中国兽医科学2017，Vol.47Issue(9)：1080-1086,7.DOI:10.16656/j.issn.1673-4696.2017.09.002
猪德尔塔冠状病毒TaqMan荧光定量PCR检测方法的建立与应用
Development and application of TaqMan real-time fluorescent PCR for detection of porcine deltacoronavirus
摘要
Abstract
The specific primer and TaqMan probe for PDCoV were designed and synthesized according to reference sequences published on GenBank,and plasmid standards were constructed.The TaqMan real time fluorescent PCR for porcine deltacoronavirus was established using standard samples and the parameters of assay were optimized including sensibility,specificity and repeatability.The results showed that no signals were detected when using PEDV,TGEV,PKV,PRRSV and FMDV as templates and indicated the assay had good specificity.The variation coefficient of cycle threshold Ctwas less than 2％ in repeated experiments using 2.66×106,2.66×105and 2.66×104copies/μL of standard samples as templates,which indicated the assay had good repeatability.The standard curve slope and correlation coefficient(R2) were-3.461 and 0.998,respectively,which indicated that there existed good linear relationship between threshold value and template concentration.Ten-fold serial standard sample dilutions were detected using this assay and the results showed that the detection limit was 2.66×101 copies/μ L plasmid DNA and indicated this assay had higher sensitivity.194 clinical swine feces were detected using this assay and the results showed the positive rate of PDCoV was 22.1％,significantly higher than that by conventional RT-PCR(ll.9％).In conclusion,this assay can be applied to clinical diagnosis and quantitative analysis for PDCoV.关键词
猪德尔塔冠状病毒/实时荧光定量PCR/病毒检测Key words
porcine deltacoronavirus/real-time fluorescent quantitative PCR/virus detection分类
农业科技引用本文复制引用
肖帅,魏彦明,刘新生,方玉珍,周鹏,张巧玲,芦延珍,董昭良,张永光,王永录..猪德尔塔冠状病毒TaqMan荧光定量PCR检测方法的建立与应用[J].中国兽医科学,2017,47(9):1080-1086,7.基金项目
国家自然科学基金项目(31602095) （31602095）
国家生猪产业体系项目(CARS-36-068) （CARS-36-068）
“十三五”国家重点研发计划(2016YFD0501505) （2016YFD0501505）
兰州兽医研究所统筹项目(Y2016CG23) （Y2016CG23）
