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.
作者
董苏洁
孔宁
秦文珍
翟焕杰
翟雪滢
杨心雨
童武
郑浩
于海
童光志
李有文
单同领
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)
出处
《中国动物传染病学报》
CAS
北大核心
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)。
