摘要
为用于非洲猪瘟病毒(African swine fever virus, ASFV)CD2v基因功能研究及区分ASFV野毒株和CD2v基因缺失毒株,本研究根据ASFV中国流行株(MK333180.1)的CD2v基因序列设计了1对特异性引物和探针,经过条件优化建立了一种以ASFV CD2v基因为靶标的能够鉴别ASFV野毒株和CD2v基因缺失毒株的TaqMan荧光定量PCR方法。结果显示,本研究建立的实时荧光定量PCR检测方法在质粒标准品为1.6×101~1.6×10^(8)拷贝/μL时,线性关系良好,线性相关系数R^(2)为0.998;该方法可以特异性检测ASFV,不与猪瘟病毒、伪狂犬病病毒、猪繁殖与呼吸综合征病毒、猪传染性胃肠炎病毒、猪圆环病毒1型和猪圆环病毒2型发生交叉反应;最低检测限为16拷贝/μL,比常规PCR的敏感性高出3个数量级;组内和组间重复性试验的变异系数均小于2%;能够有效地区分ASFV野毒株和CD2v基因缺失毒株;该方法与本实验室建立的针对ASFV p72基因的荧光定量PCR方法在检测临床样品时无统计学差异且符合率高。本研究所建立的TaqMan荧光定量PCR方法为ASFV的快速检测提供了新靶标,同时也为ASFV CD2v基因功能的研究、野毒株与CD2v基因缺失毒株的鉴别提供了有力的技术支持。
In order to study the function of CD2v gene of African swine fever virus(ASFV) and distinguish ASFV wild-type strains from CD2v gene-deletion strains, a pair of specific primers and probes were designed based on the CD2v gene sequence of ASFV Chinese epidemic strain(MK333180.1).After optimization of conditions, a TaqMan real-time quantitative PCR assay that can identify ASFV wild-type strains from CD2v gene-deletion strains was established.The results showed that the detection method established in this study has a good linear relationship when the plasmid standard was from 1.6×101 to 1.6×10^(8) copies/μL and the linear correlation coefficient R^(2) was 0.998.Moreover, it could specifically detect ASFV without cross-reaction with classical swine fever virus, pseudorabies virus, porcine reproductive and respiratory syndrome virus, porcine infectious gastroenteritis virus, porcine circovirus type 1 and porcine circovirus type 2.The minimum detection amount was 16 copies/μL,which was 3 orders of magnitude higher than the sensitivity of conventional PCR,and the coefficient of variation was less than 2% in both intra-group and intergroup repeatability tests.Furthermore,it could effectively distinguish ASFV wild strains from CD2v gene-deletion strains.Compared with the real-time PCR assay targeting the ASFVp72 gene established by our laboratory,there was no statistical difference between the two detection methods in the detection of clinical samples,and the coincidence rate was good.This study provides a new target for the rapid detection of ASFV,it also provides strong technical support for the study of the CD2v gene function,and identification of wild viruses and CD2v gene-deletion strains.
作者
韩玉
王涛
潘力
孙茂文
周萍萍
王冰
王翌
罗玉子
仇华吉
孙元
HAN Yu;WANG Tao;PAN Li;SUN Maowen;ZHOU Pingping;WANG Bing;WANG Yi;LUO Yuzi;QIU Huaji;SUN Yuan(State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China)
出处
《中国兽医学报》
CAS
CSCD
北大核心
2021年第5期847-852,共6页
Chinese Journal of Veterinary Science
基金
国家重点研发计划基金资助项目(2017YFD0500601)。
