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Diagnostic method for rifampicin resistant mycobacterium tuberculosis

A technology of Mycobacterium tuberculosis and rifampicin, applied in the field of clinical medicine, can solve the problems of cumbersome operation and expensive equipment, and achieve high reference value, high specificity, and convenient and fast identification method

Active Publication Date: 2013-03-20
广东省结核病控制中心 +1
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0003] The rapid diagnosis method of tuberculosis drug resistance is of great value to the prevention and treatment of tuberculosis. At present, my country has several patents on the detection of drug resistance of Mycobacterium tuberculosis, such as the detection kit of drug resistance gene of Mycobacterium tuberculosis and its preparation method ( 200410083826.5), the method and kit for detecting Mycobacterium tuberculosis and its drug-resistant gene mutation (200610037592), etc., the above methods have disadvantages such as cumbersome operation and expensive equipment

Method used

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Examples

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Effect test

preparation example Construction

[0027] (1) Preparation of template DNA: extract the DNA of the sample to be tested, which is a sample identified as containing Mycobacterium tuberculosis or isolated Mycobacterium tuberculosis;

[0028] (2) Loop-mediated isothermal nucleic acid amplification reaction: prepare a reaction system in a 200 μl PCR tube: 1 μl of primer mixture, 12.5 μl of reaction solution, 0.5-1 μl of DNA polymerase, 2-5 μl of template DNA, make up with sterilized deionized water to 25μl, then add 20μl of sealing solution, and react the above reaction tube at 63~65℃ for 60~90min;

[0029] The primer mixture contains the above four specific primers (SEQ ID No.1~4), wherein the concentration of inner primer 1 is 4~8 pmol / μl, the concentration of inner primer 2 is 4~8 pmol / μl, The concentration of outer primer 1 is 1 pmol / μl, and the concentration of outer primer 2 is 1 pmol / μl;

[0030] The reaction solution contains 10mM dNTP, 10×ThermoPol reaction buffer, 150mM MgSO 4 , 5mM Betaine, the volume ra...

Embodiment 1

[0036] Preparation of the loop-mediated isothermal nucleic acid amplification reaction system:

[0037] (1) Reaction solution: 10mM dNTP, 10×ThermoPol reaction buffer, 150mM MgSO 4 , 5mM Betaine, the volume ratio of the four is 8:5:3:10;

[0038] (2) Primer solution: including 4 pmol / μl internal primer 1, 4 pmol / μl internal primer 2, 1 pmol / μl external primer 1, and 1 pmol / μl external primer 2. The sequences of the four primers are:

[0039] Internal primer 1: 5'-GTGCACCCGTCGCACTACGGGCCCGACCTCCAGGCGCTG-3' (SEQ ID No.1);

[0040] Internal primer 2: 5'-GGGTCAACCCGTTCGGGTTCTGCGGTACGGCGTTTCGAT-3' (SEQ ID No.2);

[0041] Outer primer 1: 5'-GCCCGGCGGTCTGTCACGTG-3' (SEQ ID No.3);

[0042] Outer primer 2: 5'-GTCGGCGGTCAGGTACACGA-3' (SEQ ID No.4);

[0043] (3) DNA polymerase: Bst DNA polymerase, the concentration is 8U / μl;

[0044] (4) Chromogen: Fluorescent dye 1×SYBR Green I.

[0045]Use the above reaction system to diagnose rifampicin-sensitive Mycobacterium tuberculosis and r...

Embodiment 2

[0060] Preparation of the loop-mediated isothermal nucleic acid amplification reaction system:

[0061] (1) Reaction solution: 10mM dNTP, 10×ThermoPol reaction buffer, 150mM MgSO 4 , 5mM Betaine, the volume ratio of the four is 9:5:4:11;

[0062] (2) Primer solution: including 8 pmol / μl internal primer 1, 8 pmol / μl internal primer 2, 1 pmol / μl external primer 1, and 1 pmol / μl external primer 2. The sequences of the four primers are the same as in Example 1;

[0063] (3) DNA polymerase: Bst DNA polymerase, the concentration is 8U / μl;

[0064] (4) Chromogen: Fluorescent dye 1×SYBR Green I.

[0065] Use the above reaction system to diagnose rifampicin-sensitive Mycobacterium tuberculosis and rifampicin-resistant Mycobacterium tuberculosis in the following manner. The sample to be tested in this embodiment is the bronchial lavage fluid of a patient identified as Mycobacterium tuberculosis.

[0066] 1. Template DNA extraction:

[0067] 1) Take 2ml of the sample to be tested an...

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Abstract

The invention discloses a diagnostic method for rifampicin resistant mycobacterium tuberculosis. In the principle of the diagnostic method, four specific primers are designed according to the specificity of mutation loci of a mycobacterium tuberculosis rpoB gene, a deoxyribonucleic acid (DNA) template of a sample is amplified at the temperature of between 63 and 65 DEG C by using the four specific primers and DNA polymerase with strand displacement activity, the amplification efficiency can reach 109 to 1,010 copies within a short time, and color change is observed by adding SYBR Green I to determine a result. The diagnostic method has the advantages of high sensitivity, high specificity and the like, is convenient and quick, has relatively high reference value on the diagnosis and clinical administration of the rifampicin resistant mycobacterium tuberculosis, and is suitable for popularization and application.

Description

technical field [0001] The invention belongs to the field of clinical medicine and relates to a diagnostic method for pathogenic microorganisms, in particular to a ring-mediated isothermal nucleic acid amplification diagnostic method for rifampicin-resistant mycobacterium tuberculosis. Background technique [0002] The World Health Organization (WHO) lists tuberculosis, AIDS, and malaria as one of the three major infectious diseases that humans need to overcome in the 21st century. At present, the annual incidence of tuberculosis in China is about 1.3 million. Due to the increase in the number of floating population, insufficient public health resources, and insufficient public attention, about 550 million people are infected and about 130,000 people die of tuberculosis every year. my country is one of the 22 countries with a high burden of tuberculosis in the world. The epidemic of drug-resistant tuberculosis is severe. Every year, there are about 120,000 new cases of multi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
Inventor 钟球石磊周琳陈涛常彦磊叶宇鑫唐大运
Owner 广东省结核病控制中心
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