Mycobacterium tuberculosis complex rapid detection kit and preparation method thereof

A technology of Mycobacterium tuberculosis and detection kit, which is applied in biochemical equipment and methods, microorganism-based methods, and determination/inspection of microorganisms, etc. Less species and other problems, to achieve the effect of stable DNA amplification

Pending Publication Date: 2020-06-12
WEST CHINA HOSPITAL SICHUAN UNIV +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The existing nucleic acid detection methods using detection kits to detect Mycobacterium tuberculosis mostly focus on the genetic design of a certain type or several types of Mycobacterium tuberculosis, but lack of comparison and elimination of other Mycobacterium tuberculosis, which has certain advantages. limitations; such as the invention is a kit and method for identifying mycobacterial strains (application number: CN201510812703.9), comprising the following steps: (1) design and synthesis of primers and probes; (2) PCR Reaction components, (3) PCR amplification; (4) melting curve a

Method used

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  • Mycobacterium tuberculosis complex rapid detection kit and preparation method thereof
  • Mycobacterium tuberculosis complex rapid detection kit and preparation method thereof
  • Mycobacterium tuberculosis complex rapid detection kit and preparation method thereof

Examples

Experimental program
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Embodiment 1

[0072] The purpose of the present invention is achieved through the following technical solutions:

[0073] Include identification reagent, described identification reagent comprises primer pair and probe, described primer pair comprises at least one pair in ten pairs of primers, and described probe comprises at least one in ten probes; The pair of primers are: the first pair: upstream primer IS6110-F1, downstream primer IS6110-R1; the second pair: upstream primer IS6110-F2, downstream primer IS6110-F2; the third pair: upstream primer IS6110-F3, downstream primer IS6110 -F3; fourth pair: upstream primer IS6110-F4, downstream primer IS6110-R4; fifth pair: upstream primer IS6110-F5, downstream primer IS6110-F5; sixth pair: upstream primer IS6110-R6, downstream primer IS6110-R6 ; the seventh pair: upstream primer IS6110-R7, downstream primer IS6110-R7; the eighth pair: upstream primer IS6110-R8, downstream primer IS6110-R8; the ninth pair: upstream primer IS6110-R9, downstream pr...

Embodiment 2

[0089] The primer pair is changed to the second primer pair, and the corresponding probe is changed to the second probe at the same time; the remaining formulas and steps are the same as in Example 1; then the sensitivity detection can be obtained as follows: figure 2 As shown, the lowest detection limit of the second probe is: 1.0×10 - 3 pg / μL; specific detection can be obtained as Figure 15 As shown, only members of the Mycobacterium tuberculosis complex will test positive for DNA, while the remaining strains (Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, Staphylococcus aureus, Pseudomonas Bacteria) DNA are all negative; and the cycle CT difference of the repeated experiments, such as Figure 25 As shown, the difference between the CT values ​​of the second primer pair and the second probe is not more than 0.5.

Embodiment 3

[0091] The primer pair is changed to the third primer pair, and the corresponding probe is changed to the third probe; the remaining formulas and steps are the same as in Example 1; then the sensitivity detection can be obtained as follows: image 3 As shown, the lowest detection limit of the third probe is: 1.0×10 - 2 pg / μL; specific detection can be obtained as Figure 16As shown, only members of the Mycobacterium tuberculosis complex will test positive for DNA, while the remaining strains (Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, Staphylococcus aureus, Pseudomonas Bacteria) DNA are all negative; and the cycle CT difference of the repeated experiments, such as Figure 26 As shown, the difference between the respective CT values ​​of the third pair of primers and the third probe does not exceed 0.5.

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Abstract

The invention discloses a mycobacterium tuberculosis complex rapid detection kit, which comprises an identification reagent, and the identification reagent comprises at least one pair of ten pairs ofprimers and at least one of ten probes. The invention also provides a preparation method of the rapid detection kit for the mycobacterium tuberculosis complex. The preparation method comprises the following steps of: S1, designing primers and probes according to gene sequences of the mycobacterium tuberculosis complex compared in a gene pool; S2, constructing a kit program of THE mycobacterium tuberculosis complex; S3, testing the specificity and sensitivity of the kit program; S4, carrying out repeated tests on the established kit program for many times; S5, preparing a standard target gene pool; and S6, constructing a specific detection kit with high sensitivity. The invention has the beneficial effects that by utilizing the kit based on the specific primer pair and the probe provided bythe invention, mycobacterium tuberculosis can be rapidly and comprehensively detected and identified, and possibility is provided for subsequent comprehensive detection; the sensitivity test, the specificity test and the repeatability test are utilized, and meanwhile, the CT difference value is compared, so that rapid and accurate detection is achieved.

Description

technical field [0001] The invention relates to the field of detection kits for Mycobacterium tuberculosis, in particular to a rapid detection kit for Mycobacterium tuberculosis complex and a preparation method thereof. Background technique [0002] Tuberculosis is one of the major infectious diseases in the world, and my country is a big tuberculosis country in the world and it continues to grow. According to statistics, one out of every three people has Mycobacterium tuberculosis, and the number of infected people has far exceeded 400 million. , of which 90%-98% are in the incubation period. The disease is a chronic infectious disease caused by Mycobacterium tuberculosis (M.tuberculosis), Mycobacterium microti (M.microti), Mycobacterium bovis (M.bovis), etc. Organs, mostly the lungs. [0003] As early as 17,000 years ago, some scholars discovered Mycobacterium tuberculosis in the remains of bison, which is the earliest origin of tuberculosis found so far [Lu Zhiming. The ...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6851C12Q1/06C12R1/32
CPCC12Q1/689C12Q1/6851C12Q2531/113C12Q2563/107
Inventor 应斌武柯博文王睿敏
Owner WEST CHINA HOSPITAL SICHUAN UNIV
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