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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 establish a method for PCR detection of porcine circovirus 1 (PCV1) and porcine circovirus type 2 (PCV2) in attenuated live attenuated encephalitis B. The validation and preliminary application of this method are as follows. Methods According to the PCV1 (AY193712) and PCV2 sequences (AY181946) registered in Gen Bank, the primers PCV1 and PCV2 were used as template to amplify 726 bp PCV1 fragment and 433 bp PCV2 fragment respectively The annealing temperature, primer concentration and Mg2 + concentration in the PCR reaction were optimized. The PCR products were ligated into pMD18-T vector and transformed into competent E.coli DH5α. The positive clones were picked and sequenced for homology analysis. The sensitivity and specificity of the optimized PCR method were validated. PCV1 and PCV2 in three batch batches of live attenuated JE vaccine, four batches of gelatin and ten batches of trypsin were detected by this method. As a result, it was confirmed that the annealing temperature of the PCR reaction was 54 ° C, the primer concentration was 10 μmol / L, and the Mg2 + concentration was 10 mmol / L. The sequence of the target gene amplified showed 100% homology with the PCV1 and PCV2 sequences published in Gen Bank. The established PCR method can detect a minimum of 10 pg of the target gene; this method can amplify specific bands only for the PCV1 and PCV2 genomic DNA. Using this method to detect live attenuated JE vaccine seed, gelatin and trypsin, were not detected PCV1 and PCV2. Conclusion The PCR detection method of PCV1 and PCV2 in live attenuated JE vaccine has been successfully established. The method has high sensitivity and strong specificity and can be used in the working seed batch and two Detection of PCV1 and PCV2 Contamination of Swine-derived Raw Materials.