虎源传染性鼻气管炎病毒巢式PCR检测方法的建立  

Establishment of Nested PCR for Detecting Feline Herpesvirus Type 1 in Tiger(Panthera tigris)

作　　者：李林翔 韦增晖[2] 张晓明[3,4] 彭仕明[3] 陈武[3] 

机构地区：[1]苏州市动物园,苏州215001 [2]广东工业大学,广州510006 [3]广州动物园,广州510070 [4]广州海洋馆,广州510070

出　　处：《野生动物学报》2015年第4期369-372,共4页CHINESE JOURNAL OF WILDLIFE

基　　金：中国动物园协会2013年科研项目"虎源传染性鼻气管炎的快速诊断与新型疫苗的研究"

摘　　要：为了建立虎源传染性鼻气管炎病毒巢式PCR(nested PCR)的快速鉴定方法,根据虎源传染性鼻气管炎病毒(FHV-1)的gD蛋白基因序列设计套式引物,进行特异性、敏感性和重复性实验,特异性试验表明,该方法可以特异扩增出FHV-1的gD基因片断,从猫泛白细胞减少症病毒、猫支原体和阴性对照的VERO细胞均未扩增出该条带,敏感性试验表明,nested PCR检测极限是1×10~2 copies/μl,而一步法PCR的有效扩增最低极限是1×10~5 copies/μl,前者的敏感性明显高于后者,重复性实验表明,不同情况下3次重复性试验,结果相同。用nested PCR对疑似感染FHV-1的样品进行检测,实验结果与病毒分离鉴定结果一致。本研究建立的nested PCR适用于野生猫科动物FHV-1感染的快速诊断与流行病学调查。The objective of this study was to establish a nested PCR assay for detecting Feline herpesvirus type 1（FHV-1） in tiger.The primers were designed by using the gD gene of FHV- 1strain from tiger in Genbank.The repeatability,specificity and sensitivity of assay were evaluated.The specificity test showed that only the FHV-1 gD gene was amplified,but no band was obtained from other virus such as feline parvovirus,feline mycoplasma,or Vera cell for negative control.The sensitivity test showed that the detection limits were 1×10~2 copies/μl by nested PCR assay and 1x10^5 copies/μl by one step PCR assay.The sensitivity of nested PCR assay was markedly higher than that of one step PCR.The repeatability test showed that the results of three repetitive tests under different conditions were consistent.The nested PCR method was successfully used to detect suspected FHV-1 infections and results were consistent with those yielded by viral isolation and identification.The established nested PCR method can be used for a quick diagnosis and epidemiological investigation of FHV-1 infection in wild felines.

关 键 词：虎源传染性鼻气管炎病毒 巢式PCR 一步法PCR GD基因 

分 类 号：S862.65[农业科学—野生动物驯养]