Kit for detecting drug resistance of mycobacterium tuberculosis based on HRM technology and primers thereof

A Mycobacterium tuberculosis, technical detection technology, applied in the field of kits for detecting drug resistance of Mycobacterium tuberculosis, can solve the problems of low sensitivity and long detection period of drug resistance of Mycobacterium tuberculosis, and achieve high sensitivity , shorten the detection cost, and the effect of simple detection

Inactive Publication Date: 2017-06-09
SUZHOU BAIYUAN GENT CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] For this reason, the technical problem to be solved in the present invention is to overcome the problems of long detection period and low sensitivity of Mycobacterium tuberculosis resistance in the prior art, thereby providing a method based on HRM technology to quickly and accurately detect multiple drug resistance of Mycobacterium tuberculosis. Precise kits for accurate, reliable, fast and easy detection of tuberculosis drug resistance and multidrug resistance

Method used

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  • Kit for detecting drug resistance of mycobacterium tuberculosis based on HRM technology and primers thereof
  • Kit for detecting drug resistance of mycobacterium tuberculosis based on HRM technology and primers thereof
  • Kit for detecting drug resistance of mycobacterium tuberculosis based on HRM technology and primers thereof

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Experimental program
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Embodiment 1

[0060] The design and synthesis of embodiment 1 primer

[0061] Combined with previous literature reports, the genes and corresponding mutation sites used to identify the multi-drug resistance of Mycobacterium tuberculosis were determined. The drug-resistant genes of Mycobacterium tuberculosis detected were: isoniazid resistance (katG gene and inhA gene), resistance Ethambutol (embB gene) and streptomycin resistance (rpsL gene). In NCBI, the standard sequences of katG, inhA, embB and rpsL can be obtained (the sequences are from the standard strain H37Rv, NC_000962.2), primers are designed upstream and downstream of the drug resistance mutation sites of the four genes, and the mutations of the four genes The location information, mutation type, primer sequence and amplified region are shown in Table 1:

[0062] Table 1 Mutation location information, mutation type, primer sequence and amplified region of the drug resistance gene of Mycobacterium tuberculosis

[0063]

Embodiment 2

[0064] The preparation of embodiment 2 kit

[0065] This embodiment provides a kit for detecting drug resistance of Mycobacterium tuberculosis based on HRM technology, said kit including the primers for detecting drug resistance gene mutation of Mycobacterium tuberculosis based on HRM technology in the above-mentioned Example 1.

[0066] The reaction solution (10 μl system, excluding DNA template) in the non-labeled fluorescent PCR amplification process included in the kit consists of the following components:

[0067] Green-2-Go qPCR Mastermix (including EvaGreen), 5μl;

[0068] Primer F, 10 μM, 0.1 μl;

[0069] Primer R, 10 μM, 0.1 μl;

[0070] wxya 2 O, 4.3 μl;

[0071] Also include negative standard and positive standard in the test kit, the preparation method of described standard comprises the steps:

[0072] 1. According to the gene variation of each tuberculosis drug-resistant gene wild type and drug-resistant type, in combination with the primers used in the pres...

Embodiment 3

[0092] The use of embodiment 3 kits

[0093] This embodiment provides an application for mutation detection at the predetermined site of the drug-resistant gene of Mycobacterium tuberculosis using the kit of Embodiment 2, including the following steps

[0094] (1) Collect the sputum sample for liquefaction and culture, extract the genomic DNA in the sputum sample, and prepare the DNA extract of the sputum sample:

[0095] 1) Transfer 1-2mL of sputum to a processing tube in a biological safety cabinet, add 1-4 times the volume of 4% sodium hydroxide solution, vortex for 30 seconds until fully liquefied, and let stand for 15 minutes;

[0096] 2) Add PBS to neutral, transfer to a 5ml centrifuge tube, centrifuge at 6480g for 15min, discard the supernatant;

[0097] 3) Add 0.5ml PBS to step 2) and mix evenly, draw 0.1ml with a syringe, inject it into a tuberculosis culture bottle and cultivate it for 42 days;

[0098] 4) Positive samples were transferred to Roche medium and used ...

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Abstract

The invention relates to a kit for detecting the drug resistance of mycobacterium tuberculosis based on an HRM technology and primers thereof. The invention discloses the kit for detecting drug resistance genes of the mycobacterium tuberculosis and application thereof. The invention discloses a group of primers for detecting whether each of the genes of the mycobacterium tuberculosis in a to-be-tested sample generates a drug resistance mutation or not, and the sequences of the primers are shown in SEQ NO.1-SEQ NO.10. The kit containing the primers can detect the mutation situations of the four drug resistance indicator genes (embB, rpsL, inhA and katG) in DNA extracted from the to-be-tested sample. The kit provided by the invention detects the mutation situations of the four drug resistance indicator genes of the mycobacterium tuberculosis in the to-be-tested sample within a shorter time through the high resolution melting (HRM) technology and is high in result sensitivity, better in specificity, and simple and convenient in analysis process.

Description

technical field [0001] The invention belongs to biotechnology, and in particular relates to a test kit for detecting drug resistance of Mycobacterium tuberculosis based on HRM technology, primers and application thereof. Background technique [0002] Tuberculosis is one of the most threatening infectious diseases in the world today, and the emergence of multidrug-resistant tuberculosis (MDR-TB) and extensive drug-resistant tuberculosis (XDR-TB) making the epidemic situation of tuberculosis more severe. The WHO2008 report shows that the global total drug resistance rate of tuberculosis is 20.0%, and the multidrug resistance rate is 5.3%. There are about 500,000 new MDR-TB patients and 50,000 extensively drug-resistant TB patients each year. With increasing population movement worldwide, drug-resistant TB cases have been on the rise for the past 20 years, seriously threatening progress in TB control efforts. [0003] Streptomycin, isoniazid and ethambutol belong to the first...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/6858C12Q1/689C12Q2563/107C12Q2527/107
Inventor 车团结同重湘尤崇革谢小冬李琳李亚鹏
Owner SUZHOU BAIYUAN GENT CO LTD
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