猪流行性腹泻病毒实时荧光RT-RAA检测方法的建立及应用  

Establishment and application of real-time fluorescence RT-RAA detection method for PEDV

作　　者：王恋春 郑辉[2] 张慧[2] 沙洲 房琳琳 尼博[2,3] 董雅琴 魏荣[2] 崔进 郑泽中[1] WANG Lianchun;ZHENG Hui;ZHANG Hui;SHA Zhou;FANG Linlin;NI Bo;DONG Yaqin;WEI Rong;CUI Jin;ZHENG Zezhong(College of Veterinary Medicine,South China Agricultural University,Guangzhou 510642,China;China Animal Health and Epidemiology Center,Qingdao 266032,China;Key Laboratory of Animal Biosafety Risk Prevention and Control(South),Ministry of Agriculture and Rural Affairs,Qingdao 266032,China)

机构地区：[1]华南农业大学兽医学院,广州510642 [2]中国动物卫生与流行病学中心,山东青岛266032 [3]农业农村部动物生物安全风险预警及防控重点实验室(南方),山东青岛266032

出　　处：《黑龙江畜牧兽医》2024年第20期63-68,74,共7页Heilongjiang Animal Science And veterinary Medicine

基　　金：“十四五”国家重点研发计划项目“口蹄疫病毒的分子流行病学与传播机制”(2021YFD1800300)。

摘　　要：为了建立一种快速检测猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)的方法,试验基于PEDV N基因保守区域设计3对特异性引物和1个探针,通过引物的筛选和反应温度的优化建立PEDV实时荧光逆转录重组酶介导等温扩增(reverse-transcription recombinase-aided amplification,RT-RAA)检测方法,对该方法进行敏感性试验、特异性试验和重复性试验,并分别采用该方法和实时荧光定量RT-PCR检测方法对72份猪淋巴结、肝脏等组织临床样品进行检测,计算两种方法的符合率。结果表明:建立的实时荧光RT-RAA方法在41℃条件下反应20 min即可完成检测,对PEDV的最低检测限为1×10~1拷贝/μL;且仅能检测出PEDV,与传染性胃肠炎病毒(Transmissible gastroenteritis virus,TGEV)、猪轮状病毒(Porcine rotavirus,PRoV)、猪圆环病毒2型(Porcine circovirus type 2,PCV-2)、猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)、猪圆环病毒3型(Porcine circovirus type 3,PCV-3)、猪瘟病毒(Classical swine fever virus,CSFV)、猪圆环病毒4型(Porcine circovirus type 4,PCV-4)、伪狂犬病病毒(Pseudorabies virus,PRV)、猪细小病毒(Porcine parvovirus,PPV)均无交叉反应;相同拷贝浓度的质粒标准品3次重复的扩增曲线几乎重合,起峰时间与荧光值没有明显差异。对72份临床样品进行检测,该方法与实时荧光定量RT-PCR检测方法的符合率为100%。说明建立的实时荧光RT-RAA方法敏感性高,特异性和重复性良好,同时耗时短、操作简单,可用于PEDV的临床检测。In order to establish a method for the rapid detection of Porcine epidemic diarrhea virus(PEDV),in the test,three pairs of specific primers and one probe were designed based on the conserved region of PEDv N gene,and a real-time fluorescence reverse transcription recombinase-aided amplification(RT-RAA)detection method for PEDV was established through primer screening and reaction temperature optimization.Sensitivity,specificity and reproducibility tests were performed on the method;the method and real-time quantitative RT-PCR detection method were used to detect 72 clinical samples of porcine lymph nodes,livers and other tissues;the coincidence rate of both methods was calculated.The results showed that the established real-time fluorescence RT-RAA method could be detected after 20 min of reaction at 41℃.The minimum limit of detection for PEDV was 1x10'copies/μL.Only PEDV could be detected,and there was no cross-reactivity with Transmissible gastroenteritis virus(TGEV),Porcine rotavirus(PRoV),Porcine circovirus type 2(PCV-2),Porcine reproductive and respiratory syndrome virus(PRRSV),Porcine circovirus type 3(PCV-3),Classical swine fever virus(CSFV),Porcine circovirus type 4(PCV-4),Pseudorabies virus(PRV)and Porcine parvovirus(PPV).The amplification curves of the three replicates of the plasmid standard at the same copy concentration were almost identical,and there was no significant difference between the peak time and the fluorescence value.Seventy-two clinical samples were tested,and the coincidence rate between the method and the real-time RT-PCR detection method was 100%.The results indicated that the established real-time fluorescence RT-RAA method had high sensitivity,good specificity and reproducibility,and was time-short and simple to operate,which could be used for the clinical detection of PEDV.

关 键 词：猪流行性腹泻病毒 N基因 猪流行性腹泻 实时荧光RT-RAA 检测方法 

分 类 号：S855.3[农业科学—临床兽医学]