猪流行性乙型脑炎病毒一步法RT-PCR的建立及达州市猪流行性乙型脑炎病毒流行病学调查与分析  

Establishment of one-step RT-PCR for Janpanese encephalitis virus and epidemiological investigation and analysis of Janpanese encephalitis virus in Dazhou City

作　　者：何博[1] 王勤[1] 欧云文 刘俐君 HE Bo;WANG Qin;OU Yunwen;LIU Lijun(Dazhou Vocational and Technical College,Dazhou 635001,China;Kaijiang County Animal Disease Prevention and Control Center,Kaijiang 636250,China;China Animal Health and Epidemiology Center,Qingdao 266032,China;Dazhou Animal Disease Prevention and Control Center,Dazhou 635000,China)

机构地区：[1]达州职业技术学院,四川达州635001 [2]开江县动物疫病预防控制中心,四川开江636250 [3]中国动物卫生与流行病学中心,山东青岛266032 [4]达州市动物疫病预防控制中心,四川达州635000

出　　处：《黑龙江畜牧兽医》2021年第16期79-84,共6页Heilongjiang Animal Science And veterinary Medicine

基　　金：四川省科技计划项目(2020JDRC0146)。

摘　　要：为了建立一种快速检测猪流行性乙型脑炎病毒(Janpanese encephalitis virus,JEV)的病原学检测方法,并掌握达州市JEV的感染情况,试验根据GenBank中JEV E基因序列设计1对检测引物,建立一步法RT-PCR,并对退火温度(50,52,54,56,58℃)、模板添加量(1,2,4,6,8μL)、引物添加量(0.1,0.2,0.4,0.6,0.8μL)进行优化,考察方法的特异性、敏感性、重复性、符合性,同时对2017-2020年分别采集于达州市7个市(县、区)的243份样品进行JEV检测,对检测结果进行不同地区、不同年份、不同季节、不同饲养方式下猪群中JEV阳性率的比较分析。结果表明:优化后的一步法RT-PCR扩增体系为2×Step Buffer 12.5μL,上下游引物(20μmol/L)各0.4μL,RNA(68 ng/μL)4μL,H2O 7.7μL。扩增程序为50℃30 min;95℃5 min;94℃40 s,54℃40 s,72℃40 s,30个循环;72℃10 min;4℃终止反应。该方法能够特异性扩增JEV,而检测猪瘟病毒(CSFV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪伪狂犬病毒(PRV)、猪圆环病毒2型(PCV-2)、猪细小病毒(PPV)、猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)均为阴性,对JEV RNA的检测灵敏度为27.2 pg,对JEV、CSFV、PRRSV、PEDV、TGEV、PRV、PCV-2、PPV、3份JEV阳性病料、3份JEV阴性病料重复检测3次的结果完全一致,检测243份猪临床病料样品的平均阳性率为9.05%,其中不同地区阳性率为3.45%~13.04%,2017,2018,2019,2020年阳性率分别为5.77%、6.90%、10.29%和12.31%,夏季和冬季的阳性率分别为13.33%和5.17%,规模化养猪场和散养户的阳性率分别为5.94%和11.27%,说明建立的JEV一步法RT-PCR具有良好的特异性、敏感性、重复性和临床适用性,达州市JEV的流行存在个别地区感染情况较严重、2019年和2020年感染情况较2017年和2018年严重、夏季感染情况较冬季严重、散养户感染情况较规模化养猪场严重等特点。In order to establish a rapid detection method for Japanese encephalitis virus(JEV),and to master the infection situation of JEV in Dazhou City,a pair of detection primers were designed according to JEV E gene sequence in GenBank for the establishment of one-step RT-PCR.The annealing temperature(50,52,54,56,58℃),template addition amount(1,2,4,6,8μL),and primer addition amount(0.1,0.2,0.4,0.6,0.8μL)were optimized;the specificity,sensitivity,repeatability and consistency of the method were investigated.At the same time,243 samples collected from 7 cities(counties,districts)in Dazhou City from 2017 to 2020 were tested for JEV,and a comparative analysis of JEV positive infection rates in pig herds in different regions,different years,different seasons,and different feeding methods was carried out.The results showed that the optimized one-step RT-PCR amplification system was 2×Step Buffer 12.5μL,upstream and downstream primers(20μmol/L)0.4μL respectively,RNA(68 ng/μL)4μL,H2O 7.7μL.The amplification procedure was 50℃30 min;95℃5 min;94℃40 s,54℃40 s and 72℃40 s for 30 cycles;72℃10 min;the reaction was terminated at 4℃.JEV was specifically amplified by this method,while CSFV,PRRSV,PEDV,TGEV,PRV,PCV-2 and PPV were negative.The detection sensitivity of JEV RNA was 27.2 pg.The results of repeated testing of disease materials 3 times were completely consistent for JEV,CSFV,PRRSV,PEDV,TGEV,PRV,PCV-2,PPV,3 JEV-positive samples and 3 JEV-negative samples.The average positive rate of testing 243 clinical pig samples was 9.05%;the positive rates in different regions were 3.45%-13.04%;the positive rates in 2017,2018,2019 and 2020 were 5.77%,6.90%,10.29%and 12.31%,respectively.The positive rates in summer and winter were 13.33%and 5.17%,respectively.The positive rates in large-scale pig farms and free-range households were 5.94%and 11.27%,respectively.The results suggested that the established one-step RT-PCR method had good specificity,sensitivity,repeatability and clinical applicability.The prevalence of J

关 键 词：猪流行性乙型脑炎病毒 一步法RT-PCR 检测方法 流行病学调查与分析 

分 类 号：S851.3[农业科学—预防兽医学] S852.65[农业科学—兽医学]