猪流行性腹泻病毒SYBR Green I实时荧光定量PCR检测方法的建立  

Development of SYBR Green I Real-Time PCR for Detection of Porcine Epidemic Diarrhea Virus

作　　者：董苏洁 孔宁[2] 秦文珍 翟焕杰 翟雪滢 杨心雨 童武[2] 郑浩[2] 于海[2] 童光志[2] 李有文[1] 单同领[2] DONG Sujie;KONG Ning;QIN Wenzhen;ZHAI Huanjie;ZHAI Xueying;YANG Xinyu;TONG Wu;ZHENG Hao;YU Hai;TONG Guangzhi;LI Youwen;SHAN Tongling(College of Animal Science and Technology,Tarim University,Alar 843300,China;Shanghai Veterinary Research Institute,CAAS,Shanghai 200241,China)

机构地区：[1]塔里木大学动物科学与技术学院,阿拉尔843300 [2]中国农业科学院上海兽医研究所,上海200241

出　　处：《中国动物传染病学报》2024年第2期174-180,共7页Chinese Journal of Animal Infectious Diseases

基　　金：国家自然科学基金(31872478,32102665,31760741);上海自然科学基金(19ZR1469100)。

摘　　要：为了建立高效、灵敏的猪流行性腹泻病毒(PEDV)检测方法,本研究从GenBank数据库中获取PEDV N基因序列,扩增出PEDV N基因标准质粒,并在N基因的保守区域内设计了一对特异性荧光定量引物,成功建立了SYBR Green I实时荧光定量PCR检测方法。经过一系列试验表明,该检测方法线性关系良好,R^(2)值为0.99;特异性强,敏感性高,最低可检测至2.23 copies/μL,比普通PCR灵敏约100倍;重复性好,组内变异系数为0.25%~0.43%,组间变异系数为0.67%~0.97%;对于各地区96份临床样品检测出PEDV阳性率为25%。本研究建立的实时荧光定量PCR检测方法为PEDV的临床诊断、流行病学调查以及定量研究提供了有效的检测工具。In order to develop an efficient and sensitive detection method for Porcine epidemic diarrhea virus(PEDV),the PEDV N gene sequence was obtained from GenBank database and a pair of specific primers were designed from its conservative region for development of a SYBR Green I real-time PCR method.A series of experiments were performed for optimization and the optimal factors included the linear correlation coefficient at 0.99 and detection limit at 2.23 copies/μL,which was about 100 times more sensitive than conventional PCR.The coefficient of variation within groups was between 0.25%-0.43%and between groups was between 0.67%-0.97%,indicating its good repeatability.Total 96 clinical samples from various regions were tested using this method and the PEDV positive rate was 25%(24/96).The real-time PCR method developed in this study provided an effective tool for clinical diagnosis,epidemiological investigation and quantitative research of PEDV.

关 键 词：猪流行性腹泻病毒 N基因 SYBR Green I 荧光定量PCR 

分 类 号：S852.65[农业科学—基础兽医学]