同时检测PDCoV、TGEV、PoRV和PEDV的四重实时荧光RT-PCR方法的建立及应用  

Development and application of a quadruple real-time RT-PCR assay for simultaneous detection of PDCoV,TGEV,PoRV and PEDV

作　　者：高艺祥 王金凤 张倩 孙晓霞 刘立兵 顾文源[3] 马宏伟 韩庆安[3] 马增军[2] 王建昌 GAO Yixiang;WANG Jinfeng;ZHANG Qian;SUN Xiaoxia;LIU Libing;GU Wenyuan;MA Hongwei;HAN Qinan;MA Zengjun;WANG Jianchang(Technology Center of Shijiazhuang Customs,Shijiazhuang 050051,China;Hebei Province Key Laboratory of Veterinary Medicine,College of Animal Science and Technology,Hebei Normal University of Science&Technology,Qinhuangdao,Hebei 066004,China;Hebei Provincial Center for Animal Disease Prevention and Control,Shijiazhuang 050035,China)

机构地区：[1]石家庄海关技术中心,河北石家庄050051 [2]河北科技师范学院动物科技学院河北省预防兽医学重点实验室,河北秦皇岛066004 [3]河北省动物疫病预防控制中心,河北石家庄050035

出　　处：《中国兽医学报》2023年第3期448-454,461,共8页Chinese Journal of Veterinary Science

基　　金：河北省省级科技基金资助项目(19226612D)。

摘　　要：为实现对猪德尔塔冠状病毒(porcine deltacoronavirus,PDCoV)、猪传染性胃肠炎(porcine transmissible gastroenteritis virus,TGEV)、猪轮状病毒(porcine rotavirus,PoRV)和猪流性腹泻病毒(porcine epidemic diarrhea virus,PEDV)的同时鉴别检测,根据PDCoV M基因、TGEV S基因、PoRV VP6基因和TGEV N基因保守区域序列合成特异性引物和TaqMan探针,通过优化反应条件,成功建立了一种可同时检测PDCoV、TGEV、PoRV和PEDV的实时荧光RT-PCR方法。结果显示,所建立的四重实时荧光RT-PCR方法仅对PDCoV、TGEV、PoRV和PEDV出现阳性扩增,与PRV、PPV、PRRSV、CSFV、PCV2等猪常见病原无交叉反应;对PDCoV、TGEV、PoRV和PEDV的最低检出限分别为1.17×10^(3),1.13×10^(3),1.31×10^(2)和1.18×10^(2)拷贝;重复性好,组内和组间试验变异系数均小于4.5%。进一步应用所建立的同时检测PDCoV、TGEV、PoRV和PEDV的实时荧光RT-PCR方法对199份采集自河北省不同地区腹泻猪的临床样品进行检测,结果检出23份PoRV阳性(11.56%)、12份PEDV阳性(6.03%)和1份TGEV阳性(0.50%),均为单一感染;未检出PDCoV阳性。本研究所建立的四重实时荧光RT-PCR检测方法可实现对PDCoV、TGEV、PoRV和PEDV的同时鉴别检测,为实验室快速鉴别诊断猪病毒性腹泻提供了有效的技术支持。In order to realize the simultaneous detection and differentiation of porcine delta coronavirus(PDCoV),porcine transmissible gastroenteritis virus(TGEV),porcine rotavirus(PoRV)and porcine epidemic diarrhea virus(PEDV),the specific primers and TaqMan probes were synthesized based on the conserved regions of PDCoV M gene,TGEV S gene,PoRV VP6 gene and TGEV N gene,and a quadruple real-time RT-PCR assay for simultaneous detection of PDCoV,TGEV,PoRV and PEDV was established through optimizing the reaction conditions.The results showed that the developed quadruple real-time RT-PCR assay only showed positive amplifications for PDCoV,TGEV,PoRV and PEDV,and no cross-reactions with other common porcine pathogens used in this study such as PRV,PPV,PRRSV,CSFV and PCV2 were observed.The limit of detection of the quadruple real-time RT-PCR assay was 1.17×10^(3)copies for PDCoV,1.13×10^(3)copies for TGEV,1.31×10^(2)copies for PoRV and 1.18×10^(2)copies for PEDV.The assay had good reproducibility with intra-and inter-assay coefficients of variation less than 4.5%.The developed quadruple real-time RT-PCR assay was further applied to detect the PDCoV,TGEV,PoRV and PEDV in 199swine clinical samples collected from different regions of Hebei province.The detection results showed that 23of the samples were PoRV positive(11.56%),12of the samples were PEDV positive(6.03%)and one of the samples were TGEV positive(0.50%),in which all the samples were single infection and no PDCoV was detected.In conclusion,the quadruple real-time RT-PCR assay developed in this study can realize the simultaneous detection and differentiation of PDCoV,TGEV,PoRV and PEDV,and provide an effective technical support for the rapid detection and differentiation of porcine viral diarrhea in the laboratory.

关 键 词：猪德尔塔冠状病毒 猪传染性胃肠炎 猪轮状病毒 猪流性腹泻病毒 四重实时荧光RT-PCR 

分 类 号：S852.65[农业科学—基础兽医学]