猪德尔塔冠状病毒TaqMan荧光定量PCR检测方法的建立及应用  

Establishment and Application of TaqMan Fluorescence Quantitative PCR for Detecting Porcine Deltacoronavirus

作　　者：江珊 李富强[1,2,3] 李秀丽 王利丽[1,2,3] 郑丽 张莉[1,2,3] 路超 鄢明华[1,2,3] JIANG Shan;LI Fu-qiang;LI Xiu-li;WANG Li-li;ZHENG Li;ZHANG Li;LU Chao;YAN Ming-hua(Tianjin Institute of Animal Husbandry and Veterinary Science,Tianjin,300381,China;Tianjin Scientific Observation Experiment Station for Veterinary Medicine and Diagnosis Technology,Ministry of Agriculture and Rural Affairs,Tianjin,300381,China;Tianjin Engineering Research Center for Livestock and Poultry Health Breeding,Tianjin,300381,China)

机构地区：[1]天津市畜牧兽医研究所,天津300381 [2]农业农村部兽用药物与诊断技术天津科学观测实验站,天津300381 [3]天津市畜禽健康养殖技术工程中心,天津300381

出　　处：《动物医学进展》2019年第10期10-17,共8页Progress In Veterinary Medicine

基　　金：天津市高端兽药先进制造科技重大专项(17ZXGSNC00080);天津市现代农业产业技术体系岗位专家项目(ITTPRS2017003);天津市农业科学院青年科研人员创新研究与实验项目(201912)

摘　　要：为建立一种快速、灵敏、准确的猪德尔塔冠状病毒(PDCoV)分子检测方法,根据PDCoV M基因的保守序列设计并合成引物和探针,建立了PDCoV M基因Taq Man荧光定量PCR检测方法。结果表明,该方法标准曲线具有良好的线性关系,相关系数为0.998;敏感性好,最低可检测到10 copies/μL的PDCoV M基因标准品;特异性试验结果显示,该方法与猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)、猪轮状病毒(PoRV)、猪瘟病毒(CSFV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪伪狂犬病病毒(PRV)、猪圆环病毒2型(PCV2)、猪细小病毒(PPV)及大肠埃希菌K88菌株均无交叉反应;重复性试验变异系数低于2%,表明重复性良好。对121份临床样本进行检测,阳性检出率为53.7%(65/121),随机选择10份阳性样品的扩增产物进行测序分析,进一步证实扩增产物中含有PDCoV。该方法具有敏感性高、特异性强、用时短和高通量等特点,适用于PDCoV的定量检测与分子流行病学调查,为PDCoV的准确定量及进一步研究奠定了基础。In order to establish a rapid,sensitive and accurate molecular detecting method for porcine deltacoronavirus(PDCoV),primers and probe were designed and synthesized according to the conservative sequence of PDCoV M gene,and Taq Man fluorescence quantitative PCR method for detecting PDCoV M gene was established.The results showed that the standard curve of the method had a good linear relationship,and the correlation coefficient was 0.998.The method had good sensitivity,and the lowest detecting concentration of PDCoV M gene was 10 copies/μL for standard sample.The result of specificity test represented that there was no cross reaction with porcine epidemic diarrhea virus(PEDV),transmissible gastroenteritis of swine(TGEV),porcine rotavirus(PoRV),classical swine fever virus(CSFV),porcine reproductive and respiratory syndrome virus(PRRSV),pseudorabies virus(PRV),porcine circovirus 2(PCV2),porcine parvovirus(PPV),and Escherichia coli K88 strain.The variation coefficient of repeatability test was below 2%,indicating the method had good repeatability.121 clinical samples were detected by this method,and the positive detection rate was 53.7%(65/121).The amplified products of 10 positive samples were randomly selected to be sequenced and analyzed,which further confirmed that PDCoV was contained in the amplified products.This method has the characteristics of high sensitivity,good specificity,short time and high throughput,which is suitable for quantitative detection and molecular epidemiological investigation of PDCoV.It lays a foundation for accurate quantitation and further research of PDCoV.

关 键 词：猪德尔塔冠状病毒 M基因 TAQ Man探针 荧光定量PCR 

分 类 号：S852.659.6[农业科学—基础兽医学]