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The widespread occurrence of drug-resistant Mycobacterium tuberculosis places great importance on the detection of TB drug susceptibility.Conventional drug susceptibility testing (DST) is a lengthy process, thus rapid drug resistance testing methods are needed.We developed a novel enzymatic color-reaction based biochip detection assay for the identification of M.tuberculosis (TB) and non-tuberculosis mycobacteria (NTM) and the drug resistance of TB towards isoniazid (INH) and rifampicin (RIF).The process includes asymmetric multiplex PCR /templex PCR, biochip hybridization and an enzymatic color reaction, with specific software used for data acquisition and automated interpretation.The assay contained probes specific for most of the frequently observed mutations.A sensitivity of 50 copies per PCR reaction was achieved, and allele sequences could be detected in mixed samples at a proportion as low as 10%.The templex PCR technique was applied in sample preparation to avoid interference between the different primers that is observed when using conventional multiplex-PCR.The whole testing process takes four and a half hours.We applied this assay to 294 clinical specimens, including 254 M.tuberculosis and 40 non-tuberculosis mycobacteria specimens.The results were compared with those obtained using DNA sequencing and the DST method.The results showed a concordance rate of 91.34% (232/254) for RIF resistance detection (sensitivity 89.15%, specificity 93.6%) and 78.74% (200/254) for INH resistance detection (sensitivity 78.57%, specificity 78.85%).The assay is rapid, sensitive and specific, and can be used for testing of mixed samples.It can be a reliable tool of TB clinical diagnosis and treatment.