聚合酶链反应-反向斑点杂交鉴定分枝杆菌菌种

目的 建立一种简便、快速、敏感和特异的分枝杆菌菌种鉴定方法。方法以１６ＳｒＤＮＡ为靶序列，采用聚合酶链反应（ＰＣＲ）－反向斑点杂交检测２５种分枝杆菌１１种分枝杆菌标准株、１２０株分枝杆菌临床分离株和２６份痰标本。结果 分枝杆菌、非分枝杆菌标准株经ＤＮＡ扩增，分枝杆菌均出现５７８ｂｐＤＮＡ片段，非分枝杆菌除假白喉棒状杆菌可见同样片段外，其余菌种均未见扩增。敏感性试验可检测出１００ｆｇ结核分枝杆菌ＤＮ

PCR-reverse dot blot hybridization assay for rapid identification of mycobacterium species

Objective To establish a simple, rapid, sensitive, and specific method for identification of mycobacterium species. Method Based on the gene sequence of 16S rDNA, the standard strains of 25 mycobacteria and 11 nonmycobateria, 10 clinical isolates of mycobacteria, and 26 sputum specimens were detected by PCR reverse dot blot hybridization assay. Results 578 bp DNA fragment was amplified from all mycobacteria tested and was not found in all nonmycobacteria besides corynebacterium pseudodiphtheriticum. PCR assay detected 100 fg of M. tuberculosis DNA. Among 17 oligonucleotide probes, pMyc, pTub2, pAvi, pInt, pKan, pScr, pMar, pXen, pSzu, pGor, pFor2, pChe, pTer, pNon were specific respectively for mycobacterium spp, M. tuberculosis complex, M. avium, M. intracellulare, M. Kansasii complex, M. scrofulaceum, M. marinum, M. xenopi, M. Szulgai, M. gordonae, M. fortuitum, M. chelonae, M. terrae, M. nonchromogenicum, with pTub1. pFor1. pSme, cross hybridization occurred. The results of 10 clinical isolates tested by this assay were in agreement with those obtained by conventional identification methods. Twenty six smear positive sputum specimens were determined to be M. tuberculosis complex by this procedure. Conclusion A 16s rDNA PCR reverse dot blot hybridization is of value in rapid, effective identification of mycobacterium species.