (sputum, known to contain Mycobacterium tuberculosis (MTB), provided by the Tuberculosis Laboratory of the Institute of Infectious Disease Control and Prevention, Chinese Center for Disease Control and Prevention, and informed by the patient) into 1.5ml Eppendorf, Centrifuge at 9500×g for 15 minutes, and discard the supernatant.

[0076] ② Add 100 μl of lysate (containing 50mM Tris-HCl, pH 8, 50mM KCl, 2.5mM MgCl 2 , 0.45% Tween 20, 0.45% Nonidet P-40, 10 μg proteinase K).

[0077] ③ After shaking evenly, place in a water bath at 56°C for 3 hours.

[0078] ④ Inactivate proteinase K at 95°C for 10 minutes.

[0079] ⑤ Centrifuge at 9500×g for 5 minutes.

[0080] Transfer the supernatant to a new Eppendorf tube and use it as a DNA template.

[0081] 2. MDA-PCR two-step reaction procedure.

[0082] According to step 2 in Example 2,...

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Abstract

The invention discloses a method for detecting mycobacterium tuberculosis and special-purpose primers therefor. The special-purpose primers are a pair of primers for assisting mycobacterium tuberculosis detection. A nucleotide sequence of one of the special-purpose primers is shown in the sequence 1 in the sequence table and a nucleotide sequence of the other of the special-purpose primers is shown in the sequence 2 in the sequence table. An experiment proves that the special-purpose primers are used in a novel multiple displacement amplification (MDA)-polymerase chain reaction (PCR) two-step method and mycobacterium tuberculosis nucleic acids in sputum is enriched by a MDA complete-genome isothermal amplification technology and mycobacterium tuberculosis is detected by a specific primer-PCR combined technology, and thus short detection time, high sensitivity and high accuracy are realized.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for detecting mycobacterium tuberculosis and special primers thereof. Background technique [0002] Tuberculosis is a chronic infectious disease that seriously endangers people's health. Since the 1990s, tuberculosis has once again "resurrected" around the world. The Chinese government has always paid more attention to the prevention and control of tuberculosis, and has introduced a series of policies and measures. After years of hard work, the rising trend of the tuberculosis epidemic in my country has been curbed, and both the morbidity and mortality have declined. But the tuberculosis epidemic in my country is still very serious. According to WHO estimates, there are about 1.3 million new tuberculosis patients in the world every year, about 130,000 people die of tuberculosis every year, and nearly 100,000 cases of multidrug-resistant tuberculosis (MDR-TB) every year. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C12R1/32
Inventor 朱宝利万康林武娜张媛媛王志云
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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