PCR-reverse dot hybridization technique for detection of Mycobacterium tuberculosis drug resistance gene mutation

Objective To discuss the effect of PCR-reverse dot blot hybridization technique for detection of Mycobacterium tuberculosis and drug-resistant gene mutation, and understand Mycobacterium tuberculosis drug resistance gene mutation in Shaoxing area and its relationship with the drug resistance. Methods According to the characteristics of Mycobacterium tuberculosis gene, we used reverse dot blot hybridization technique construct specific PCR reaction system, and 10 strains of non Mycobacterium tuberculosis, 3 strains of other strains (Staphylococcus aureus, Escherichia coli and Acinetobacter. baumannii sp.) and 1 strain of Mycobacterium tuberculosis (H37Rv) to validate the reaction system. The system was also used to detect five kinds of resistance mutation detection of the gene (rpoB, katG, InhA, embB and rpsL) of 125 acid fast bacilli smear positive sputum samples and 150 strains of Mycobacterium tuberculosis sourcing from the Shaoxing Municipal Hospital. Results By construction of PCR reverse dot blot hybridization system, the detection result of 10 strains of non Mycobacterium tuberculosis and 3 strains of other strains were negative. H37Rv standard strains could be detected, but they were not detected in the resistant mutations.Among 275 cases of tuberculosis patients, 201 cases (73.1%) were sensitive to four kinds of anti-TB drugs;20 patients (7.3%) were single drug; 54 cases were multi-drug resistance (19.6%) . 133 resistance mutations loci were detected in 275 samples, of which there were 41, 51, 30 and 11 resistant mutation loci of rifampicin, isoniazid, streptomycin and ethambutol respectively. The top 5 gene mutation detection loci Mycobacterium tuberculosis of were 315M, 43M, S531L, 15M and H526Y. Conclusions The drug resistance of Mycobacterium tuberculosis in Shaoxing area is mainly based on the multi drug resistance. Construction of PCR-reverse dot blot hybridization system for detecting Mycobacterium tuberculosis is of specificity and high sensitivity, which can quickly detect the resistance gene of Mycobacterium tuberculosis. Key words: Mycobacterium tuberculosis; Drug resistance; Gene mutation