A combination of four first-line drug resistance gene specific fragments of Mycobacterium tuberculosis and its application

Abstract

The invention provides a mycobacterium tuberculosis drug resistance detection related nucleic acid fragment combination of four first-line drugs including rifampin, isoniazid, streptomycin and ethambutol for tuberculosis, and an application thereof. Specifically, the invention provides a mutated nucleic acid fragment combination of rifampin drug resistance-related gene rpoB, isoniazid drug resistance-related gene katG, inhA promoter and ahpC promoter, streptomycin drug resistance-related genes rpsL and rrs, and ethambutol drug resistance-related gene embB, oligonucleotide for specifically detecting mutation, a kit prepared from the oligonucleotide, and related drug resistance detection methods.

Description

[0001] field of invention

[0002] The present application generally relates to the technical field of detection of drug resistance of pathogenic bacteria. Specifically, this application relates to the detection of drug resistance of Mycobacterium tuberculosis to four first-line drugs for tuberculosis, rifampicin, isoniazid, streptomycin and ethambutol.

[0003] Background of the invention

[0004] At present, tuberculosis is still one of the major public health problems in most developing countries, and the emergence of multi-drug resistant tuberculosis (MDR-TB) and extensive drug-resistant tuberculosis (extensive drug resistant tuberculosis, XDR-TB) making the epidemic situation of tuberculosis 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.

[0005] Rifampic...

Examples

Embodiment 1

[0258] Example 1. Preparation of Rifampicin Drug Sensitivity Detection Membrane Strips

[0259] 1) Applying Clone Manager (Professional Suite) to design a probe sequence for the mutation site of rpoB, a rifampicin resistance-related gene, the probe sequence is SEQ ID NO: 15-26;

[0260] 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.13-14;

[0261] 3) Synthesize each probe with the sequence shown in SEQ ID NO.13-26 according to the prior art, and label the 5' end of the probe with an amino group;

[0262] 4) Designing and synthesizing a biotin sequence probe (SEQ ID NO.12), labeling with amino group at the 5' end and biotin labeling at the 3' end;

[0263] 5) Dilute the newly synthesized probe with TE buffer to make the final concentration 10 pmol / μL;

[0264] 6) Incubate the nylon membrane (Biodyne C membrane) with freshly prepared 16% (w / v) EDAC at room tem...

Embodiment 2

[0274] Example 2. Detection of rifampicin resistance of Mycobacterium tuberculosis

[0275] In this example, performance tests were performed on the prepared rifampicin drug susceptibility detection membrane strip.

[0276] Acquisition and identification of resistant strains

[0277] Thirty-eight rifampicin-resistant strains were obtained from sputum samples of 38 tuberculosis patients from Fujian and Anhui provincial tuberculosis prevention institutions, and the drug-resistant phenotype was determined by the following tests.

[0278] 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 saline. -2 mg / ml and 10 -4 mg / ml bacteria solution, inoculate 10 -3 mg and 10 -5 mg bacteria solution on the culture medium containing anti-tuberculosis drugs, cultured at 37°C for 4 weeks...

Embodiment 3

[0311] Example 3. Detection of Rifampicin, Isoniazid, Streptomycin and Ethambutol resistance of Mycobacterium tuberculosis and comparison with other testing methods

[0312] This embodiment tested the effect of the rifampicin, isoniazid, streptomycin and ethambutol drug susceptibility detection film strip prepared according to the application on a larger scale, and compared with the conventional solid medium ratio method (Table type detection) and the results obtained by DNA sequencing method for cross-comparison.

[0313] Prepare rifampicin drug susceptibility detection membrane strips as described in Example 1, and prepare isoniazid, streptomycin and ethambutol drug susceptibility detection membrane strips in a similar manner, for the wild type used in each drug The probes and mutation detection probes are respectively: SEQ ID NO:27-38, SEQ ID NO:39-45, SEQ ID NO:46-53.

[0314] The detection method is generally as described in Example 2, wherein the forward and reverse amp...

[0001] field of invention

[0002] The present application generally relates to the technical field of detection of drug resistance of pathogenic bacteria. Specifically, this application relates to the detection of drug resistance of Mycobacterium tuberculosis to four first-line drugs for tuberculosis, rifampicin, isoniazid, streptomycin and ethambutol.

[0003] Background of the invention

[0004] At present, tuberculosis is still one of the major public health problems in most developing countries, and the emergence of multi-drug resistant tuberculosis (MDR-TB) and extensive drug-resistant tuberculosis (extensive drug resistant tuberculosis, XDR-TB) making the epidemic situation of tuberculosis 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.

[0005] Rifampic...

Examples

Embodiment 1

[0258] Example 1. Preparation of Rifampicin Drug Sensitivity Detection Membrane Strips

[0259] 1) Applying Clone Manager (Professional Suite) to design a probe sequence for the mutation site of rpoB, a rifampicin resistance-related gene, the probe sequence is SEQ ID NO: 15-26;

[0260] 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.13-14;

[0261] 3) Synthesize each probe with the sequence shown in SEQ ID NO.13-26 according to the prior art, and label the 5' end of the probe with an amino group;

[0262] 4) Designing and synthesizing a biotin sequence probe (SEQ ID NO.12), labeling with amino group at the 5' end and biotin labeling at the 3' end;

[0263] 5) Dilute the newly synthesized probe with TE buffer to make the final concentration 10 pmol / μL;

[0264] 6) Incubate the nylon membrane (Biodyne C membrane) with freshly prepared 16% (w / v) EDAC at room tem...

Embodiment 2

[0274] Example 2. Detection of rifampicin resistance of Mycobacterium tuberculosis

[0275] In this example, performance tests were performed on the prepared rifampicin drug susceptibility detection membrane strip.

[0276] Acquisition and identification of resistant strains

[0277] Thirty-eight rifampicin-resistant strains were obtained from sputum samples of 38 tuberculosis patients from Fujian and Anhui provincial tuberculosis prevention institutions, and the drug-resistant phenotype was determined by the following tests.

[0278] 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 saline. -2 mg / ml and 10 -4 mg / ml bacteria solution, inoculate 10 -3 mg and 10 -5 mg bacteria solution on the culture medium containing anti-tuberculosis drugs, cultured at 37°C for 4 weeks...

Embodiment 3

[0311] Example 3. Detection of Rifampicin, Isoniazid, Streptomycin and Ethambutol resistance of Mycobacterium tuberculosis and comparison with other testing methods

[0312] This embodiment tested the effect of the rifampicin, isoniazid, streptomycin and ethambutol drug susceptibility detection film strip prepared according to the application on a larger scale, and compared with the conventional solid medium ratio method (Table type detection) and the results obtained by DNA sequencing method for cross-comparison.

[0313] Prepare rifampicin drug susceptibility detection membrane strips as described in Example 1, and prepare isoniazid, streptomycin and ethambutol drug susceptibility detection membrane strips in a similar manner, for the wild type used in each drug The probes and mutation detection probes are respectively: SEQ ID NO:27-38, SEQ ID NO:39-45, SEQ ID NO:46-53.

[0314] The detection method is generally as described in Example 2, wherein the forward and reverse amp...

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