猪凸隆病毒猪嵴病毒猪捷申病毒猪轮状病毒多重荧光RT-PCR方法的建立与初步应用  

Development and application of multiplex fluorescence RT-PCR for detection of porcine torovirus,porcine kobuvirus,porcine teschovirus and porcine rotavirus

作　　者：赵冉 蔡振鸿 彭小莉 宋娜杰 陈雅婷 陈琼 孔繁德 石文志 刘文媛 许秋贝 何扬州 ZHAO Ran;CAI Zhen-hong;PENG Xiao-li;SONG Na-jie;CHEN Ya-ting;CHEN Qiong;KONG Fan-de;SHI Wen-zhi;LIU Wen-yuan;XU Qiu-bei;HE Yang-zhou(Xiamen Animal Disease Prevention and Control Center,Xiamen 361009,China;Xiamen Siming District Urban Utilities Center,Xiamen 361005,China;Xiamen Customs Technology Center,Xiamen 361000,China;Xiamen Zeesan Biotech Co.,Ltd.,Xiamen 361000,China)

机构地区：[1]厦门市动物疫病预防控制中心,福建厦门361009 [2]厦门市思明区市政中心,福建厦门361005 [3]厦门海关技术中心,福建厦门361000 [4]厦门致善生物科技股份有限公司,福建厦门361000

出　　处：《中国兽医科学》2023年第7期816-822,共7页Chinese Veterinary Science

基　　金：厦门市科技计划项目(3502Z20226027)。

摘　　要：为建立一种同时检测猪凸隆病毒、猪嵴病毒、猪捷申病毒和猪轮状病毒的高特异性、高灵敏性的检测方法,根据这4种病毒的保守基因序列分别合成相应的特异性引物及TaqMan探针,采用One-Step法进行反应体系的优化,建立多重荧光RT-PCR方法,评估该方法的特异性、敏感性、重复性,并用于本地猪场腹泻样品中上述4种病毒的流行病学调查。结果显示:该方法特异性好,可特异性地检测上述4种病毒的单一模板及混合模板,其他对照株均无扩增信号;灵敏度高,上述4种病毒的最低检出限均为101copies/μL;重复性好,批内及批间重复试验变异系数均小于3%。所建立方法还具有高通量、低污染和操作简便的优点,3 h内即完成全市猪场265份腹泻样品的检测,大大提高了检测效率。流行病学调查显示,厦门市养猪场猪嵴病毒、猪捷申病毒、猪轮状病毒和猪凸隆病毒这4种病毒均具有较高的检出率,阳性率依次为67.92%、65.28%、36.60%和27.55%。该研究将为猪腹泻病鉴别诊断及防控提供重要的技术支持。In order to establish a method with high specificity and sensitivity detecting porcine torovirus(PToV),porcine kobuvirus(PKV),porcine teschovirus(PTV)and porcine rotavirus(PoRV),four pairs of specific primers and TaqMan probes were synthesized based on their respective conservative gene sequences.By optimizing the reaction system with One-Step reagent,a multiplex fluorescence RT-PCR method for detection of PKV,PTV,PoRV,and PToV was established.The specificity,sensitivity and repeatability of the method were evaluated,and it was used for epidemiological investigation of the above 4 viruses in diarrhea samples from local pig farms.The results showed that the method was good specificity and repeatability and can specifically detect the single template and mixed template of four viruses above,and no amplification signals were found in other control strains.The lowest detection limit for all four viruses were 101 copies/μL.The variable coefficient of intra-and inter-batch repeated tests were less than 3%.The method is high throughput,low pollution and simple operation.The detection of 4 diarrheal viruses in 265 diarrheal samples from 53 local pig farms was completed within 3 h,greatly improving the detection efficiency.Epidemiological investigation showed that PKV,PTV,PoRV and PToV had high detection rates in Xiamen pig farms,with positive rates of 67.92%,65.28%,36.60%and 27.55%.This study will provide important technical support for the differential diagnosis,pre-vention and control of porcine diarrheal disease.

关 键 词：猪凸隆病毒 猪嵴病毒 猪捷申病毒 猪轮状病毒 多重荧光RT-PCR 

分 类 号：S852.651[农业科学—基础兽医学]