Specific fragment composition of mycobacterium tuberculosis drug-resistant gene of four second-line drugs and application thereof

A technology of Mycobacterium tuberculosis and nucleic acid fragments, applied in the field of drug resistance detection of Mycobacterium tuberculosis

Inactive Publication Date: 2015-01-14
ICDC CHINA CDC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The German HAIN company already has a GenoType MTBDRs1 kit based on linear hybridization technology that can detect gyrA mutations associated with fluoroquinone drug resistance, but this kit is limited to the detection of fluoroquinone drug resistance in second-line drugs

Method used

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  • Specific fragment composition of mycobacterium tuberculosis drug-resistant gene of four second-line drugs and application thereof
  • Specific fragment composition of mycobacterium tuberculosis drug-resistant gene of four second-line drugs and application thereof
  • Specific fragment composition of mycobacterium tuberculosis drug-resistant gene of four second-line drugs and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0172] Example 1. Preparation of fluoroquinolones, kanamycin, amikacin and capreomycin sensitivity detection film strips

[0173] 1) Use Clone Manager (Professional Suite) to target the mutation site of the fluoroquinolone resistance-related gene gyrA, kanamycin, amikacin, and capreomycin resistance-related gene rrs, and the mutation site of the eis promoter Point design probe sequence, the probe sequence is SEQ ID NO:8-24;

[0174] 2) Using Clone Manager (Professional Suite) to design the Mycobacterium tuberculosis complex species-specific 16S rRNA probe sequence, the probe sequence is SEQ ID NO.6-7;

[0175] 3) According to the prior art, synthesize sequences such as each probe shown in SEQ ID NO.6-24, and make an amino label at the 5' end of the probe;

[0176] 4) Design and synthesize a biotin sequence probe (SEQ ID NO.5), labeling with amino group at the 5' end and biotin labeling at the 3' end;

[0177] 5) Dilute the newly synthesized probe with TE buffer to make the f...

Embodiment 2

[0188] Example 2. Detection of fluoroquinolone, kanamycin, amikacin and capreomycin resistance of Mycobacterium tuberculosis

[0189] In this example, performance tests were performed on the prepared fluoroquinolone, kanamycin, amikacin and capreomycin sensitivity detection membrane strips.

[0190] Acquisition and identification of resistant strains

[0191] 14 strains of Mycobacterium tuberculosis were obtained from sputum specimens of 14 tuberculosis patients from Fujian, Tibet and Gansu provincial tuberculosis control institutions, among which 11 strains were resistant to quinolones, 3 strains were resistant to both quinolones and kanamycin, The resistant phenotype was determined by the assay described below.

[0192] The drug susceptibility test was carried out by traditional Roche medium ratio method. Take 1 ring of Mycobacterium tuberculosis grown on Roche's medium for 4-5 weeks, grind the bacteria to make 1 McFarland turbidity, and then adjust it to 10 with normal ...

Embodiment 3

[0223] Example 3. Detection of fluoroquinolones, kanamycin, amikacin and capreomycin resistance of Mycobacterium tuberculosis and comparison with other testing methods

[0224] The present embodiment tested the effect of the fluoroquinolones prepared according to the application, kanamycin, amikacin and capreomycin sensitivity detection membrane strips on a larger scale, and compared with the conventional solid medium ratio method ( Phenotype detection) and the results obtained by DNA sequencing method for cross-comparison.

[0225] Examples 1 and 2 show the preparation method and detection method of film strips for detecting drug susceptibility to fluoroquinolones, kanamycin, amikacin and capreomycin.

[0226] Using the fluoroquinolones, kanamycin, amikacin and capreomycin sensitivity test strips prepared by this application to analyze the gyrA gene mutation of 214 clinical isolates of Mycobacterium tuberculosis, the results showed that 59 strains of fluoroquinolones Mutatio...

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Abstract

The invention provides a nucleotide acid fragment composition related to mycobacterium tuberculosis drug resistance detection of four second-line drugs, namely a fluoroquinolone drug, aminoglycoside drug kanamycin and amikacin (amikacin) and cyclopeptide drug capreomycin which are used for treating tuberculosis, and application thereof, and particularly provides a promotor mutation nucleotide acid fragment composition of a drug-resistant related gene gyrA of the fluoroquinolone drug and drug-resistant related genes rrs and eis of the aminoglycoside drug kanamycin and amikacin (amikacin) and the cyclopeptide drug capreomycin, a specific mutation detection oligonucleotide probe, a kit prepared through the specific mutation detection oligonucleotide probe and a related drug resistance detection method.

Description

field of invention [0001] The present application generally relates to the technical field of detection of drug resistance of pathogenic bacteria. Specifically, this application relates to the tuberculosis branch of four second-line drugs for tuberculosis, fluoroquinolones, aminoglycosides kanamycin and amikacin (amikacin) and cyclic peptides capreomycin Bacillus resistance detection. Background of the invention [0002] Currently, tuberculosis is still one of the major public health problems in most developing countries, while multidrug resistant tuberculosis (MDR-TB) and extensive drug resistant tuberculosis (XDR-TB) are The emergence of tuberculosis has made the epidemic situation more severe. China is one of the 22 countries with a high burden of tuberculosis in the world, and the number of new tuberculosis patients every year ranks second in the world, especially the epidemic of drug-resistant tuberculosis is becoming more and more serious. [0003] Fluoroquinolones,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/32
CPCC12Q1/68C12Q1/689C12Q1/6834C12Q2600/106C12Q2600/156C12Q2531/113
Inventor 万康林李桂莲赵丽丽
Owner ICDC CHINA CDC
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