摘要
为了快速同步检测猪德尔塔冠状病毒(PDCoV)、猪嵴病毒(PKV)和猪呼肠孤病毒(MRV),参考GenBank中PDCoV M、PKV 5’UTR和MRV L1基因保守序列,设计了特异性引物和探针并优化了反应条件,建立了同时检测PDCoV、PKV和MRV的三重荧光定量PCR方法。该方法可特异性检出PDCoV、PKV和MRV,与PRRSV、CSFV、PCV2、PRV、PEDV、TGEV、RV等病原无交叉反应,3种病毒的最低检测量分别为2.7×10^(1)copies/μL、1.22×10^(1)copies/μL和1.26×10^(2)copies/μL,组内及组间变异系数为0.001~0.019。应用所建立的方法与行业标准及文献报道方法同时检测142份临床样本,结果显示与行业标准及文献报道方法阳性符合率为78%~94.92%。本研究建立的三重荧光定量PCR方法特异性强、敏感性高、重复性好,为临床PDCoV、PKV和MRV的鉴别诊断和流行病学调查提供了快速敏感的技术手段。
In order to rapidly and simultaneously detect porcine deltacoronavirus(PDCoV),porcine kobuvirus(PKV)and mammalian orthoreovirus(MRV),the specific primers and probes were designed accord-ing to the conserved region of PDCoV M gene,PKV 5′UTR gene and MRV L1 gene respectively and the reaction conditions were optimized,a triple real-time fluorescence quantitative PCR assay for PDCoV,PKV and MRV was established successfully.The assay could detect PDCoV,PKV and MRV specifically,and there was no cross reaction to PRRSV,CSFV,PCV2,PRV,PEDV,TGEV,RV and other pathogens.The lowest limit of PDCoV,PKV and MRV was 2.7×101 copies/μL,1.22×101 copies/μL and 1.26×102 copies/μL,respectively.The variation coefficient of the intra-and inter-assay ranged from 0.001—0.019.The comparison results between using the methods reported by the trade standard and documents and this method showed that the positive coincidence rate was 78%—94.92%(142 samples).It showed that the method established in this study has strong specificity,high sensitivity and good repeatability,which provided a valuable tool for the rapid diagnosis and epidemiological investigations of co-infection of PDCoV,PKV and MRV.
作者
王利丽
李富强
董明奇
任卫科
路超
张莉
江珊
田向学
鄢明华
WANG Li-li;LI Fu-qiang;DONG Ming-qi;REN Wei-ke;LU Chao;ZHANG Li;JIANG Shan;TIAN Xiang-xue;YAN Ming-hua(Institute of Animal Science and Veterinary,Tianjin Academy of Agricultural Sciences,Tianjin 300381,China;Tianjin Observation and Experiment Site of National Animal Health,Tianjin 300381,China;Tianjin Key Laboratory of Animal Molecular Breeding and Biotechnology,Tianjin 300381,China;Tianjin Engineering Research Center of Animal Healthy Farming,Tianjin 300381,China;Harbin National Engineering Research Center of Veterinary Biologics Corp,Harbin 150028,China)
出处
《中国兽医科学》
CAS
CSCD
北大核心
2023年第7期823-830,共8页
Chinese Veterinary Science
基金
中央级公益性科研院所基本科研业务费院级统筹项目:动物疫病数据中心
天津市重大推广项目(ITTPRS2021003)。
