Primer, probe and kit for detecting gene mutation sites related to drug resistance of mycobacterium tuberculosis and treatment drugs

A technology of Mycobacterium tuberculosis and mutation sites, applied in the field of medical testing, can solve the problems of increasing the risk of PCR contamination and false positives, not covering the detection of pyrazinamide and streptomycin resistance, and long experiment cycle

Inactive Publication Date: 2021-09-14
HAINAN MEDICAL COLLEGE
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, two rounds of PCR are required in the detection process, which has a long experimental cycle and increases the risk of contamination and false positives during the PCR experiment; in addition, this patented technology can only simultaneously detect the isoniazid-resistant katG gene of Mycobacterium tuberculosis and inhA gene, the rpoB gene for rifampicin resistance and the gyrA gene for fonoquinone resistance, which did not cover the resistance detection of the first-line anti-tuberculosis drugs pyrazinamide and streptomycin

Method used

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  • Primer, probe and kit for detecting gene mutation sites related to drug resistance of mycobacterium tuberculosis and treatment drugs
  • Primer, probe and kit for detecting gene mutation sites related to drug resistance of mycobacterium tuberculosis and treatment drugs

Examples

Experimental program
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Effect test

Embodiment 1

[0247] The known sample 1 contains isoniazid and rifampicin-resistant Mycobacterium tuberculosis, which is detected by the method of the present invention, and the specific steps are as follows:

[0248] a. Extract the total DNA in the sample;

[0249] b. First use the total DNA of the sample extracted in step a as a template to perform PCR. Add the following substances to the PCR tube: 2×Multiplex PCRMasterMix mixture (including four deoxyribonucleotides, PCR buffer, Taq DNA polymerase) 12.5 μL, IS6110-F, IS6110-R, IS6110-R, 16S-F, 16S-R, katG-F, katG-R, inhA-F, inhA-R, rpoB-F, rpoB-R, gyrA-F, gyrA-R, rpsL-F, rpsL-R, rrs- F, rrs-R, pncA-F and pncA-R each 0.125 μL, DNA template 1 μL and ultrapure water 9.25 μL.

[0250] The sequences of each primer are as follows:

[0251] IS6110-F: biotin-CATGTCCGGAGACTCCAGTT

[0252] IS6110-R: GGTACCTCCTCGATGAACCA

[0253] 16S-F: biotin-TAGATACCCTGGTAGTCC

[0254] 16S-R: CGACACGAGCTGACGACA

[0255] katG-F: biotin-CTCGGCGATGAGCGTTACA ...

Embodiment 2

[0335] Known sample 2 contains fonoquinones and streptomycin-resistant Mycobacterium tuberculosis, which is detected by the method of the present invention, and the steps are the same as in Example 1, and the results are as shown in Table 1. The fluorescence value of the probe IS6110 of the mycobacterium-related gene IS6110 is 5892.4, and the fluorescence value of the 16S probe 16S is 4403, which is greater than 100, indicating that it contains Mycobacterium tuberculosis.

[0336] The fluorescence value of the mutant probe katG-315ACC at the 315 amino acid mutation site of the isoniazid resistance-related gene katG gene in this sample is 2268, which is greater than 100, and is more than ten times the fluorescence value of the mutant probe katG-315AGC. Therefore, this sample does not contain drug resistance mutations related to katG-315 amino acid. The fluorescence value of the wild-type probe inhA--15C at the 15th amino acid mutation site of the inhA gene related to isoniazid ...

Embodiment 3

[0343] Known sample 3 contains pyrazinamide drug-resistant Mycobacterium tuberculosis, and is detected by the method of the present invention, and concrete steps are identical with example 1, and the result of fluorescence detection value is as shown in table 1, and this sample Mycobacterium tuberculosis is relevant The fluorescence value of the probe IS6110 of the gene IS6110 is 4988.3, ​​and the fluorescence value of the 16S probe 16S is 3891, which is greater than 100, indicating that it contains Mycobacterium tuberculosis.

[0344] The fluorescence value of the wild-type probe katG-315AGC at amino acid 315 of the isoniazid resistance-related gene katG gene in this sample is 1923.5, which is greater than 100, and is more than ten times the fluorescence value of the mutant probe katG-315ACC, so this sample is not Contains drug resistance mutations related to katG-315 amino acid. The fluorescence value of the wild-type probe inhA--15C at the -15 amino acid mutation site of th...

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Abstract

The invention relates to the technical field of medical detection, in particular to a primer, a probe and a kit for detecting mycobacterium tuberculosis and drug-resistant gene mutation sites. The detection primer and the probe comprise DNA (deoxyribonucleic acid) sequences as shown in SEQ ID NO: 1 to SEQ ID NO: 68. The drug resistance type of mycobacterium tuberculosis is detected based on a Luminex liquid chip, whether the mycobacterium tuberculosis exists in a sample or not can be detected, four first-line antituberculous drugs including isoniazide, rifampicin, pyrazinamide and streptomycin and a second-line antituberculous drug, namely fluoroquinolone, are covered, mutation sites are comprehensive, the detection speed is high, only 3.5 hours are needed to complete one-time detection, and as many as 96 samples can be detected at the same time at one time. Therefore, the detection process is simplified, the experimental period is shortened, the detection accuracy and sensitivity are improved, and clinical application is facilitated.

Description

technical field [0001] The invention relates to the technical field of medical detection, in particular to a detection primer, probe and kit for Mycobacterium tuberculosis and drug-resistant gene mutation sites. Background technique [0002] Tuberculosis (TB) is one of the top ten causes of death in the world caused by a single source of infection (Mycobacterium tuberculosis). About 1.7 billion people in the world are infected with Mycobacterium tuberculosis, and about 100 million people are diagnosed with tuberculosis every year. . Emergence of multidrug resistance (MDR) and extensive drug resistance (XDR) in Mycobacterium tuberculosis is considered one of the most challenging threats to TB control, with approximately half a million new cases of rifampicin-resistant TB in 2018 (78% of which were MDR-TB). Now anti-tuberculosis drugs such as isoniazid, rifampin, pyrazinamide, streptomycin and fonoquinones have drug resistance problems. At present, the key to the treatment ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6837C12Q1/6827C12Q1/04C12N15/11
CPCC12Q1/689C12Q1/6837C12Q1/6827C12Q2600/156C12Q2531/113C12Q2563/107C12Q2563/149
Inventor 尹飞飞黄艺杜江杜永国张丽媛刘昌江田秀颖王高玉彭箬岩胡小媛郎雨萌薛丽颖陈锦龙李天阳
Owner HAINAN MEDICAL COLLEGE
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