摘要
依据GenBank公布的猪圆环病毒2型Cap基因序列,在保守区域设计特异性引物和TaqMan探针,优化反应体系,建立评价猪圆环病毒2型灭活疫苗中病毒含量的实时荧光定量PCR检测方法,对方法的特异性、敏感性和重复性进行试验,并验证灭活剂用量和灭活时间对检测结果的影响。结果显示:该方法只对猪圆环病毒2型基因有特异性扩增,其他3种对照病毒基因的扩增结果均为阴性;检测灵敏度达到102.0 TCID50/mL,比普通PCR方法高100倍;方法的重复性好,对同一样品进行10次检测,变异系数为2.28%;不同灭活剂用量和灭活时间对结果的影响较小,不会因各厂家使用的灭活剂用量和灭活时间不同,影响疫苗对比实验的公平性。该研究成功建立了一种评价猪圆环病毒2型灭活疫苗中病毒含量的实时荧光定量PCR检测方法,用于猪圆环病毒2型灭活疫苗样品中病毒抗原含量的定量,其结果可以反映不同猪圆环病毒2型灭活疫苗样品中抗原含量差异,为研究猪圆环病毒2型灭活疫苗病毒抗原含量评估方法提供了新的思路。
A real-time fluorescence quantitative PCR assay to quantify the antigen concentration of inactivated porcine circovirus type 2 vaccines was developed based on a set of primers and TaqMan probe designed targeting the Cap gene sequence of porcine circovirus type 2 from GenBank.After optimizing the reaction system,the specificity,sensitivity and repeatability of the established assay were assessed.Subsequently,the impacts of adding amount of inactivator and inactivation time on detection performance of the established assay were investigated.The results showed that positive amplification product was exclusively obtained for target gene of porcine circovirus type 2 by using the established real-time fluorescence quantitative PCR assay,while the other three kinds of porcine origin viruses had no expected amplification products;the sensitivity of the established assay reached up to 102.0 TCID50/mL,which was 100 times higher than that of the ordinary PCR assay;the established assay had good repeatability with the variable coefficient of 2.28%in 10 repeated tests for the same one sample;the different adding amount of inactivator and inactivation time had limited impacts on detection performance of the established assay,in other words,the creditability of the inactivated vaccine comparison test was not affected by the adding amount of inactivator recommended by varied vaccine producers and different inactivation time.In this study,a real-time fluorescence quantitative PCR assay to quantify the antigen concentration of inactivated porcine circovirus type 2 vaccines is successfully developed.This established assay can quantitatively discriminate the antigen concentration of different inactivated porcine circovirus type 2 vaccine samples,providing novel insight into the development of quantitative evaluation method for antigen concentration of inactivated porcine circovirus type 2 vaccines.
作者
李雪峰
康斌
赵炳武
武玉梅
戴伶俐
董鹏
张建春
张春阳
斯琴高娃
LI Xue-feng;KANG Bin;ZHAO Bing-wu;WU Yu-mei;DAI Ling-li;DONG Peng;ZHANG Jian-chun;ZHANG Chun-yang;Siqingaowa(Jinhe Youben Biological Products Co.,Ltd.,Hohhot 011517,China;Inner Mongolia Academy of Agricultural and Animal Husbandry Sciences,Hohhot 010031,China;Hangzhou Youben Animal Vaccine Co.,Ltd.,Hangzhou 310018,China)
出处
《畜牧与饲料科学》
2021年第1期8-13,共6页
Animal Husbandry and Feed Science
关键词
实时荧光定量PCR
猪圆环病毒2型
灭活疫苗
Cap基因
real-time fluorescence quantitative PCR assay
porcine circovirus type 2
inactivated vaccines
Cap gene
