Yang Young Geun, Song Man Ki, Park Su Jeong, Kim Suhng Wook
Department of Clinical Laboratory Science, College of Health Sciences, Korea University, Seoul, Korea.
J Microbiol Biotechnol. 2007 Oct;17(10):1616-21.
We have established a SYBR Green-based realtime PCR method using AnyDirect solution, which enhances PCR from whole blood, for direct amplification of the virA gene of Shigella flexneri and the invA gene of Salmonella typhimurium from human feces without prior DNA purification. When we compared the efficiency of conventional or realtime PCR amplification of the virA and invA genes from the supernatant of boiled feces supplemented with S. flexneri and S. typhimurium in the presence or absence of AnyDirect solution, amplification products were detected only in reactions to which AnyDirect solution had been added. The detection limit of real-time PCR was 1 x 10(4) CFU/g feces for S. flexneri and 2 x 10(4) CFU/g feces for S. typhimurium this sensitivity level was comparable to other studies. Our real-time PCR assay with AnyDirect solution is simple, rapid, sensitive, and specific, and allows simultaneous detection of S. flexneri and S. typhimurium directly from fecal samples without prior DNA purification.
我们建立了一种基于SYBR Green的实时PCR方法，该方法使用AnyDirect溶液，可增强全血的PCR反应，用于直接从人粪便中扩增弗氏志贺菌的virA基因和鼠伤寒沙门氏菌的invA基因，无需事先进行DNA纯化。当我们比较在有或没有AnyDirect溶液的情况下，从添加了弗氏志贺菌和鼠伤寒沙门氏菌的煮沸粪便上清液中对virA和invA基因进行常规PCR或实时PCR扩增的效率时，仅在添加了AnyDirect溶液的反应中检测到扩增产物。实时PCR对弗氏志贺菌的检测限为1×10⁴CFU/g粪便，对鼠伤寒沙门氏菌的检测限为2×10⁴CFU/g粪便，该灵敏度水平与其他研究相当。我们使用AnyDirect溶液的实时PCR检测方法简单、快速、灵敏且特异，无需事先进行DNA纯化即可直接从粪便样本中同时检测弗氏志贺菌和鼠伤寒沙门氏菌。
