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Kit for detecting nontuberculous mycobacteria, detection method and application thereof

A detection kit and technology of mycobacteria, applied in the field of molecular biology, can solve the problems of different clinical manifestations, difficulties in identification and differentiation, etc., and achieve the goals of shortening detection time, predicting disease risk and health management, and improving detection throughput Effect

Active Publication Date: 2022-04-29
广州蔚捷生物医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Different pathogenic strains of NTM, affected tissues and organs have different clinical manifestations, and it is difficult to quickly identify and distinguish the specific species of NTM clinically.

Method used

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  • Kit for detecting nontuberculous mycobacteria, detection method and application thereof
  • Kit for detecting nontuberculous mycobacteria, detection method and application thereof
  • Kit for detecting nontuberculous mycobacteria, detection method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0098] Example 1 Design and synthesis of primers and probes

[0099] According to the Mycobacterium avium complex, Mycobacterium abscessus and Mycobacterium fortuitum among the non-tuberculous mycobacteria published by Genebank, the minor species include Mycobacterium kansasii, Mycobacterium chelonis, Mycobacterium goldenscens, Mycobacterium toadiensis, Mycobacterium simianum, Mycobacterium sulja, Mycobacterium marmoe, Mycobacterium haemophilus, Mycobacterium intracellulare, Mycobacterium scrofula, Mycobacterium asiatica and human internal standard The genome sequence of the gene, compare and analyze the differences of each genome sequence, and design probes and primers.

[0100] The final designed primer pairs and corresponding probe sequences are as follows:

[0101] Primer pairs for NMT:

[0102] Upstream primer NMT-F: 5'-AGAAGACCGACGACGTBGC-3' (SEQ ID NO: 1);

[0103] Downstream primer NMT-R: 5'-RTCTSGGTGAYCTTYTCSAC-3' (SEQ ID NO: 2);

[0104] Probe sets for NMT:

[0...

Embodiment 2

[0121] Example 2 A fluorescent quantitative PCR-HRM detection kit for non-tuberculous mycobacteria

[0122] The components of the non-tuberculous mycobacteria fluorescent quantitative PCR-HRM detection kit in this embodiment are as shown in Table 3 and Table 4:

[0123] Table 3 Components of Kit 1

[0124]

[0125] Table 4 Components of Kit 2

[0126]

[0127] Note: After the plasmid was dissolved with 1×TE buffer, the concentration was determined by UV spectrophotometry, and then diluted to 1×10 with 1×TE buffer. 5 copies / μL.

[0128] Wherein, PCR reaction systems 1 and 2 include the NMT primer pair and NMT probe set in Example 1, as well as human internal standard gene primers and probes.

[0129] Wherein, the nucleotide sequence of the human internal standard gene primer is:

[0130] Upstream primer: human internal standard gene F: 5'-TAGGGAGTATATAGGTTGGGGAAGTT-3' (SEQ ID NO: 10);

[0131] Downstream primer: human internal standard gene R: 5'-AACACACAATAACAAACACA...

Embodiment 3

[0148] Example 3 Preparation of standard positive samples and PCR-HRM detection

[0149] (1) Extraction of DNA in standard positive sample nucleic acid:

[0150] The samples to be tested were Mycobacterium avium, Mycobacterium abscessus and Mycobacterium fortuitum, and the secondary species were Mycobacterium kansasii, turtle clam Mycobacterium chelonae, Mycobacterium gordonae, Mycobacterium xenopi, Mycobacterium simiae, Mycobacterium szulgai, Malmo Mycobacterium malmoense, Mycobacterium haemophilum, Mycobacterium intercelleulare, Mycobacterium scrofulaceum, and Mycobacterium arisiense (approximately 2.0 ×10 5 copies / mL) and human internal standard gene.

[0151] Reagents and instruments for DNA extraction of samples to be tested: Nucleic acid extraction was performed using the magnetic bead method nucleic acid extraction or purification kit of Guangzhou Weijie Biomedical Technology Co., Ltd. The nucleic acid extraction instrument is Weijie Auto-Extractor32 nucleic acid ex...

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Abstract

The invention discloses a kit for detecting nontuberculous mycobacteria, a detection method and application thereof, the rapid and accurate detection and identification of 14 nontuberculous mycobacteria are realized through a designed specific primer combination probe group, melting curve analysis is carried out on an amplification product obtained by carrying out multiple fluorescent PCR amplification on a primer group, and the detection result is accurate. The melting temperature is obtained, and the 14 non-tuberculosis mycobacteria can be rapidly and accurately detected according to the melting temperature. The PCR-HRM detection method provided by the invention has the advantages of high detection flux, obvious shortening of the detection time of the nontuberculous mycobacteria, high authenticity, high accuracy, strong specificity, high sensitivity and strong anti-interference ability, and the sensitivity of the PCR-HRM detection method provided by the invention is 10-20 times higher than that of clinical general requirements.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a kit for detecting non-tuberculous mycobacteria, a detection method and an application thereof. Background technique [0002] Nontuberculous mycobacteria (NTM) are mycobacteria other than Mycobacterium tuberculosis (MTB) complex and Mycobacterium leprae within the genus Mycobacterium. Nontuberculous mycobacteria mainly include Mycobacterium avium, Mycobacterium abscessus and Mycobacterium fortuitum, and the minor species include Mycobacterium kansasii, tortoise Mycobacterium chelonae, Mycobacterium gordonae, Mycobacterium xenopi, Mycobacterium simiae, Mycobacterium szulgai, Mar Mycobacterium malmoense, Mycobacterium haemophilum, Mycobacterium intercelleulare, Mycobacterium scrofulaceum and Mycobacterium arisiense. NTM can invade human lungs, lymph nodes, bones, joints, skin and soft tissues and other tissues and organs, and can cause systemic disseminated...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6851C12Q1/04C12N15/11
CPCC12Q1/689C12Q1/6851C12Q2600/16C12Q2600/166C12Q2531/113C12Q2537/143C12Q2563/107C12Q2527/107C12Q2545/101C12Q2545/113
Inventor 朱兴彪张友兵徐贵峰马星星方洁赵京鹏
Owner 广州蔚捷生物医药科技有限公司
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