Kim Hong, Kim Sun-Hyun, Shim Tae-Sun, Kim Mi-na, Bai Gill-Han, Park Young-Gil, Lee Sueng-Hyun, Cha Chang-Yong, Kook Yoon-Hoh, Kim Bum-Joon
Department of Microbiology and Liver Research Institute, College of Medicine, Seoul National University, 28 Yongon-dong, Chongno-gu, Seoul 110-799, South Korea.
J Microbiol Methods. 2005 Aug;62(2):199-209. doi: 10.1016/j.mimet.2005.02.010.
A method based on PCR-restriction fragment length polymorphism analysis (PRA) using a novel region of the hsp65 gene was developed for the rapid and exact identification of mycobacteria to the species level. A 644 bp region of hsp65 in 62 mycobacteria reference strains, and 4 related bacterial strains were amplified, and the amplified DNAs were subsequently digested with restriction enzymes, namely, AvaII, HphI, and HpaII. Most of the mycobacteria species were easily differentiated at the species level by the developed method. In particular, the method enabled the separation of M. avium, M. intracellulare and M. tuberculosis to the species level by AvaII digestion alone. An algorithm was constructed based on the results and a blind test was successfully performed on 251 clinical isolates, which had been characterized by conventional biochemical testing. Our results suggest that this novel PRA offers a simple, rapid, and accurate method for the identification of mycobacteria culture isolates at the species level.
开发了一种基于PCR-限制性片段长度多态性分析（PRA）的方法，该方法使用hsp65基因的一个新区域，用于将分枝杆菌快速准确地鉴定到种水平。对62株分枝杆菌参考菌株和4株相关细菌菌株中的hsp65基因的一个644bp区域进行扩增，随后用限制性内切酶AvaII、HphI和HpaII对扩增的DNA进行酶切。通过所开发的方法，大多数分枝杆菌菌种在种水平上易于区分。特别是，该方法仅通过AvaII酶切就能将鸟分枝杆菌、胞内分枝杆菌和结核分枝杆菌区分到种水平。基于这些结果构建了一种算法，并对251株临床分离株成功进行了盲测，这些临床分离株已通过传统生化检测进行了鉴定。我们的结果表明，这种新型PRA为在种水平上鉴定分枝杆菌培养分离株提供了一种简单、快速且准确的方法。
