Dual channel real-time PCR melting curve analysis-based assay for detecting of anti-tuberculosis drugresistant mutations in Mycobacterium tuberculosis

Objective Based on dual channel melting curve analysis-based assay,we developed a method to rapidly detect the drug-resistant mutations in Mycobacterium tuberculosis through real-time PCR. Methods According to the common first-line drug-resistant mutations of Mycobacterium tuberculosis,we designed six dual-labeled fluorescence probes to rapidly detect the drug-resistant mutations through real-time PCR melting curve after amplifications of drug-resistant related gene region of DNA.The targets include rpoB 81 bp core region,katG315,inh A promoter,ahpC promoter and embB306.To validate the sensitivity and specificity of our method,we performed real-time PCR assays to detect drug-resistant mutations in 76 clinical MDR-TB samples,which were collected by Shanghai CDC in 2008. Results In the validation,this method successfully detected drug-resistant mutations in all 76 clinical MDR-TB samples.The ΔTmof mutations were from 1.8 to 14.4℃.Comparing with the sequencing data,all mutations covered by the six probes were detected with 100% sensitivity and 100% specificity (rpoB,80/80;inh A,7/7; katG315,59/59; ahpC,8/8; embB306,27/27).This method can successfully detect drug-resistant mutations from 100 copies/μl DNA samples. Conclusions A widely applicable real-time PCR assay to detect first line drug-resistant mutations of Mycobacterium tuberculosis has benn developed.This method has proven to have the advantages of high sensitivity,specificity and low risk of contamination.It can be used in rapid diagnosis of clinical drug-resistant tuberculosis and the evaluation of laboratory drug sensitivity test.(Chin J Lab Med,2013,36:63-67) Key words: Real-time polymerase chain reaction; Mycobacterium tuberculosis; Mutation