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Mycobacterium tuberculosis drug resistance gene locus, primer group and detection method based on MassARRAY nucleic acid mass spectrum platform

A drug-resistant gene and Mycobacterium tuberculosis technology, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, and microbial measurement/testing, can solve problems such as difficulty in getting started, high cost, and lack of promotion, and achieve individuality The effects of standardization, lower testing costs, and precise drug use

Pending Publication Date: 2022-05-17
SHANGHAI CITY JIADING DISTRICT CENT HOSPITAL +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Next-generation sequencing technology for detecting tuberculosis drug resistance has advantages such as high sensitivity and reusable sequencing data analysis. However, due to factors such as high cost and difficulty in getting started, it has not been promoted in some areas with limited resources.

Method used

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  • Mycobacterium tuberculosis drug resistance gene locus, primer group and detection method based on MassARRAY nucleic acid mass spectrum platform
  • Mycobacterium tuberculosis drug resistance gene locus, primer group and detection method based on MassARRAY nucleic acid mass spectrum platform
  • Mycobacterium tuberculosis drug resistance gene locus, primer group and detection method based on MassARRAY nucleic acid mass spectrum platform

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] A kit for detecting tuberculosis drug resistance-related genes, including a primer set for tuberculosis drug resistance-related gene mutations, information on 25 sites of 7 genes related to tuberculosis drug resistance, as shown in Table 1, and the primer set is shown in Table 1 2 shown;

[0087] PCR amplification primers

[0088] According to the 55 gene mutation types of the selected 7 genes, the amplification primers for each gene mutation type were designed, and the amplified region of the amplification primer was a piece of DNA sequence including the mutation site. The amplification primer has 15 or more bases that completely match the target gene sequence at the 3' end, and a universal tag sequence of 10 bases (ACGTTGGATG) at the 5' end. The specific primers and extension primers are shown in Table 2 respectively:

[0089] Table 1 Drug resistance gene mutation information

[0090]

[0091] Table 2 PCR amplification primers and extension primer sequences (TER...

Embodiment 2

[0117] Using the reagents and methods in Example 1, 60 cases of clinical samples that have undergone generation sequencing were tested, and some sites of 6 samples (S234, y11, y54, y81, y126, y252) were selected, see mass spectrometry for details Analyzing the peak map and comparing the peak map of the first generation sequencing ( Figure 1-12 ), it can be seen that the method of the present invention can detect the genotype of the target locus quickly and accurately, with strong flexibility and short detection cycle. Clinicians can guide medication according to the test results. Among the seven genes involved in the present invention, the genes related to rifampicin resistance include rpoB, the genes related to isoniazid resistance include katG and inhA, and the genes related to fluoroquinolones Drug resistance-related genes include gyrA and gyrB, genes eis and rrs related to second-line anti-TB drugs.

[0118] According to the above interpretation, the relevant information ...

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Abstract

The invention relates to a drug-resistant site of mycobacterium tuberculosis and a detection method thereof. The invention discloses a mycobacterium tuberculosis drug-resistant gene locus, a primer group and a detection method based on a MassARRAY nucleic acid mass spectrum platform. The detection method comprises the following steps: designing amplification primers covering drug-resistant loci such as rpoB, inhA, katG, eis, rrs, gyrB and gyrA and extension primers covering drug-resistant loci such as rpoB, inhA, katG, eis, rrs, gyrB and gyrA; preparing a nucleic acid template, preparing a corresponding extension mixed reaction solution, and carrying out PCR amplification reaction to obtain a target product; and carrying out SAP enzyme digestion reaction and extension reaction: carrying out desalination treatment on the obtained extension amplification product, carrying out sample application, and analyzing by using analysis software of a mass spectrometer. The method is high in flux, good in flexibility, easy to implement and high in cost performance.

Description

technical field [0001] The invention relates to drug resistance of Mycobacterium tuberculosis, in particular to drug-resistant sites of Mycobacterium tuberculosis and a detection method thereof. Background technique [0002] Tuberculosis is one of the top ten causes of death in the world, and the number of tuberculosis patients in China ranks third in the world, second only to India and Indonesia. Drug resistance of tuberculosis means that the pathogenic bacteria of tuberculosis produce resistance to tuberculosis drugs during the growth, which makes some patients develop drug resistance during the process of receiving drug treatment and fall into the dilemma of treatment. Although the treatment of drug-resistant tuberculosis has made great progress in recent years, about 5.7% of new and 25.6% of treated tuberculosis cases suffer from multidrug-resistant (multi drug-resistant, MDR) tuberculosis. [0003] Currently, the diagnosis of drug-resistant TB mainly includes the follo...

Claims

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Application Information

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IPC IPC(8): C12Q1/6858C12Q1/689C12N15/11C12R1/32
CPCC12Q1/6858C12Q1/689C12Q2600/106C12Q2600/156C12Q2531/113C12Q2535/125C12Q2521/525C12Q2565/627Y02A50/30
Inventor 余艳芳孙亚蒙赵开顺徐军屠春林谢倩凤
Owner SHANGHAI CITY JIADING DISTRICT CENT HOSPITAL
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