猪Hokovirus TaqMan荧光定量PCR方法的建立与应用  

Development and application of TaqMan-based real-time PCR assay for the detection of porcine Hokovirus

作　　者：李彬[1] 杜露平[1] 何孔旺[1] 刘浩飞[1] 温立斌[1] 张雪寒[1] 郭容利[1] 茅爱华[1] 倪艳秀[1] 周俊明[1] 吕立新[1] 俞正玉[1] 王小敏[1] 胡屹屹[1] 祝昊丹[1] 于洋[1] 

机构地区：[1]江苏省农业科学院兽医研究所农业部兽用生物制品工程技术重点实验室,江苏南京210014

出　　处：《中国预防兽医学报》2013年第6期460-463,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：江苏省农业科技自主创新资金(CX(11)2060)

摘　　要：为建立检测PHoV的TaqMan荧光定量PCR方法,本研究根据PHoV的V P2基因序列设计引物和探针,以梯度稀释的含有V P2基因的重组质粒作为标准品,进行定量PCR反应。结果显示,该方法的检测灵敏度为10拷贝;而且该检测方法特异性较好,与猪的其他病毒核酸均无交叉反应;批内和批间的变异系数低于3.29%,表明该方法的重复性较好。对华东地区采集的225份临床样品进行检测,结果显示,PHoV的阳性率为14.2%。本研究建立的荧光定量PCR方法灵敏度高、特异性好,可以为PH oV的流行病学调查和发病机制等研究提供可靠的工具。Porcine Hokovirus （PHoV） was a recently discovered parvovirus from pigs. To establish a rapid and sensitive method for detection of PHoV, the TaqMan-based real-time PCR assay was developed with a pair of primers and a probe targeted the PHoV VP2 gene. The recombinant plasmid containing the VP2 gene was constructed to establish the standard curve. The results shown that the real-time PCR was specific for PHoV with a detection limit of 10 copies, but no amplifications from other related porcine virus, and the inter-and intro-variation was less than 3.29%. Furthermorre, a total of 225 clinical samples collected from Eastern of China were tested by the real-time PCR, and the positive rate of PHoV was 14.2%. The assay for the detection and quantification of PHoV was highly sensitive and specific which provide a reliable protocol for the epidemiological survey and pathogenetic study of PHoV infection.

关 键 词：猪Hokovirus TaqMan荧光定量PCR 检测 

分 类 号：S852.65[农业科学—基础兽医学]