Application Of Multiplex PCR In Rapid Identification Of M.tuberculosis And Detection Of Drug Resistance

ObjectivesTo establish and evaluate a multiplex PCR technology for rapid identification of M.tuberculosis and its resistance to provide a scientific basis for clinical application.MethodsTake 119 suspected Mtb isolates from the Dali Prefecture Center for Disease Control and prevention from June 2015 to June 2017.Firstly,the strains were identified using traditional culture in nitrobenzoic acid(PNB)/Thiophene-2-carboxylic acid hydrazide(TCH)culture medium.Then primers were designed for the984 bp fragment of the Mycobacterium Tuberculosis Complex-specific insertion sequence IS6110(Insertion sequence 6110,IS6110)and the 396 bp fragment of the M.tuberculosis protein 40(MTP40)for multiplex PCR which identified rapidly in vitro amplification.The coincidence rates of the two methods were compared and the sensitivity and specificity of the multiplex PCR method were calculated using the traditional culture method as a standard.The 107 strains identified as M.tuberculosis by both methods were tested for susceptibility to Rifampicin(RFP)and Isoniazid(INH)using the traditional antimicrobial susceptibility test firstly.The primers were designed for the rpoB gene in the rifampin resistance-determining region and the katG gene and inhA gene in the isoniazid resistance-determining region,then these three pairs of primers were simultaneously amplified in vitro using a multiplex PCR method.The multiplex PCR amplification products were sent to the gene company for sequencing.The sequencing results were compared with the standard gene sequences of the NCBI website Genbank database to obtain the gene mutations of the isolates.Taking the traditional proportional susceptibility test as the reference standard,the multiplex PCR method was evaluated by SPSS 11.0 statistical software.ResultsThe 107 strains were identified as M.tuberculosis strains and 4 strains were M.bovis by traditional culture method among 119 suspected Mtb.Multiplex PCR also identified 107 strains as M.tuberculosis,and 4 strains were other species Non-M.tuberculosis in the Mycobacterium tuberculosis complex.Compared with the traditional culture method,the sensitivity and the specificity are 100%,respectively of the multiplex PCR method.Among the 107 M.tuberculosis strains,25 strains were drug-resistant strains,and 82 strains were drug-susceptible strains that the drug resistance rate was 23.4%(25/107).While the results of multiplex PCR showed that 26 strains were resistant strains and 81 strains were drug-susceptible strains.The drug resistance rate was24.3%(26/107).Of the 26 resistant strains detected by multiplex PCR,24 strains were identified as resistant by the traditional proportional susceptibility test and 2 strains were sensitive.One strain was identified as drug resistant by drug susceptibility,and multiplex PCR was identified as sensitive.The sensitivity and specificity of multiplex PCR for the detection of M.tuberculosis drug resistance were 96%and 97.6%,respectively.The recombination rate of the two methods was 97.2%.The two methods were paired?~2 test,P>0.05,the difference was not statistically significant.ConclusionMultiplex PCR can be used for identification of M.tuberculosis and detection of drug resistance.The results are consistent with traditional methods.However,the multiplex PCR method is simpler and faster than the traditional method,and can provide a scientific basis for clinical identification of M.tuberculosis and detection of drug resistance rapidly.