摘要
为了建立特异、灵敏及快速的猪星状病毒3型(PAstV3)检测方法并应用于临床检测,根据GenBank登录的PAstV 3序列,在ORF1b保守区设计特异性引物和TaqMan探针;经过反应体系和反应条件优化,对建立的实时荧光定量RT-PCR方法进行敏感性、特异性测定,并初步应用于临床粪便样品的检测。结果显示,该方法标准曲线线性关系良好,R2值达到0.997 1;与多种常见的猪病病毒均无交叉反应,特异性良好。与常规PCR相比,该检测方法敏感性是常规PCR方法的100倍,敏感性较高,批内、批间重复性试验的变异系数均小于2.0%,重复性良好。使用该方法对临床收集的30份疑似PAstV3粪便样本进行检测,阳性率为40%,检出率高于常规PCR方法。上述结果表明,该方法的建立为PAstV3的诊断及流行病学调查提供了快速、准确的检测手段。
In order to build a specific,sensitive and rapid detection method for PAstV3 detection,the PAstV3 gene sequences in Genbank were used and the conserved region in ORF1b was selected to design specific primers and TaqMan probe.Clinical stool samples were collected and preliminary detected by this newly established real-time RT-PCR method after reaction systems and conditions optimization.This detection method established in this study has a good linear relationship with the standard curve,with R2 value up to 0.9971.The sensitivity is 100 times higher than conventional PCR method,The variation co-efficient of in-batch and inter-batch repeatability test is less than 2.0%,indicating good repeatability.The detection results of clinical sam ples showed that the positive rate of this method is higher than conventional PCR method.The establishment of this method provides a rapid detection means for PAstV3 laboratory diagnosis and epidemiological investigation.
作者
刘心
张文超
董覃婷
刘燚
陈樱
韦祖樟
欧阳康
刘欢
黄伟坚
LIU Xin;ZHANG Wen-chao;DONG Qin-ting;LIU Yi;CHEN Ying;WEI Zu-zhang;OUYANG Kang;LIU Huan;HUANG Wei-jian(College of Animal Science and Technology,Guangxi University,Nanning 530005,China;The First Affiliated Hospital of Guangxi University of Chinese Medicine,Nanning 530024,China)
出处
《中国兽医科学》
CAS
CSCD
北大核心
2020年第11期1373-1378,共6页
Chinese Veterinary Science
基金
国家自然科学基金项目(31760735)
广西研究生教育创新计划项目(YCBZ2019004)。
