中国人兽共患病学报2011，Vol.27Issue(4)：297-302,310,7.
基因1型狂犬病病毒一步法荧光定量RT-PCR检测方法的建立
Establishment of the one-step real-time RT-PCR assay for detection of genotype 1 lyssavirus
摘要
Abstract
In present study, the one-step real-time RT-PCR(qRT-PCR) was developed to detect the rabies virus using a set of degenerate primers and TaqMan probe directed to the leader sequences and 3’ ends of N gene.The specificity assay showed that the method could only detect the classical rabies virus, not the other six lyssaviruses from genotype 2 to 7, and other viruses such as CDV, CPV, CAV, VSV and JEV.The detection limit of the established method was 4.68 TCID50.To validate the method, 29 fresh and 5 decomposed dog brain specimens submitted to our laboratory for rabies confirmation were detected in comparison with OIE golden diagnostic standard methods FAT and MIT and routinely-used nested RT-PCR.In the result, the real-time RT-PCR and nested RT-PCR yield 12/29 and 5/5 positive detection respectively for fresh and decomposed samples, showing 100％ similarity, while FAT was positive in 12/29 fresh and 1/5 decomposed samples, and MIT positive in 12/29 fresh and 0/5 decomposed samples.All positive samples tested by FAT and MIT were also positive in the real-time RT-PCR, but the later was more sensitive than FAT and MIT and particularly useful for decomposed brain tissue.This study shows that the established real-time RT-PCR has good specificity, sensitivity, low cross-contamination and easier operation,which rendering the method is very applicable in clinical diagnosis of classical rabies.关键词
狂犬病病毒/基因I型/一步法荧光定量RT-PCR分类
农业科技引用本文复制引用
许运斌,邵明富,范金红,孙彦伟,席进,涂长春..基因1型狂犬病病毒一步法荧光定量RT-PCR检测方法的建立[J].中国人兽共患病学报,2011,27(4):297-302,310,7.基金项目
公益性行业(农业)科研专项经费项目(200803014) （农业）
国家科技支撑计划课题(2010BAD04B01) （2010BAD04B01）
广东省动物狂犬病综合防控研究基金 （）
