PDCoV、PEAV、TGEV及PEDV“一步法”多重荧光RT-PCR的建立和应用  

Establishment and Application of"One-step"Multiplex Fluorescent RT-PCR for PDCoV,PEAV,TGEVand PEDV

作　　者：赵冉 蔡振鸿 彭小莉 陈琼 李传勇 孔繁德 许秋贝 宋娜杰 陈雅婷 石文志 刘文媛 Zhao Ran;Cai Zhenhong;Peng Xiaoli;Chen Qiong;LiChuanyong;Kong Fande;Xu Qiubei;Song Najie;Chen Yating;Shi Wenzhi;Liu Wenyuan(Xiamen Center for Animal Disease Prevention and Control,Xiamen,Fujian 361009,China;Siming Civic Center,Xiamen,Fujian 361005,China;Xiamen Customs Technology Center,Xiamen,Fujian 361000,China;Xiamen Zeesan Biotech Co.,Ltd.,Xiamen,Fujian 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：《中国动物检疫》2023年第7期88-94,共7页China Animal Health Inspection

基　　金：厦门市科技计划项目(3502Z20226027)。

摘　　要：为快速鉴别检测猪冠状病毒,使用一步法试剂,对猪δ冠状病毒、猪肠道α冠状病毒、猪传染性胃肠炎病毒以及猪流行性腹泻病毒的检测方法进行优化,建立了同时检测上述4种病毒的“一步法”多重荧光RTPCR。随后对该方法的特异性、敏感性、重复性进行评估,并将其用于厦门市规模猪场上述4种腹泻病毒感染的流行病学调查。结果显示:该方法的敏感性高,对上述4种病毒阳性质粒的最低检出限均可达101copies/μL;特异性好,对4种病毒的单一及混合模板进行检测时,均可在相应荧光通道中获得良好的扩增曲线,而对其他病毒和细菌无扩增信号;重复性好,批间试验变异系数小于1.1%。使用该方法对从厦门市53家猪场采集的265份腹泻样品进行检测,4 h即可完成全部检测,猪δ冠状病毒和猪流行性腹泻病毒的场阳性率分别为18.87%和5.66%,未检出猪肠道α冠状病毒和猪传染性胃肠炎病毒。结果表明,本研究建立的方法敏感性高,特异性及重复性好,操作简便、耗时短,适用于临床大批量样本的检测,可为防控猪腹泻疾病提供有力的技术支持。In order to quickly identify and detect porcine coronavirus,the detection method for porcine deltacoronavirus(PDCoV),porcine enteric alphacoronavirus(PEAV),porcine transmissible gastroenteritis(TGEV)and porcine epidemic diarrhea virus(PEDV)was optimized by one-step reagent,and a“one-step”multiplex fluorescent RT-PCR assay was established to simultaneously detect the above four viruses,then applied for the epidemiological investigation on the four diarrhea viruses prevalent in intensive farms in Xiamen after evaluation on its specificity,sensitivity and repeatability.The results showed that,for the method,its minimum detection limit for positive plasmids of the four viruses could be up to 1o'copies/μL due to its high sensitivity:;good amplification curves could be obtained from corresponding fluorescence channels due to its good specificity when detecting the single or mixed template of the four viruses rather than other viruses and bacteria;and the coefficient of variation(CV)of batch tests was less than 1.1%due to its good repeatability.265 diarrhea samples from 53 swine farms across the city were detected by the method,and all tests were completed within 4 hours.The farm positive rates of PDCoV and PEDV were 18.87%and 5.66%,respectively.No PEAV or TGEV was detected.In conclusion,the method established in the study,with the advantages of high sensitivity,good specificity,repeatability,rapidity and user-friendly control,was appropriate for large amount of clinical samples,and technically supported to fight against swine diarrhea diseases.

关 键 词：多重荧光RT-PCR PDCoV PEDV TGEV PEAV 

分 类 号：S855.3[农业科学—临床兽医学]