摘要
针对我国狂犬病临床诊断的现实需求,本研究根据狂犬病病毒基因组核苷酸保守序列,设计巢式引物,对引物工作浓度和退火温度进行了优化,并对RT-PCR敏感性、特异性和重复性进行了检验。结果显示,巢式RT-PCR方法最低能够检测0.01MILD50/30μL的脑组织病毒量,对阳性样品和非狂犬病感染样品的3次重复检测结果均与狂犬病诊断OIE推荐方法(荧光抗体染色法)的确定结果完全一致;在针对203份临床样品的检测中,FAT疑似样品通过MIT和巢式RT-PCR进一步确诊,巢式RT-PCR显示出较高的敏感性和特异性。以上表明,本研究建立的狂犬病病毒核酸巢式RT-PCR方法高度敏感和特异,可以用于FAT和MIT疑似样品的辅助检测,为试剂盒的研发与应用奠定基础。
A set of primers, based on highly conserved regions of the nucleoprotein gene,was de-signed for nested reverse transcription-PCR for the detection of rabies virus from multiple sam-pies. The detective limit was 0.01 MILD50/30μL for brain tissue cultured virus and not reaction to some other dog viruses. The sensitivity, specificity and repeatability of this method were confirmed by FAT. In the application,the nested RT-PCR was proved to be fully reaction to a panel of iso-lates from humans, dogs, sheep, deer and ferret badgers in China and more suitable for detection of decomposed naturally infected brains. The results indicate that the new RT-PCR can be used as a candidate method for detection kit production.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2013年第8期1227-1231,共5页
Chinese Journal of Veterinary Science
基金
国家公益性行业(农业)科研专项经费资助项目(201203056)
