文献类型：期刊文献

中文题名：猪圆环病毒3型PCR-ELISA检测方法的研究与应用

英文题名：Research and application of PCR-ELISA detection method for porcine circovirus type 3

作者：李鹏[1];孙延举[2];雷梦瑶[2];孙国鹏[1];银梅[2];苏为为[1];王选年[1];刘兴友[1];王利平[1]

机构：[1]新乡学院生命科学与基础医学学院,河南新乡453003;[2]河南科技学院动物科技学院,河南新乡453003

第一机构：新乡学院

年份：2022

卷号：42

期号：11

起止页码：2152-2157

中文期刊名：中国兽医学报

外文期刊名：Chinese Journal of Veterinary Science

收录：CSTPCD;;北大核心:【北大核心2020】；CSCD:【CSCD_E2021_2022】；

基金：河南省科技厅科技攻关资助项目(202102110094);河南省现代农业产业技术体系资助项目(HARS2212G3);河南省新乡市科技攻关资助项目(GG2019018)。

语种：中文

中文关键词：PCV3;PCV2;PCR-ELISA

外文关键词：PCV3;PCV2;PCR-ELISA

摘要：为建立一种检测猪圆环病毒3型(porcine circovirus type 3,PCV3)的PCR-ELSIA方法,根据GenBank上报道的PCV3全基因组序列,针对其高度保守区域设计1对特异性引物,上、下游引物的5′端分别标记生物素和地高辛。经PCR扩增获得大量带有标记的目的基因,依靠生物素-链霉亲和素的非共价结合作用固定目的基因于载体上,再通过羊抗DIG-HRP的显色反应,建立PCR-ELISA检测方法。结果表明,该方法与猪繁殖与呼吸综合征病毒、猪瘟病毒、猪细小病毒和猪圆环病毒2型无交叉反应,特异性良好。PCR-ELISA方法最低检测限为5.58×10^(2)copies/μL,敏感性比常规PCR高100倍(常规PCR的检测限为5.58×10^(4)copies/μL)。PCR-ELISA方法批内和批间重复性检测的变异系数分别为1.90%~5.20%和3.89%~9.03%。对132份临床样品进行PCV3检测,PCR-ELSIA检出率为9.10%(12/132),高于常规PCR的检出率(4.55%,6/132)。综上,该方法具有特异、灵敏、安全高效的优点,可为PCV3的诊断、防控和流行病学调查提供新的技术手段。
To establish a PCR-ELSIA method for the detection of porcine circovirus type 3(PCV3),a pair of specific primers was designed for its highly conserved region based on the complete genome sequence of PCV3 reported on GenBank,and the 5′ends of the forward and reverse primers were labeled with biotin and digoxin,respectively.A large number of labeled target genes were obtained by PCR amplification,and the target gene was immobilized on the vector by relying on the non-covalent binding of biotin-streptavidin,and then a PCR-ELISA assay was established by the chromogenic reaction of sheep anti-DIG-HRP.The results showed that the method had no cross reaction with porcine reproductive and respiratory syndrome virus,classical swine fever virus,porcine parvovirus and porcine circovirus 2,with good specificity.The lowest detection limit of PCR-ELISA was 5.58×10^(2)copies/μL,the sensitivity was 100-fold higher than that of conventional PCR,and the detection limit of conventional PCR was 5.58×10^(4)copies/μL.The coefficient of variation of intra-batch and inter-batch repeatability of PCR-ELISA was 1.90%-5.20%and 3.89%-9.03%,respectively.A total of 132 clinical samples were screened for PCV3,and the PCR-ELSIA detection rate was 9.10%(12/132),which was higher than the detection rate of conventional PCR(4.55%,6/132).In summary,this method has the advantages of specificity,sensitivity,safety and efficiency,and can provide new technical means for the diagnosis,prevention and control,and epidemiological investigation of PCV3.