A novel enzymatic color reaction based biochip detection assay for RIF and INH drug resistance of clinical mycobacterial specimens

摘要：

　　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.

关键词： Mycobacterium tuberculosis drug resistance biochip enzymatic color-reaction mixed sample templex PCR

作者: Wenfei Xue Xiaoli Yu Shulin Zhang Jinfu Peng Danqing Jiang Bingbing Ding Bin Zhu Yao Li

作者单位: State Key Laboratory of Genetic Engineering,Institute of Genetics,School of Life Science,Fudan Unive School of Biology and Pharmaceutical Engineering,Wuhan Polytechnic University,68 Xuefu south Road,Ch Department of Medical Microbiology and Parasitology,Shanghai Jiao Tong University School of Medicine Baio Technology Limited Company,Shanghai 200233,PR China

会议类型: 国内会议

会议名称: 中华医学会结核病分会2013年学术大会

会议地点: 重庆

会议语种:英文

页码: 244-256

在线出版日期: 2013-07-18（万方平台首次上网日期，不代表论文的发表时间）