Drug-resistance gene film chip for detecting mycobacterium tuberculosis

A technology of Mycobacterium tuberculosis and drug-resistant genes, which is applied in the direction of nucleotide library, microbe-based method, microbiological determination/testing, etc., can solve the problem of slow growth of Mycobacterium tuberculosis, failure to meet clinical detection, low detection rate of sputum culture, etc. question

Inactive Publication Date: 2009-11-18
GUANGXI MEDICAL UNIVERSITY
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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

Method used

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  • Drug-resistance gene film chip for detecting mycobacterium tuberculosis
  • Drug-resistance gene film chip for detecting mycobacterium tuberculosis
  • Drug-resistance gene film chip for detecting mycobacterium tuberculosis

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Embodiment Construction

[0088] The technical solutions of the present invention will be further described below through specific examples.

[0089] 1. The membrane chip used for detection is 2cm wide and 6.5cm long.

[0090] 2. Equipment required for detection: PCR instrument, shaker, hybridization furnace, constant temperature water bath.

[0091] 3. Required reagents:

[0092] 20×SSC solution: 175.3g NaCl, 88.2g sodium citrate, dissolved in 900ml water to adjust the pH to 7.0, add water to make up to 1L, and sterilize under high temperature and high pressure.

[0093] 10% SDS: Dissolve 100g of electrophoretic grade SDS in 900ml of water, heat to 68°C to aid dissolution, add a few drops of concentrated hydrochloric acid, adjust the pH to 7.2, add water to make up to 1L.

[0094] Solution A (2×SSC, 0.1% SDS, pH7.4), sterilized under high temperature and high pressure.

[0095] Liquid B (0.5×SSC, 0.1% SDS, pH7.4), sterilized under high temperature and high pressure.

[0096] Solution C (0.1M sodiu...

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Abstract

A drug-resistance gene film chip for detecting mycobacterium tuberculosis is prepared by designing 54 probe spotting on a nylon film by aiming at the mutant sites of mycobacterium tuberculosis rpoB, kagG, embB, inhA, ahpC, gyrA, rrs, rpsL and pncA gene; 12 pairs of specific primers with biotin-labeled at 5' end are utilized; a sample DNA is subjected to triple PCR and amplified to form a large amount of gene fragment products with biotin; the amplified products and the probes on the film chip carry out specific hybridization; and then film washing, enzyme-linking and color reaction are carried out, thus preparing the chip. The gene film chip and the detection method thereof can detect the common gene mutations of the mycobacterium tuberculosis on the drug resistance of drugs such as isoniazid, rifampicin, streptomycin, ethambutol, pyrazinamide, quinolone and the like at one step, and are applicable to extracorporeal detection sputum sample, clinical isolation strains and mycobacterium tuberculosis multi-drug resistant gene in the organization sample.

Description

technical field [0001] The invention relates to a gene membrane chip and a detection method thereof, in particular to a genotype membrane chip for detecting multi-drug resistance of mycobacterium tuberculosis. It can be applied to detect the MDR gene of Mycobacterium tuberculosis in sputum samples, clinical isolates and tissue samples in vitro. Background technique [0002] At present, the research on the molecular mechanism of drug resistance of Mycobacterium tuberculosis mainly focuses on the target sites of various anti-tuberculosis drugs and the mutations of related genes. The types of gene mutations of Mycobacterium tuberculosis that have been discovered are mainly point mutations, which are essentially insertions, deletions or substitutions of one or several nucleotides within a gene, resulting in wrong nucleotide codes being translated into wrong amino acid sequences , leading to resistance of Mycobacterium tuberculosis to anti-tuberculosis drugs. The anti-tuberculo...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C40B40/06C12R1/32
Inventor 何敏韦世录何晓
Owner GUANGXI MEDICAL UNIVERSITY
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