摘要
为实现对猪流行性腹泻病毒(PEDV)和猪传染性胃肠炎病毒(TGEV)的快速鉴别诊断,根据PEDV的N基因和TGEV的S基因序列设计引物,并以猪β-肌动蛋白(ACTB)作为内参对照,建立了PEDV和TGEV的TaqMan探针双重荧光定量PCR检测方法。结果显示:该方法对PEDV、TGEV和ACTB的检测灵敏度分别为1、10和10 copies/μL;PEDV和TGEV敏感度为10^(0.02)TCID_(50)和10_(0.05)TCID_(50),高于常规RT-PCR,批内和批间变异系数均小于1%。与猪德尔塔冠状病毒、猪急性腹泻综合征冠状病毒、猪轮状病毒、乙型脑炎病毒、塞内卡谷病毒、猪繁殖与呼吸综合征病毒、脑心肌炎病毒、猪瘟病毒、伪狂犬病毒和猪圆环病毒2型均无交叉反应。采集124份临床病料样品进行检测,PEDV阳性率为42.74%,TGEV阳性率为8.87%,检测结果与该病毒单一荧光RT-PCR方法的检测结果均一致,证明该方法具有较高特异性和敏感性,可用于PEDV和TGEV临床病料检测。
In order to rapidly and differentially diagnose two important pathogens of porcine epidemic diarrhea virus(PEDV)and porcine transmissible gastroenteritis virus(TGEV),PCR primers and probes were designed based on the S genes of PEDV N and TGEV to establish a double fluorescent quantitative PCR method for PEDV and TGEV.Porcineβ-actin(ACTB)was used as an internal control.The results showed that the detection sensitivity of this method for PEDV,TGEV and ACTB was 1,10 and 10 copies/μL,respectively.The sensitivity of the method for PEDV and TGEV was 10_(0.02)TCID_(50)and 10^(0.05)TCID_(50),respectively,which were higher than those of conventional RT-PCR.The intra-and inter-batch coefficients of variation of this method were all below 1%.The method had no cross-reaction with porcine delta coronavirus,swine acute diarrhea syndrome coronavirus,porcine rotavirus,Japanese encephalitis virus,Seneca virus,porcine reproductive and respiratory syndrome virus,encephalomyocarditisvirus,swine fever virus,pseudorabies and porcine circovirus type 2.The method was used to detect the virus in all the 124 clinical disease material samples.The results showed that the positive rates of PEDV and TGEV were 42.74%and 8.87%respectively,which were consistent with those detected by a single fluorescent RT-PCR for PEDV or TGEV.The present results indicated that the method of virus detection established in this study had high specificity and efficiency,and might be used for detection of PEDV and TGEV in clinical samples.
作者
梁霄
孙萌
王鹏程
白娟
王先炜
刘星
姜平
LIANG Xiao;SUN Meng;WANG Pengcheng;BAI Juan;WANG Xianwei;LIU Xing;JIANG Ping(Nanjing Agriculture University/Key Laboratory of Animal Bacteriology,Ministry of Agriculture and Rural Affairs,Nanjing 210095,China)
出处
《畜牧与兽医》
北大核心
2022年第6期119-125,共7页
Animal Husbandry & Veterinary Medicine
基金
国家生猪产业技术体系专项(CARS-35)
国家重点研发计划项目(2021YFD1801102)。
