Preparation method and application of mc-arms-mmb triple technology in drug-resistant tuberculosis diagnostic kit

A diagnostic kit, technology for tuberculosis, applied in the directions of microorganism-based methods, biochemical equipment and methods, determination/examination of microorganisms, etc.

Active Publication Date: 2021-04-06
FUJIAN MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] At present, few domestic and foreign researchers in the detection of drug-resistant Mycobacterium tuberculosis combine phenotypic bacterial culture with genotypic PCR

Method used

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  • Preparation method and application of mc-arms-mmb triple technology in drug-resistant tuberculosis diagnostic kit
  • Preparation method and application of mc-arms-mmb triple technology in drug-resistant tuberculosis diagnostic kit
  • Preparation method and application of mc-arms-mmb triple technology in drug-resistant tuberculosis diagnostic kit

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Embodiment 1: micro-colony culture method

[0074] Taking the standard strain of Mycobacterium tuberculosis H37Ra as the research object, and taking the standard McFarland turbidimetry as the reference, the strains were respectively prepared into 0.5McF (approximate concentration of bacteria 1.5×10 8 cfu / mL), 1.0McF (approximate concentration of bacteria 3×10 8 cfu / mL), 2.0McF (approximate concentration of bacteria 6×10 8 cfu / mL) McFarland concentration, observe the growth state of micro-colony after 14h and 24h of culture on the filter membrane of Middle brook7H10 solid medium containing penicillin with different concentrations of bacterial solution, and explore the optimal conditions for micro-colony culture, the results are shown in figure 1 .

Embodiment 2

[0075] Embodiment 2: the preparation of standard substance

[0076] 1. Using genetic engineering technology, clone the DNA fragments of IS6110 (92bp) and rpoB (163bp) and katG (149bp) into the PMD-19T vector, and confirm the construction of the standard recombinant plasmid through PCR and sequencing identification.

[0077] 2. Through gradient dilution of the plasmid concentration, the sensitivity experiment was done by PCR reaction and 3% agarose gel electrophoresis, and it was confirmed that the lower limit of detection of Mycobacterium tuberculosis by ordinary PCR was 10 0 copies / ml.

[0078] 3. Using diluted recombinant plasmids with different concentrations to perform real-time PCR reactions, construct positive standards, and formulate standard curves, laying the foundation for the detection of Mycobacterium tuberculosis by molecular beacon fluorescent quantitative PCR technology. Embodiment 3: Fluorescence quantitative PCR (molecular beacon) carries out accounting ampli...

Embodiment 3

[0078] 3. Using diluted recombinant plasmids with different concentrations to perform real-time PCR reactions, construct positive standards, and formulate standard curves, laying the foundation for the detection of Mycobacterium tuberculosis by molecular beacon fluorescent quantitative PCR technology. Embodiment 3: Fluorescence quantitative PCR (molecular beacon) carries out accounting amplification and hybridization detection sample DNA:

[0079] The method for detecting whether Mycobacterium tuberculosis exists in a biological sample according to the present invention comprises the following steps:

[0080] a) collecting biological samples and carrying out micro-colony test b) preparing DNA template c) performing fluorescent quantitative PCR amplification on the DNA template obtained in step b, wherein the primers and probes used are as follows:

[0081] TbIS6110 F:ACGCCTACGT CGCAGGATC

[0082] TbIS6110 R: GGGTCCAGAT GGCTTGCTC

[0083] Tb rpoB F:CGCTGTCGGGGTTGACACA

[008...

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Abstract

The invention discloses a preparation method and application of a MC-ARMS-mMB triplet technology in a drug-resistant tuberculosis diagnosis kit, and describes the MC-ARMS-mMB (microcolony-amplification-impeding mutation system-multiplex molecular beacon) triplet technology , a diagnostic kit for rapid culture of Mycobacterium tuberculosis and its drug-resistant bacteria. At the same time, the preparation method of the diagnostic kit was clarified; and a scientific presentation was made on its clinical application. It is particularly important to point out the clinical application value and better development prospect of the diagnostic kit.

Description

[0001] 1. Technical field [0002] The field of the present invention includes the following: [0003] (1) The field of molecular biology. [0004] Bacterial biometrics research field. [0005] Infectious disease diagnostic medical research field. [0006] 2. Background technology [0007] (1) Molecular biology: the main body of this application is the molecular beacon probe of Mycobacterium tuberculosis. The unique sequence rpoB, katG315 and IS6110 of Mycobacterium tuberculosis were selected as target sequences to design primers and probes to detect Mycobacterium tuberculosis. [0008] (2) Study on the biological characteristics of bacteria: After the microcolony culture of Mycobacterium tuberculosis, it is proved that the inoculated bacteria are live bacteria or drug-resistant bacteria. [0009] (3) Medical research on the diagnosis of infectious diseases. [0010] The treatment of tuberculosis has become a therapeutic challenge, not only because of the innately high lev...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12R1/32
Inventor 朱玲
Owner FUJIAN MEDICAL UNIV
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