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CN-121992125-A - Primer probe composition for detecting drug resistance of mycobacterium tuberculosis complex rifampicin and isoniazid and application of primer probe composition

CN121992125ACN 121992125 ACN121992125 ACN 121992125ACN-121992125-A

Abstract

The invention belongs to the technical field of immunological detection, and particularly relates to a primer probe composition for detecting rifampicin and isoniazid resistance of a mycobacterium tuberculosis complex and application thereof. According to the invention, a multicolor melting curve method is adopted, the mutation detection of the rifampicin resistance gene is carried out by detecting the mutation of the resistance determining region of 507-533 sites of the mycobacterium tuberculosis complex rpoB gene, the mutation detection of the isoniazid resistance gene is carried out by detecting the mutation of the ahpC promoter region, the inhA promoter region and the katG315 codon, and the resistance condition of the detected strain can be judged according to the Tm value of the melting curve. The results of the examples show that the rifampicin resistance detection and isoniazid resistance detection in the invention realize the detection of the mutation of one drug in one tube without separating two tubes, and can independently detect the drug resistance of one drug or the drug resistance of two drugs in two tubes according to different conditions when in use, thereby being flexible in application and wider in application market.

Inventors

  • LI LEI
  • Tan Miaoying
  • GUO HUA
  • CHEN HUIPING

Assignees

  • 珠海贝索生物技术有限公司

Dates

Publication Date
20260508
Application Date
20260409

Claims (10)

  1. 1. A primer probe composition for detecting the drug resistance of mycobacterium tuberculosis complex rifampicin and isoniazid, which is characterized by comprising an A-tube composition and a B-tube composition; Wherein the A-tube composition comprises a primer pair for amplifying the rpoB gene and a probe set for detecting the mutation of the rpoB gene; The B-tube composition comprises a primer pair for amplifying the ahpC promoter region, a primer pair for amplifying the inhA promoter region, a primer pair for amplifying the katG315 codon, and a probe set for detecting mutations of the ahpC promoter region, the inhA promoter region and the katG315 codon.
  2. 2. The primer probe composition according to claim 1, wherein the nucleotide sequences of the primer pair for amplifying rpoB gene in the a-tube composition are shown in SEQ ID No.1 and SEQ ID No. 2; in the A tube composition, the nucleotide sequence of the probe set for detecting rpoB gene mutation is shown as SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 5.
  3. 3. The primer probe composition according to claim 1, wherein the nucleotide sequences of the primer pair for amplifying the ahpC promoter region in the B-tube composition are shown in SEQ ID No.6 and SEQ ID No. 7; In the B tube composition, the nucleotide sequences of the primer pair for amplifying the inhA promoter region are shown in SEQ ID NO.10 and SEQ ID NO. 11; In the B tube composition, the nucleotide sequences of the primer pair for amplifying katG315 codon are shown in SEQ ID NO.13 and SEQ ID NO. 14.
  4. 4. The primer probe composition according to claim 1, wherein the nucleotide sequence of the probe for detecting the mutation of the ahpC promoter region in the B-tube composition is shown in SEQ ID No.8 and SEQ ID No. 9; in the B tube composition, the nucleotide sequence of a probe for detecting the mutation of the inhA promoter region is shown as SEQ ID NO. 12; In the B-tube composition, the nucleotide sequence of the probe for detecting the katG315 codon mutation is shown in SEQ ID NO. 15.
  5. 5. A kit for the detection of drug resistance of mycobacterium tuberculosis complex rifampicin and isoniazid, comprising the primer probe composition of any one of claims 1-4.
  6. 6. The kit of claim 5, further comprising a PCR amplification buffer, a DNA polymerase, a positive control, and a negative control.
  7. 7. A method for detecting the non-diagnostic purpose of the drug resistance of mycobacterium tuberculosis complex rifampicin and isoniazid, characterized by comprising the following steps: s1, extracting DNA in a sample to be detected as a template; S2, performing fluorescent quantitative PCR amplification and melting curve analysis on the template by using the primer probe composition according to any one of claims 1-4; s3, judging the drug resistance of the sample to be tested according to the Tm value in the melting curve.
  8. 8. The method according to claim 7, wherein the fluorescent quantitative PCR amplification in step S2 is performed in 95℃for 3 min,1 cycle, 95℃for 10min, 62℃for 20S, 72℃for 40S, 50 cycles, 95℃for 1 min,1 cycle, 45℃for 5 min,1 cycle; The melting curve program is that the temperature is raised to 95 ℃ at 45 ℃ and fluorescence signals are collected at 0.2 ℃ per second, and the temperature is 45 ℃ and 15 s and 1 cycle.
  9. 9. The method of claim 7, wherein the final concentration of the a-tube composition in the PCR amplification system is: The primer with the nucleotide sequence shown as SEQ ID NO.1 is 0.1 mu M; the primer with the nucleotide sequence shown as SEQ ID NO.2 is 1.6 mu M; the probes with nucleotide sequences shown as SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5 are all 0.4 mu M.
  10. 10. The method of claim 7, wherein the final concentration of the B-tube composition in the PCR amplification system is: The primers shown in the nucleotide sequences SEQ ID NO.6, SEQ ID NO.10 and SEQ ID NO.13 are all 0.08 mu M; The primer with the nucleotide sequence shown as SEQ ID NO.7 is 0.8 mu M; The primer with the nucleotide sequence shown as SEQ ID NO.11 is 1.6 mu M; The primer with the nucleotide sequence shown as SEQ ID NO.14 is 1.6 mu M; The probes with the nucleotide sequences shown as SEQ ID NO.8 and SEQ ID NO.9 are all 0.4 mu M; the probe with the nucleotide sequence shown as SEQ ID NO.12 is 0.8 mu M; the probe with the nucleotide sequence shown in SEQ ID NO.15 was 0.8. Mu.M.

Description

Primer probe composition for detecting drug resistance of mycobacterium tuberculosis complex rifampicin and isoniazid and application of primer probe composition Technical Field The invention belongs to the technical field of immunological detection, and particularly relates to a primer probe composition for detecting rifampicin and isoniazid resistance of a mycobacterium tuberculosis complex and application thereof. Background Tuberculosis is a serious public health problem worldwide, and multi-drug-resistant tuberculosis (MDR-RESISTANT TUBERCULOSIS, MDR-TB) and widely-resistant tuberculosis (extensively drug-RESISTANT TUBERCULOSIS, XDR-TB) are one of the problems to be solved in clinical urgent need at present, so that rapid and accurate detection of Mycobacterium Tuberculosis (MTB) in specimens and rapid diagnosis of drug-resistant tuberculosis are of great importance for effective epidemic control. Along with the elucidation of the drug-resistant molecular mechanism of the mycobacterium tuberculosis, a rapid molecular drug sensitivity test (drug susceptibility test, DST) and a novel phenotype method are established and are widely applied clinically, and a more perfect and standard drug resistance detection method and flow of the mycobacterium tuberculosis are required to be established, so that tuberculosis clinicians, inspectors and prevention and control personnel can properly apply the detection method to correctly interpret the detection result, further the diagnosis and treatment level is improved, and the control of drug-resistant tuberculosis is accelerated. Rifampicin is one of the main antitubercular drugs, and most rifampicin resistant strains are resistant to isoniazid at the same time, so that the rapid and convenient detection technology of rifampicin and drug-resistant gene mutation has important significance for controlling multi-drug-resistant tuberculosis. About one-fourth of tuberculosis patients die due to rifampicin resistant mycobacterium tuberculosis, wherein more than 96% of rifampicin resistance caused by rpoB mutation is related to clinically 95% of rifampicin resistant mycobacterium tuberculosis in the resistance determining region (RIFAMPIN RESISTANCE DETERMINING region, RRDR) in mycobacterium tuberculosis rpoB gene, which is located at 507-533, and rifampicin after mutation cannot be effectively bound to the β subunit of DNA-dependent RNA polymerase, thereby resulting in the generation of rifampicin resistance. Isoniazid (isoniazid.1nh), an important first-line antitubercular drug, is a WHO recommended DOTs therapeutic drug, and can also be used for the prophylactic treatment of latent tuberculosis infection. Isoniazid resistance involves multiple genes of Mtb, and currently there are 20 known INH resistance-related genes, including metabolic enzyme activity, cell wall synthesis and efflux pump-related coding and regulatory genes. Since the amount of information on the gene obtained by conventional PCR amplification is limited, the previous studies have reported that the gene is mainly concentrated on part of the gene and on high frequency mutation sites such as katG 315 and inhA promoter region-15 mutation. Meanwhile, the number of samples and the representativeness are limited, and the mutation information of other INH drug resistance related genes with low occurrence frequency is less, so that no systematic and complete research on all reported known INH drug resistance related gene mutation conditions is performed on clinical strains at present. Along with the development of molecular biology theory and technology, the drug-resistant molecular mechanism of the mycobacterium tuberculosis is gradually elucidated, and a molecular drug sensitivity detection kit based on the mutation detection of the drug-resistant related target genes of the mycobacterium tuberculosis is also gradually developed. Currently, there are 16 major DST agents approved by the national drug administration. The kit comprises 5 imported reagents, 3 BACTEC MGIT 960 systems for phenotype DST, bact/ALERT 3D and Mycobacterium drug sensitivity detection plates sendititre@MYCOTB, 2 GeneXpert MTB/RIF and Geno Type MTBDRplus for molecular DST, 11 domestic reagents, 2 Mycobacterium drug sensitivity detection kits (culture method) and MTB drug sensitivity kits for phenotype drug sensitivity detection, 9 for molecular DST, including DNA microarray chip method, PCR-linear probe hybridization method, fluorescent PCR melting curve method, PCR-gene sequencing method and the like, for example, xiamen is good, a fusion curve mutant gene detection product is mainly, a PCR-reverse point hybridization method is adopted for a sub-biological product, hangzhou you Dai, suzhou Kong and Yipeng (Xuzhou) are combined by fluorescent PCR and melting curve technology, so that a kit product integrating identification and drug sensitivity detection is formed and output in the near two years. The multicolor probe melting curve method