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Method and kit for detecting isoniazid resistance of mycobacterium tuberculosis under constant temperature condition

A technology for Mycobacterium tuberculosis and isoniazid, which is applied in the directions of microorganism-based methods, biochemical equipment and methods, and microorganism determination/inspection, etc. short effect

Pending Publication Date: 2022-05-13
GUANGZHOU DEAOU MEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there is still a lack of effective methods for detecting isoniazid resistance of Mycobacterium tuberculosis using loop-mediated isothermal amplification technology for multiple targets at the same time

Method used

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  • Method and kit for detecting isoniazid resistance of mycobacterium tuberculosis under constant temperature condition
  • Method and kit for detecting isoniazid resistance of mycobacterium tuberculosis under constant temperature condition
  • Method and kit for detecting isoniazid resistance of mycobacterium tuberculosis under constant temperature condition

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Embodiment 1 provides primers, probes

[0066] This example provides a combination of primers for detecting isoniazid resistance of Mycobacterium tuberculosis, including primer set I, primer set II, molecular beacon probe katG-M and molecular beacon probe inhA-M.

[0067] Primer set I is used to specifically amplify fragments on the nucleic acid sequence of the katG gene (the length of the amplicon is about 300bp), including the outer primer KatG-F3, the outer primer KatG-B3, the inner primer KatG-FIP, and the inner primer KatG-BIP , loop primer KatG-LB;

[0068] Primer set II is used to specifically amplify fragments on the nucleic acid sequence of the inhA gene (the length of the amplicon is about 300bp), including the outer primer InhA-F3, the outer primer InhA-B3, the inner primer InhA-FIP, and the inner primer InhA-BIP , loop primer InhA-LB;

[0069] The 5' end of the molecular beacon probe katG-M is modified with FAM, and the 3' end is modified with BHQ1; the 5'...

Embodiment 2

[0073] Example 2 Kit

[0074] This embodiment provides a kit, which includes the primer combination described in Example 1, a negative quality control product, a positive quality control product, Bst DNA polymerase and a reaction solution.

[0075] The positive quality control product is a T vector clone containing katG gene and inhA gene DNA fragments; the negative quality control product is deionized water. The reaction solution was mixed with 10mM dNTPs, 10×ThermoPol reaction buffer and 100mM MgSO 4 The aqueous solution is prepared by mixing uniformly according to the volume ratio of 7:5:3.

Embodiment 3

[0076] Example 3 Detection of Mycobacterium tuberculosis isoniazid resistance

[0077] This embodiment provides a method for detecting whether Mycobacterium tuberculosis has isoniazid resistance in a sample to be tested, comprising the following steps:

[0078] (1) Take a sputum sample suspected to contain Mycobacterium tuberculosis, extract DNA in the sputum sample by pyrolysis, and use the extracted DNA as a template to be tested in subsequent steps;

[0079] (2) Prepare the following reaction system: each 1.0 μmol / L of outer primer KatG-F3, outer primer KatG-B3, outer primer InhA-F3, outer primer InhA-B3, inner primer KatG-FIP, inner primer KatG-BIP, inner primer Primer InhA-FIP, internal primer InhA-BIP each 2.0 μmol / L, loop primer KatG-LB, loop primer InhA-LB each 2.0 μmol / L, molecular beacon probe katG-M, molecular beacon probe inhA-M Each 0.5 μmol / L, the reaction solution described in Example 2 was used to make up to 22 μL; Bst DNA polymerase 1 μL (8U), the template to...

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Abstract

The invention relates to the technical field of biological detection, in particular to a method and a kit for detecting isoniazid resistance of mycobacterium tuberculosis under a constant temperature condition. The invention provides a primer combination which comprises an inner primer, an outer primer and a loop primer which are used for specifically amplifying a KatG gene and an InhA gene, and a molecular beacon probe. The invention also provides a corresponding kit and a detection method. The detection method provided by the invention can be used for detecting the isoniazid drug resistance of the mycobacterium tuberculosis on the to-be-detected sample under a constant temperature condition, and has the advantages of high detection speed, high sensitivity and strong specificity.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a method and a kit for detecting isoniazid resistance of mycobacterium tuberculosis under constant temperature conditions. Background technique [0002] Mycobacterium tuberculosis (Mycobacterium tuberculosis) is elongated and straight or slightly curved. It is the pathogenic bacterium that causes human tuberculosis infection. The lethality rate of a single infectious pathogen is higher than that of HIV, and it is one of the ten leading causes of death in the world. According to the World Health Organization's 2020 tuberculosis report, about a quarter of the world's population is infected with Mycobacterium tuberculosis. China is one of the three countries with the highest proportion of tuberculosis burden in the world, and continuous research and innovation are needed in tuberculosis prevention, screening and treatment. [0003] Isoniazid, also known as 4-pyridinyl ...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6844C12N15/11C12R1/32
CPCC12Q1/689C12Q1/6844C12Q2600/106C12Q2600/156C12Q2531/119C12Q2537/1376C12Q2527/107C12Q2563/107C12Q2525/307
Inventor 陈皇佑何潘敏
Owner GUANGZHOU DEAOU MEDICAL TECH CO LTD