文献类型：期刊文献

中文题名：猪瘟病毒PCR-ELISA检测方法的初步应用

英文题名：Preliminary Application of PCR-ELISA Detection Method in Swine Fever Virus

作者：王利平[1];金前跃[2];刘兴友[1];王选年[1];谭东鹤[1];李鹏[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]河南省农业科学院动物免疫学重点实验室,河南郑州450002

第一机构：新乡学院

年份：2023

卷号：44

期号：7

起止页码：64-71

中文期刊名：家畜生态学报

外文期刊名：Journal of Domestic Animal Ecology

收录：CSTPCD;;北大核心:【北大核心2020】；

基金：河南省重点研发与推广专项(科技攻关)(202102110094,212102310335);新乡市科技攻关项目(GG2019018,GG2020018)。

语种：中文

中文关键词：猪瘟病毒;PCR-ELISA;荧光定量PCR;间接免疫荧光

外文关键词：classical swine fever virus;PCR-ELISA;fluorescence quantification PCR;indirect immunofluorescence

摘要：猪瘟(classicalswinefever,CSF)是危害猪健康的重要传染病之一,具有急性、热性和高度接触性等特性,给世界养猪业造成了巨大的经济损失。PCR-ELISA检测方法是在PCR和ELISA基础上创新的一种新技术,其灵敏性和准确性都高于单纯的PCR检测和ELISA检测。根据GenBank中猪瘟石门株序列,设计特异性引物,分别在5'端标记生物素和地高辛,经过PCR扩增后,加入到包被有链霉亲和素的ELISA反应板上,然后通过DIG-HRP抗体进行显色反应,建立PCR-ELISA检测方法。结果表明:PCR-ELISA最低检测到1.81×10^(4)拷贝/μL,普通PCR最低检测到1.81×10^(6)拷贝/μL,因此PCR-ELISA灵敏性比普通PCR高约100倍;对27份未知样品进行检测,普通PCR检测出阳性样品15份,阳性率为55.5%,PCR-ELISA检出20份阳性样品,阴性7份,阳性率为74%,阳性检出率增加18.5%;用建立的Real-TimePCR进行验证,检测结果与PCR-ELISA一致,两者的符合度为100%;然后用PK15细胞对普通PCR未检测出的阳性样品进行病毒分离培养,通过间接免疫荧光(IFA)及普通PCR检测,结果显示该5份样品均为阳性。说明本研究所建立的PCR-ELISA方法具有灵敏、特异、准确、快速、安全等优势,可应用于CSFV的快速诊断,为CSFV的流行病学监测提供有力保障。
Classical swine fever(CSF)is one of the important infectious diseases endangering pig health,which has the characteristics of acuteness,heat and high contact,causing huge economic losses to the global pig industry.PCR-ELISA is a new technology based on PCR and ELISA.Its sensitivity and accuracy are higher than PCR or ELISA.According to the sequence of shimen strain of classical swine fever in GenBank,specific primers were designed to label biotin and digoxin at the 5'end respectively.After PCR amplification,they were added to the ELISA reaction plate coated with Streptavidin,and then color reaction was carried out by DIG-HRP antibody to establish a PCR-ELISA detection method.The sensitivity test results of PCR-ELISA showed that the lowest detection rate of PCR-ELISA is 1.81%×10^(4)copies/μL.The lowest detection rate of PCR is 1.81%×10^(6)copies/μL.The sensitivity of PCR-ELISA is about 100 times higher than that of ordinary PCR.Among 27 unknown samples,15 positive samples were detected by ordinary PCR,the positive rate was 55.5%,20 positive samples were detected by PCR-ELISA,7 samples were negative,the positive rate was 74%,the positive rate increased by 18.5%.The results of Real-time PCR and PCR-ELISA were consistent,and the coincidence was 100%.Then PK15 cells were used to isolate and culture the positive samples which were not detected by ordinary PCR.The results of IFA and ordinary PCR showed that the five samples were all positive.Therefore,the PCR-ELISA established in this study has the advantages of sensitivity,specificity,accuracy,rapidity and safety,which can be applied to the rapid diagnosis of CSFV and provide a strong guarantee for the epidemiological monitoring of CSFV.