PCR-ELISA based method for detecting mycobacterium tuberculosis resistance gene

A technology of Mycobacterium tuberculosis and drug-resistant genes, which is applied to the determination/testing of microorganisms, biochemical equipment and methods, etc., to achieve high sensitivity, increase sensitivity, and low price

Inactive Publication Date: 2012-05-23
SHANGHAI JIAOTONG UNIV SCHOOL OF MEDICINE
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Problems solved by technology

However, the traditional tuberculosis drug susceptibility testing method is a phenotypic drug resistance testing method, which is based on the culture of Mycobacterium tuberculosis, and usually takes 4 to 6 weeks, which is far from meeting the needs of clinical treatment.

Method used

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  • PCR-ELISA based method for detecting mycobacterium tuberculosis resistance gene
  • PCR-ELISA based method for detecting mycobacterium tuberculosis resistance gene

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preparation example Construction

[0028] 1. Preparation of template DNA:

[0029] For the DNA extraction of Mycobacterium tuberculosis in the specimen to be tested, take the clinical mycobacterium strain and place it in a 1.5 ml centrifuge tube, dissolve it in 0.5 ml TE solution, centrifuge at 14000 r / min for 5 min, discard the supernatant; add 100 μL of 1% TritonX-100 lysate, vortex to mix, 100 ° C water bath for 15 min, immediately ice bath for 3 min; 140000 r / min, 10 min, take the supernatant.

[0030] 2. PCR reaction:

[0031] a. Prepare 3×50 μL reaction system: In each reaction tube, 2 μL of primers rpoB-F and rpoB-R, 5 μL of 10×Buffer, 0.5 μL of template DNA, 2 μL of dNTPs, 2 U of Taq enzyme, the total volume is 50 μL. The target gene of the primer is rpoB gene (GenBank accession number: L27989)

[0032] The 5' ends of the downstream primers were all labeled with digoxigenin, and the sequences of the primers were:

[0033] rpoB-F 5'-GTGGTCGCCGCGATCAAG-3'

[0034] rpoB-R 5'-CACGTCGCGGACCTCCAG-3'

[0...

Embodiment 1

[0060] Specimens to be tested: 1 case of Escherichia coli strain, 1 case of Mycobacterium tuberculosis H37Rv strain, 3 cases of non-tuberculous mycobacteria were Mycobacterium avium, Mycobacterium intracellulare and Mycobacterium smegmatis; 1 case of clinical tuberculosis Mycobacterium rpoB gene wild-type strains, 2 cases of clinical Mycobacterium tuberculosis rpoB gene mutant strains.

[0061] 1. Preparation of template DNA:

[0062] Take the branched strain and put it in a 1.5 ml centrifuge tube, dissolve it in 0.5 ml TE solution, centrifuge at 14000 r / min for 5 min, discard the supernatant; add 100 μL of 1% TritonX-100 lysate, vortex to mix, and place in a 100°C water bath 15 min, immediately ice bath for 3 min; 140000 r / min, take the supernatant.

[0063] 2. PCR reaction:

[0064] a. Prepare 3×50 μL reaction system: In each reaction tube, 2 μL of primers rpoB-F and rpoB-R, 5 μL of 10×Buffer, 0.5 μL of template DNA, 2 μL of dNTPs, 2 U of Taq enzyme, the total volume is 50...

Embodiment 2

[0076] 1. Preparation of bacterial DNA 156 strains of Mycobacterium tuberculosis clinical isolates were derived from clinical inpatients. Mycobacterium tuberculosis was cultured in accordance with the "National Regulations for the Bacteriology of Diagnosis of Tuberculosis". Mycobacteria were initially identified and tested for drug susceptibility. Take clinical mycobacterium strains and place them in a 1.5 ml centrifuge tube, dissolve them in 0.5 ml TE solution, centrifuge at 14000 r / min for 5 min, discard the supernatant; add 100 μL of 1% TritonX-100 lysate, vortex to mix, and store at 100°C Water bath for 15 min, immediately ice bath for 3 min; 140000 r / min, take the supernatant.

[0077] 2. PCR amplification

[0078] a. Prepare 3×50 μL reaction system: In each reaction tube, 2 μL of primers rpoB-F and rpoB-R, 5 μL of 10×Buffer, 0.5 μL of template DNA, 2 μL of dNTPs, 2U of Taq enzyme, the total volume is 50 μL. The target gene of the primers is the mutation hotspot of the ...

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Abstract

The invention provides a PCR-ELISA (polymerase chain reaction enzyme-linked immunosorbent assay) based method for detecting a mycobacterium tuberculosis resistance gene. The method comprises the steps of: (1) designing 11 probes directed at rpoB gene mutation sites, and applying the probes on a 96-microplate; (2) amplifying the DNA of each detection sample in 3 reaction tubes simultaneously, adding primers rpoB-F and rpoB-R into each tube, marking the 5' end of a downstream primer in each primer pair with digoxin, conducting PCR amplification so as to obtain a lot of gene segment products corresponding to mycobacterium tuberculosis and containing digoxin markers; (3) subjecting the amplification products and the probes to hybridization so as to make target genes equipped with digoxin markers and the probes combined together, washing off uncombined segments, conducting an ELISA reaction, and measuring an OD (optical density) value, thus obtaining a result. By means of one hybridization experiment, the method of the invention can rapidly obtain the drug resistance information of mycobacterium tuberculosis to a plurality of clinical isolates, so that detection of the drug resistance of mycobacterium tuberculosis to isolates can be shortened to 6-8h, thus fully showing the superiority of the method.

Description

technical field [0001] The invention relates to the field of biomedical in vitro diagnostic reagents, in particular to a method for detecting drug-resistant genes of Mycobacterium tuberculosis based on PCR-ELISA. Background technique [0002] Drug susceptibility testing of Mycobacterium tuberculosis has been a very important technique in the bacteriological diagnosis of tuberculosis for a long time. However, the traditional tuberculosis drug susceptibility testing method is a phenotypic drug resistance testing method, which is based on the culture of Mycobacterium tuberculosis, and usually takes 4 to 6 weeks, which is far from meeting the needs of clinical treatment. [0003] With the development of molecular biology diagnostic technology based on PCR technology and the gradual elucidation of the molecular mechanism of tuberculosis drug resistance, the detection method of tuberculosis drug resistance gene based on molecular biology technology has also been developed rapidly...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04
Inventor 张舒林唐神结孙战强张颖王洪海郭晓奎
Owner SHANGHAI JIAOTONG UNIV SCHOOL OF MEDICINE
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