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Multidrug-resistant mycobacterium tuberculosis non-fluorescent DNA (deoxyribonucleic acid) microarray detection method and kit

A Mycobacterium tuberculosis detection method technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems that cannot meet the needs of clinical detection, slow growth of Mycobacterium tuberculosis, and reduce the affinity of rifampicin, etc. It achieves the effects of convenient monitoring and quality control, clear and accurate test results, and reduced testing costs

Inactive Publication Date: 2012-10-10
FUDAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The detection results of this method are reliable, but the biggest disadvantage is that the culture conditions are high, and the growth of Mycobacterium tuberculosis is slow, which often takes 6-8 weeks; at the same time, the detection rate of sputum culture is low, only 30%. The rate is only about 30%, which cannot meet the needs of clinical testing
The rpoB gene is mutated under the drug selection pressure of rifampicin to change the conformation of the encoded rpoB subunit, thereby reducing the affinity with rifampicin

Method used

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  • Multidrug-resistant mycobacterium tuberculosis non-fluorescent DNA (deoxyribonucleic acid) microarray detection method and kit
  • Multidrug-resistant mycobacterium tuberculosis non-fluorescent DNA (deoxyribonucleic acid) microarray detection method and kit
  • Multidrug-resistant mycobacterium tuberculosis non-fluorescent DNA (deoxyribonucleic acid) microarray detection method and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1: Preparation of probe synthesis and gene chip

[0055] Purchase aldehyde-modified glass slides. Design 16 oligonucleotide probes, modify the 5' ends of the probes with amino groups, and commission related companies to synthesize the probes. The synthesized probe was diluted into an aqueous solution with a concentration of 100 μM, and mixed with 2× spotting buffer (product of Shanghai Bio-Technology Co., Ltd.) in equal proportions. Use the spotting instrument of Affymetrix Company to spot the chip array according to the method described in the manual, and place it at room temperature for 3 hours for later use. The probe information is shown in Table 4.

Embodiment 2

[0056] Example 2: Extraction of Mycobacterium tuberculosis Genomic DNA

[0057] (1) Take the isolates from clinical samples identified as positive for Mycobacterium tuberculosis after culture as samples, inactivate the bacteria at 80°C for 2 hours, and use a commercial bacterial genomic DNA extraction kit to extract the sample DNA. The obtained genomic DNA can be It can be directly used as a PCR reaction template, and can also be stored at -20°C for future use.

[0058] (2) Take 2 mL of clinically obtained sputum, add 2.5 times the volume of 4% NaOH, and incubate at 37°C for 30 minutes. The liquefied sputum was centrifuged to remove the supernatant. The obtained precipitate was extracted with a commercial bacterial genomic DNA extraction kit, and the obtained genomic DNA could be directly used as a PCR reaction template or stored at -20°C for future use.

Embodiment 3

[0059] Example 3: Primer synthesis, PCR reaction and product labeling

[0060] Download the promoter sequences of Mycobacterium tuberculosis rpoB gene, katG gene and inhA gene from the NCBI database, use Primer Primer 5 software to design forward and reverse primers and entrust Jerui Biotech to synthesize them (reverse primers rpoB-R, katG-R, inhA The 5' end of -R is labeled with biotin), and the primer information is shown in Table 1.

[0061] The synthesized primers were dissolved in water and diluted to 10 μM. Mix a pair of forward and reverse primers with Taq DNA polymerase, 10×PCR buffer, dNTP, UNG enzyme, dUTP, MgCl 2 The solution, pure water and the amplification template obtained in Example 2 were mixed to prepare a PCR amplification system for the target fragment of rpoB / katG / inhA gene. The PCR amplification system formula is as follows:

[0062] PCR system (rpoB / katG / inhA) Volume (μL) 10×PCR buffer 5 dNTPs (2 μM) 1 Forward primer (10 μ...

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Abstract

The invention belongs to the field of biotechnological detection, and particularly relates to a multidrug-resistant mycobacterium tuberculosis detection method and a kit. The kit comprises PCR (polymerase chain reaction) forward and reverse amplification primers (a biotin label is arranged at the 5' end of the forward amplification primer) of the nucleic acid segments of the major drug-resistant gene of mycobacterium tuberculosis rifampicin and isoniazide and an oligonucleotides probe sequence (an amino mark is arranged at the 5' end); and the mutation conditions of the loca 511, 516, 526, 531 and 533 of the rpoB gene, the locus 315 of the katG gene and the locus 15 of the inhA gene can be detected at the same time in a reaction unit. The kit provided by the invention can quickly detect the mutation condition of the major drug-resistant gene of mycobacterium tuberculosis rifampicin and isoniazide, has low requirements on instrument and expenses, is easy to operate and can be used for detecting the drug-resistant gene of the mycobacterium tuberculosis.

Description

technical field [0001] The invention belongs to the field of biotechnology detection, and in particular relates to a detection method and kit for multidrug-resistant Mycobacterium tuberculosis, in particular to a DNA microarray hybridization detection method for rifampicin and isoniazid resistance gene mutations and the method prepared by using the method kit. Background technique [0002] Tuberculosis is one of the most threatening infectious diseases to humans in the world today, and it has been on the rise in the world since the mid-1980s. According to the World Health Organization, my country is one of the 22 countries with a high burden of tuberculosis in the world, and the number of active tuberculosis patients ranks second in the world. At present, the tuberculosis infection rate of all age groups in my country is 44.5%, and about 550 million people in the country have been infected by tuberculosis bacteria, which is higher than the infection rate of 1 / 3 of the globa...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12R1/32
Inventor 薛文斐朱滨张舒林李瑶张墨翰吴海
Owner FUDAN UNIV
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