摘要
本研究旨在建立一种灵敏、快速检测猪δ冠状病毒(porcine deltacoronavirus,PDCoV)的TaqMan荧光定量RT-PCR方法。参照GenBank中PDCoV有关基因序列,设计一对特异性引物用于扩增PDCoV M 基因。将测序正确的基因片段克隆入pMD18-T载体,构建重组质粒作为建立标准曲线的病毒模板。设计合成一对特异性引物与TaqMan探针,进行反应条件和反应体系的优化,建立快速检测PDCoV的TaqMan实时荧光定量RT-PCR方法,并进行该方法的灵敏性、特异性与重复性验证。结果表明,该方法能有效扩增1.0×10^1 ~1.0×10^9 拷贝·μL^-1 的PDCoV标准质粒,建立的标准曲线呈现良好的线性关系。该方法的检测灵敏度为1.0×10^1 拷贝·μL^-1;对猪流行性腹泻病毒、猪传染性胃肠炎病毒、猪伪狂犬病病毒等病原不发生交叉反应,具有很好的特异性;重复性试验结果显示变异系数(CV)小于1%,重复性良好。对2017—2018年期间收集的河南省不同猪场的100份腹泻病料进行检测,PDCoV的阳性检出率为23%(23/100),与PDCoV SYBR Green Ⅰ荧光定量RT-PCR检测方法的符合率为100%。人工感染PDCoV的仔猪,取攻毒后不同时间的粪便样品,应用本研究建立方法与常规RT-PCR方法对样品进行检测,TaqMan荧光定量RT-PCR检测方法的灵敏度远远优于常规RT-PCR。上述结果表明,本研究所建立的TaqMan荧光定量RT-PCR检测方法能够灵敏、特异地检测PDCoV,可用于临床PDCoV的检测。
This experiment was conducted to develop a TaqMan based real-time fluorescent RT-PCR for the detection of PDCoV. Based on the M gene sequence of PDCoV in GenBank,a pair of primers was designed,and M gene of PDCoV was amplified and cloned into pMD18-T vector to get the recombinant plasmid. Then another pair of primers and a TaqMan probe was designed,and a TaqMan based real-time fluorescent RT-PCR assay was developed by optimizing the conditions and systems of reaction. Sensitivity,specificity and reproducibility assay were determined. The results showed that this assay could detect the PDCoV between 1.0×10^1-1.0×10^9 copies·μL^-1 with a good linear relation,and the sensitivity limit was 1.0×10^1 copies·μL^-1 . There were no cross-reaction with porcine epidemic diarrhea virus (PEDV),transmissible gastroenteritis virus (TGEV),pseudorabies virus (PRV) and other porcine viruses. The CV (Coefficient of Variation) of intra-assay and inter-assay were no more than 1%. The TaqMan based real-time RT-PCR assay was then used to detect 100 diarrhea clinical samples of porcine that collected from 2017 to 2018,and the PDCoV positive ratio was 23%,and the coincidence rate with SYBR Green Ⅰ real-time PCR assay was 100%. Piglets were inoculated with PDCoV,and fecal samples were collected and detected by TaqMan real-time RT-PCR and common RT-PCR assay,the results showed that the sensitivity of the former was much better than the latter. This assay provides a high sensitivity,specificity and reproducibility method for detection of PDCoV.
作者
郑兰兰
朱静静
王盼
舒燕
梁青青
李炳晓
王超群
魏战勇
ZHENG Lanlan;ZHU Jingjing;WANG Pan;SHU Yan;LIANG Qingqing;LI Bingxiao;WANG Chaoqun;WEI Zhanyong(College of Animal Husbandry and Veterinary,Henan Agricultural University,Zhengzhou 450002,China;Henan Animal Food Safety Key Laboratory,Zhengzhou 450002,China;Xuchang Customs,Xuchang 461000,China)
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2019年第6期1261-1267,共7页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
国家重点研发计划(2018YFD0501205)
许昌市基础与前沿项目(JC2018005)
