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Molecular beacon probe for rapid detection of rifampin-resistant mycobacterium tuberculosis and detection method thereof

A molecular beacon probe, the technology of Mycobacterium tuberculosis, which is applied in the fields of biochemical equipment and methods, determination/inspection of microorganisms, DNA/RNA fragments, etc. Safety, difficult to promote and use, etc., to achieve the effect of suitable for popularization and use, high sensitivity and strong specificity

Inactive Publication Date: 2019-06-14
北京岱美仪器有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main disadvantage of culture-based drug susceptibility testing is that it takes a long time. First, the identification of Mycobacterium tuberculosis needs to be carried out on the basis of isolation and culture. This process takes 4-8 weeks, and then the follow-up traditional solid ratio is carried out on this basis. method (4-8 weeks) or liquid drug susceptibility (1-2 weeks), and the live bacteria operation is performed, which is not safe for people and the environment
The fluorescent quantitative PCR melting curve represented by nucleic acid-based molecular biology detection requires the peak shape and Tm value of the melting peak to interpret the results, which increases the difficulty of basic-level experimental staff and is not easy to promote and use at the grass-roots level.

Method used

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  • Molecular beacon probe for rapid detection of rifampin-resistant mycobacterium tuberculosis and detection method thereof
  • Molecular beacon probe for rapid detection of rifampin-resistant mycobacterium tuberculosis and detection method thereof
  • Molecular beacon probe for rapid detection of rifampin-resistant mycobacterium tuberculosis and detection method thereof

Examples

Experimental program
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preparation example Construction

[0041](1) Preparation of template DNA: extract the DNA of the sample to be tested, which is a sputum sample identified as containing Mycobacterium tuberculosis or isolated Mycobacterium tuberculosis or non-tuberculosis mycobacterium;

[0042] For the detection of a sample, tube A and tube B are required to be tested at the same time

[0043] (2) Real-time fluorescent quantitative PCR reaction: For tube A: 25 μL of PCR amplification system: 12.5 μL 2× amplification buffer, 1 μL rpoB-F, 1 μL rpoB-R, 0.8 μL Beacon1, 0.8 μL Beacon2, 0.8 μL Beacon3, 3.1 μL water, 5.0 μL sample DNA.

[0044] For tube B: PCR amplification system 25 μL: 12.5 μL 2× amplification buffer, 1 μL rpoB-F, 1 μL rpoB-R, 0.8 μL Beacon4, 0.8 μL Beacon5, 3.9 μL water, 5.0 μL sample DNA.

[0045] (3) PCR amplification program: Touchdown cycle program: 94°C 10S, 66°C 15S (1°C drop per cycle), 72°C 15S, a total of 10 cycles; then 40 cycles, 94°C 10S, 56°C 30S ( Simultaneously carry out FAM, HEX and TEXRED three ki...

Embodiment 1

[0049] Preparation of real-time fluorescent quantitative PCR amplification system:

[0050] (1) For tube A: PCR amplification system 25 μL: 12.5 μL 2× amplification buffer, 1 μL rpoB-F, 1 μL rpoB-R, 0.8 μL Beacon1, 0.8 μL Beacon2, 0.8 μL Beacon3, 3.1 μL water, 5.0 μL sample DNA.

[0051] For tube B: PCR amplification system 25 μL: 12.5 μL 2× amplification buffer, 1 μL rpoB-F, 1 μL rpoB-R, 0.8 μL Beacon4, 0.8 μL Beacon5, 3.9 μL water, 5.0 μL sample DNA.

[0052] (2) PCR amplification program: Touchdown cycle program: 94°C 10S, 66°C 15S (1°C drop per cycle), 72°C 15S, a total of 10 cycles; then 40 cycles, 94°C 10S, 56°C 30S ( Simultaneously carry out FAM, HEX and TEXRED three kinds of fluorescence detection), 72 ℃ 15S.

[0053] Rifampin-sensitive Mycobacterium tuberculosis and rifampicin-resistant Mycobacterium tuberculosis were diagnosed using the above reaction system as follows. The sample to be tested in this embodiment is a sputum sample suspected to contain Mycobacteriu...

Embodiment 2

[0066] Preparation of real-time fluorescent quantitative PCR amplification system:

[0067] (1) For tube A: PCR amplification system 25 μL: 12.5 μL 2× amplification buffer, 1 μL rpoB-F, 1 μL rpoB-R, 0.8 μL Beacon1, 0.8 μL Beacon2, 0.8 μL Beacon3, 3.1 μL water, 5.0 μL sample DNA.

[0068] For tube B: PCR amplification system 25 μL: 12.5 μL 2× amplification buffer, 1 μL rpoB-F, 1 μL rpoB-R, 0.8 μL Beacon4, 0.8 μL Beacon5, 3.9 μL water, 5.0 μL sample DNA.

[0069] (2) PCR amplification program: Touchdown cycle program: 94°C 10S, 66°C 15S (1°C drop per cycle), 72°C 15S, a total of 10 cycles; then 40 cycles, 94°C 10S, 56°C 30S ( Simultaneously carry out FAM, HEX and TEXRED three kinds of fluorescence detection), 72 ℃ 15S.

[0070] Use the above reaction system to diagnose rifampicin-sensitive Mycobacterium tuberculosis and rifampicin-resistant Mycobacterium tuberculosis in the following manner. The sample to be tested in this embodiment is the sputum sample of a patient who has ...

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Abstract

The invention discloses a molecular beacon probe for rapid detection of rifampin-resistant mycobacterium tuberculosis and a detection method thereof. The molecular beacon probe comprises Beacon 1, Beacon 2, Beacon 3, Beacon 4 and Beacon 5. All 81 sites of a rifampin-resistant rpoB gene are covered by the five probes, so that the rifampin-resistant mycobacterium tuberculosis can be detected. The molecular beacon probe has high detection sensitivity and specificity, and can rapidly and specifically detect the rifampicin-resistant mycobacterium tuberculosis. The invention also discloses a detection method of the molecular beacon probe for rapid detection of the rifampin-resistant mycobacterium tuberculosis.

Description

technical field [0001] The invention relates to a molecular beacon probe for rapidly detecting rifampicin-resistant mycobacterium tuberculosis and a detection method thereof, belonging to the technical field of detection of mycobacterium tuberculosis. Background technique [0002] Tuberculosis is an ancient disease that seriously endangers human health and development. Although the invention and clinical application of anti-tuberculosis drugs have played an important role in the control of tuberculosis, the situation is still not ideal. With the widespread emergence of drug-resistant tuberculosis, the situation of tuberculosis prevention and control is becoming increasingly severe. According to the "Global Tuberculosis Report 2018" released by the World Health Organization, China ranks second among countries with a high burden of tuberculosis in the world, with approximately 900,000 new tuberculosis patients each year. Drug-resistant TB remains a public health crisis and t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6851C12Q1/04C12N15/11
Inventor 马彩霞畅志强
Owner 北京岱美仪器有限公司
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