Method and kit for detecting ethambutol resistance mutation of Mycobacterium tuberculosis

A technology of Mycobacterium tuberculosis and ethambutol, which can be applied to the determination/testing of microorganisms, biochemical equipment and methods, material stimulation analysis, etc., and can solve problems such as troublesome and high cost

Active Publication Date: 2011-11-02
XIAMEN UNIV +1
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  • Abstract
  • Description
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Problems solved by technology

Since the ends of the two probes that make up the FRET probe have to be blocked to avoid reactions, the cost of synthesis will be relatively high and it will be troublesome.

Method used

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  • Method and kit for detecting ethambutol resistance mutation of Mycobacterium tuberculosis
  • Method and kit for detecting ethambutol resistance mutation of Mycobacterium tuberculosis
  • Method and kit for detecting ethambutol resistance mutation of Mycobacterium tuberculosis

Examples

Experimental program
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Embodiment 1

[0069] Design and synthesis of embodiment 1 primers and probes

[0070] The DNA of Mycobacterium tuberculosis samples was extracted according to the routine DNA extraction method in the laboratory. According to the embB gene sequence of Mycobacterium tuberculosis, 3 pairs of primers and 4 probes were designed, and a double-tube dual-color detection system was established to detect the mutations of 6 codons 306, 368, 378, 380, 406 and 497 on the embB gene. The specific sequences of the primers are as follows:

[0071]

[0072] Primers and probes were synthesized by Shanghai Sangon Biotechnology Co., Ltd. The primers and probes of the present invention can also be oligonucleotide sequences derived from the above 6 primer sequences and 4 probes, with a difference of no more than 8 bases and the antisense complementary sequences of each sequence, or their variants, or partial sequences thereof.

Embodiment 2E

[0073] Embodiment 2EMB drug-resistant mutation detection kit detects EMB drug-resistant mutation

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Abstract

The invention relates to techniques for detecting drug resistance mutation of Mycobacterium tuberculosis and provides a method and kit for detecting ethambutol resistance mutation of Mycobacterium tuberculosis, which have the advantages of simple design, simplified operation, high analysis sensitivity and low cost. The method comprises the following steps of: extracting DNA of a Mycobacterium tuberculosis sample; designing primers and probes by using the primer design software PrimerPremier 5 according to the embB gene sequences of Mycobacterium tuberculosis, constructing a PCR (polymerase chain reaction) system, performing PCR, performing melting curve analysis and detection result judgment, and determining whether the mutation occurs in the regions which are covered by the probes according to the Tm change of the analyzed melting curve. By adopting three pairs of primers and four probes and constructing the double-tube double-color system, the purpose of detecting the mutations of six codons 306, 368. 378, 380, 406 and 497 on the embB gene is achieved.

Description

technical field [0001] The present invention relates to the detection technology of the drug-resistant mutation of Mycobacterium tuberculosis, in particular to a detection method and kit for the drug-resistant mutation of Mycobacterium tuberculosis ethambutol. The method is a double-labeled self-quenching probe-based Probe melting curve analysis technique for detection of ethambutol resistance mutations in Mycobacterium tuberculosis. Background technique [0002] Ethambutol is one of the drugs for the treatment of tuberculosis. It is an arabinose analogue, which affects the formation of mycolic acid-arabinogalactan-leucan complex in the cell wall and exerts an anti-mycobacterial effect. According to the results of the national tuberculosis epidemiological survey in 2000, the average drug resistance rate of ethambutol was 1.3%. At present, its drug resistance detection still needs to be confirmed by culture and drug sensitivity test, the detection cycle is long, the workload...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/02G01N21/64
Inventor 李庆阁胡思玉陈晓云付军那巴古·耶罕姆
Owner XIAMEN UNIV
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