乙型脑炎减毒活疫苗中猪圆环病毒PCR检测方法的建立及验证  

Development and verification of a PCR assay for porcine circovirus in live attenuated Japanese encephalitis vaccine

作　　者：黄敏[1] 吕冰凌[1] 袁良玉[1] 易维维 吴朔华 曲芙莲 潘海龙[1] 

机构地区：[1]成都生物制品研究所有限责任公司,四川成都610023

出　　处：《中国生物制品学杂志》2016年第12期1346-1348,1354,共4页Chinese Journal of Biologicals

摘　　要：目的建立乙型脑炎减毒活疫苗中猪圆环病毒1型(porcine circovirus 1,PCV1)和猪圆环病毒2型(PCV2)PCR检测方法,并进行验证及初步应用。方法参照Gen Bank中登录的PCV1(AY193712)及PCV2序列(AY181946)设计引物,以提取的PCV1和PCV2基因组DNA为模板,分别经PCR扩增726 bp的PCV1片段和433 bp的PCV2片段,并对PCR反应中的退火温度、引物浓度、Mg2+浓度参数进行优化。将PCR扩增产物分别与p MD18-T载体连接,连接产物转化感受态大肠埃希菌DH5α,挑取阳性克隆,测序并进行同源性分析。对优化的PCR方法进行灵敏度和特异性验证,并用该方法检测3批乙型脑炎减毒活疫苗工作种子批、4批明胶和10批胰蛋白酶中的PCV1和PCV2。结果确定PCR反应的退火温度为54℃,引物浓度为10μmol/L,Mg2+浓度为10 mmol∕L。扩增的目的基因测序结果与Gen Bank中发布的PCV1及PCV2序列的同源性均为100%。建立的PCR方法最低可检出10 pg的目的基因;该方法只对PCV1和PCV2基因组DNA能扩增出特异性条带。用该方法检测乙型脑炎减毒活疫苗工作种子批、明胶和胰蛋白酶,均未检出PCV1和PCV2。结论成功建立了乙型脑炎减毒活疫苗中PCV1和PCV2的PCR检测方法,该方法具有较高的灵敏度和较强的特异性,可用于乙型脑炎减毒活疫苗工作种子批及两种猪源性原材料的PCV1和PCV2污染检测。Objective To develop, verify and preliminarily apply a PCR assay for porcine circovirus types 1（PCV1）and2（PCV2） in live attenuated Japanese encephalitis vaccine（JEV）. Methods Primers were designed according to the sequences of PCV1（AY193712）and PCV2（AY181946）in Gen Bank, based on which the PCV1 and PCV2 fragments, at lengths of 726 and 433 bp, were amplified by PCR using the extracted PCV1 and PCV2 genomic DNAs as templates respectively. The temperature for annealing, primer concentration and magnesium ion concentration in PCR assay were optimized. The PCR products were inserted into vector p MD18-T respectively, and the constructed recombinant plasmids were transformed to E. coli DH5α. The positive clones were screened, sequenced and analyzed for homology. The optimized PCR assay was verified for sensitivity and specificity, and used for tests for PCV1 and PCV2 in three batches of working seeds JEV, four batches of gelatin and ten batches of trypsin. Results The optimal temperature for annealing,primer concentration and magnesium ion concentration in PCR assay were 54 ℃, 10 μmol / L and 10 mmol / L respectively. Sequencing proved that both the homologies of amplified PCV1 and PCV2 genes were 100% to those in Gen Bank. The minimum detection limit of the developed PCR assay was 10 pg. Specific DNA bands were amplified by the developed PCR assay only from the genomes of PCV1 and PCV2. No PCV1 and PCV2 DNAs were found in the working seeds of JEV, gelatin and trypsin. Conclusion A highly sensitive and specific PCR assay for PCV1 and PCV2 in JEV was developed, which might be used for test for PCV1 and PCV2 in the working seeds of JEV and in two raw materials from porcine origin.

关 键 词：乙型脑炎减毒活疫苗 猪圆环病毒1型 猪圆环病毒2型 聚合酶链反应 

分 类 号：R373.31[医药卫生—病原生物学] R392.33[医药卫生—基础医学]