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Method and kit for detecting drug resistance gene mutant type of tuberculous bacillus (TB)

A technology of Mycobacterium tuberculosis and drug resistance gene, applied in the field of molecular biology, can solve the problems of low throughput, low resolution, high background signal, etc., and achieve the effect of reducing usage, simple and stable reagent consumables

Inactive Publication Date: 2013-07-03
北京宏微特斯生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0016] However, the current molecular detection methods are mainly based on restriction fragment length polymorphism analysis, PCR sequencing, and gene chips, which generally have defects such as low throughput, low resolution, high background signal, and high cost.

Method used

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  • Method and kit for detecting drug resistance gene mutant type of tuberculous bacillus (TB)
  • Method and kit for detecting drug resistance gene mutant type of tuberculous bacillus (TB)
  • Method and kit for detecting drug resistance gene mutant type of tuberculous bacillus (TB)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] The design of embodiment 1 primer

[0060] (1) PCR amplification primers:

[0061] According to the drug-resistant gene mutations of the five TB drugs selected to be detected, design specific amplification primers for each drug-resistant gene, and the amplification primers can amplify a DNA sequence including the mutation site. The amplification primer has at least 15 bases at the 3' end that completely match the gene sequence it is targeting, and has a tag sequence of 10 bases (acgttggatg) at the 5' end to distinguish the amplification primer from the extension primer.

[0062] (2) Mass spectrometry extension primer:

[0063] Design an extension primer, the length of the extension primer is 17-28 bases, and its 3' end is located at the last base of the drug-resistant mutation site, the extension primer only extends one base when the extension reaction occurs, and the extension The base is the resistance mutation site. Wherein, the molecular weight difference between...

Embodiment 2

[0076] Preparation of detection template

[0077] The cultured Mycobacterium tuberculosis standard strain CMCC 93009 strain and INH drug-resistant clinical isolate were counted and diluted to 10 5 Bacteria / ml, 10 4 Bacteria / ml, 10 3 Bacteria / ml concentration gradient, each concentration is prepared into a mixture of different proportions: (1) 100% CMCC 93009 bacterial strain, (2) 5% INH drug-resistant clinical isolates added to 93009 bacterial strain, (3) 93009 bacterial strain Add 25% INH resistant clinical isolates, (4) 93009 strains add 50% INH resistant clinical isolates, (5) 93009 strains add 75% INH resistant clinical isolates, (6) 100% INH resistant clinical isolates. DNA was extracted from the above-mentioned mixtures of different concentrations and different ratios (using Tiangen Bacterial DNA Kit for extraction, and the operation method was extracted according to the instructions of the kit).

Embodiment 3

[0078] The detection method of the drug-resistant gene mutant type of embodiment 3TB medicine

[0079] (1) Using the DNA extracted in Example 2 as a template, use the PCR amplification primers in Example 1 to amplify by PCR to obtain the target sequence amplification product. See Table 4 for the PCR amplification reaction system. Among them, all reagents were purchased from Sequenom Company.

[0080] Table 4: PCR amplification reaction system

[0081]

[0082]

[0083] The PCR reaction conditions were 94°C for 15 minutes; denaturation at 94°C for 20 seconds, annealing at 56°C for 30 seconds, extension at 72°C for 1 minute, and a total of 45 cycles of amplification; finally, extension at 72°C for 3 minutes. In this example, the DNA extracted in Example 2 was used as the DNA template for PCR amplification. At the same time, sterile double distilled water was used as a negative control. The control sample and the test sample were reacted and tested according to the same...

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Abstract

The invention provides a method and a kit for detecting a drug resistance gene mutant type of tuberculous bacillus (TB). Compared with the prior art, the invention has the following advantages: 1) reagents and consumables used in the invention are simple and stable, and expensive reagents like fluorescent dyes and special enzyme are not needed; 2) a reaction can be carried out in a microscale system, and usage of a sample and a variety of consumables is reduced; 3) since mass spectrometry is used for direct detection of molecular weight (a mass-to-charge ratio) of DNA and direct determination of types of bases (i.e., no need for signal conversion in any manner), it is theoretically practicable that identification can be realized as long as one copied amplified DNA fragment is amplified, so high sensitivity is obtained; and 4) since mass spectrometry also has the characteristics of automatic and high-flux detection and the like, combination of mass spectrometry and multi-primer extension enables a plurality of drug resistance sites to be simultaneously detected in one reaction system, thereby substantially reducing workload and increasing detection flux.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a multiplex PCR amplification technique and a time-of-flight mass spectrometry genotyping system (MassArray). And more specifically, the present invention relates to a method and a kit for detecting drug-resistant gene mutants of Mycobacterium tuberculosis. Background technique [0002] Mycobacterium tuberculosis (M.tuberculosis, TB), commonly known as Mycobacterium tuberculosis, is the pathogenic bacteria that causes tuberculosis. It can invade all organs of the body, but tuberculosis is the most common. Tuberculosis is still an important infectious disease, and it is estimated that 1 / 3 of the world's population is infected with Mycobacterium tuberculosis. According to the WHO, about 8 million new cases occur each year and at least 3 million people die from the disease. [0003] Antibiotics are the main means of treating tuberculosis, and the first-line anti-tubercu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12R1/32
Inventor 陈唯军赵雁林赵金银王胜芬刘利成逄宇王鹏志周杨吴伟力刘琦徐磊
Owner 北京宏微特斯生物科技有限公司
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