Kit for detecting genotype of mycobacterium tuberculosis clinical isolation strain quickly

A Mycobacterium tuberculosis detection kit technology, applied in the field of medical testing, can solve the problem of insufficient resolution of Mycobacterium tuberculosis strains, and achieve the effect of convenient and efficient amplification conditions

Inactive Publication Date: 2011-06-15
FUDAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, although MIRU-12 has a relatively good resolution in the United States, the resolution of

Method used

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  • Kit for detecting genotype of mycobacterium tuberculosis clinical isolation strain quickly
  • Kit for detecting genotype of mycobacterium tuberculosis clinical isolation strain quickly
  • Kit for detecting genotype of mycobacterium tuberculosis clinical isolation strain quickly

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1. Identification of Mycobacterium tuberculosis

[0070] 1. Template DNA preparation (boiling method)

[0071] (1) Scrape 1-2 inoculum rings of Mycobacterium tuberculosis H37Rv strain from the solid medium, resuspend in 100uL TE, and inactivate at 80°C for 30 minutes.

[0072] (2) The inactivated strains are taken out of the P3 laboratory for the following operations:

[0073] Boil at 100°C for 1 minute, immediately place on ice for 2 minutes, centrifuge at 12,000r / min for 10 minutes, take the supernatant and put it in another sterile EP tube, and store at -20°C. The supernatant is the template DNA for PCR amplification.

[0074] 2. Identify whether the clinical isolates are Mycobacterium tuberculosis

[0075] According to the sequence analysis, we designed 2 pairs of primers to identify whether the clinical isolates are mycobacteria. These two pairs of primers are: Mtb 1 / Mtb 2 and IS6110-P1 / IS6110-P2. In a reaction system, when the strain to be identified i...

Embodiment 2

[0084] The identification of embodiment 2 Mycobacterium tuberculosis substrains

[0085] The identification of the Beijing type of Mycobacterium tuberculosis is carried out through the PCR reaction of three primers (RD105-P1 / RD195-P2 / RD105-P3), that is, three different primers are added to the same reaction system, and according to the amplification product The size of the genotype of the detected strain was judged. For the Beijing-type Mycobacterium tuberculosis, the amplified product is 781bp, and if a product of 1495bp is amplified, it indicates that the Mycobacterium tuberculosis is not a Beijing-type Mycobacterium tuberculosis.

[0086] (1) PCR amplification: the volume of each PCR reaction system is 20ul: containing 1ul DNA template and 19ul PCR reaction mixture for identifying the Beijing strain of Mycobacterium tuberculosis (including specific primers for identifying the Beijing strain of Mycobacterium tuberculosis (RD105-P1 / RD195- P2 / RD105-P3), dNTP, Taq enzyme, magn...

Embodiment 3

[0094] Example 3 Carry out 7-site VNTR typing

[0095] (1) PCR amplification: the volume of each PCR reaction system is 20 ul: containing 1 ul of DNA template and 19 ul of Mycobacterium tuberculosis Beijing strain identification PCR reaction mixture (including specific primers for identifying each site of Mycobacterium tuberculosis (VNTR3820, Qub11b, Qub18, Qub11a, MIRU26, Qub26 and Mtub21), dNTPs, Taq enzymes, magnesium ions and corresponding buffer systems). (2) The PCR reaction amplifies the corresponding target sequence according to the following conditions:

[0096] a.VNTR3820: 5 minutes at 94°C

[0097]

[0098] 72°C for 7 minutes

[0099] b. Qub11b, Qub11a, Qub18, Mtub21, Miru26, Qub26

[0100] 94°C for 5 minutes

[0101]

[0102] 72°C for 7 minutes

[0103] (3) Agarose gel electrophoresis of the amplified product:

[0104] Use 1% agarose gel electrophoresis, voltage 150V, electrophoresis time 90-120 minutes.

[0105] According to the relative positions of th...

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Abstract

The invention belongs to the field of medical tests and provides a kit for detecting the genotype of a mycobacterium tuberculosis clinical isolation strain quickly. The kit comprises a detection probe designed aiming at an IS6110 insertion sequence. In a mycobacterium tuberculosis typing method involved in the invention, deoxyribose nucleic acid (DNA) of a sample strain is extracted, polymerase chain reaction (PCR) amplification is performed by taking the detection probe as a primer, and if the obtained product is consistent with a mycobacterium tuberculosis DNA amplification product, a sample comprises mycobacterium tuberculosis. The method is high in resolution ratio of the mycobacterium tuberculosis clinical isolation strain spreading in China currently, the genotype result of every strain is output in a digitization mode, and the genotype results can be compared among different laboratories conveniently. Moreover, a large number of equipment is not needed in the method, the kit can be operated simply, and the price is relatively low. The kit provides a powerful tool for exploring and researching the prevailing and spreading law of tuberculosis in China.

Description

technical field [0001] The invention belongs to the field of medical inspection, and relates to a method for identifying mycobacterium tuberculosis, in particular to a rapid genotype detection kit for clinically isolated strains of mycobacterium tuberculosis. Background technique [0002] Tuberculosis is an infectious disease that threatens human health for a long time, and its pathogen is Mycobacterium tuberculosis. According to the statistics of the World Health Organization, one-third of the world's population is infected with Mycobacterium tuberculosis, of which 9 million new tuberculosis patients are diagnosed each year, and 1.6 million people die of tuberculosis. China is one of the 22 countries with a high burden of tuberculosis in the world. The number of tuberculosis patients ranks second in the world. The number of tuberculosis infections in the country is as high as 500 million, and there are 4.5 million tuberculosis patients. There are 1.45 million new cases ever...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04
Inventor 李霞张璐陈婧王春香高建恩高谦
Owner FUDAN UNIV
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