人用疫苗及生产用细胞中猪圆环病毒PCR检测方法的建立及验证

Development and verification of a PCR assay for porcine circovirus in vaccines for human use and cells used for production

目的建立人用疫苗及生产用细胞中猪圆环病毒1型(porcine circovirus 1,PCV1)和2型(PCV2)PCR检测方法,并进行验证。方法参照Gen Bank中登录的PCV1(KC447455)和PCV2(AY578327)序列,全基因组合成PCV1和PCV2基因序列,以合成的PCV1和PCV2全基因组序列DNA为模板,进行PCR扩增,对PCR反应中的退火温度进行优化。对优化后的PCR方法进行灵敏度和特异性验证,并用该方法检测疫苗生产用细胞KMB_(17)、Vero工作种子批、脊髓灰质炎减毒活疫苗(oral poliomyelitis vaccine,OPV)收获液、冻干甲型肝炎减毒活疫苗(freeze-dried hepatitis A vaccine,f HAV)成品、肠道病毒71型(enterovirus 71,EV71)灭活疫苗收获液及成品、Sabin株脊髓灰质炎灭活疫苗(inactivated poliomyelitis vaccine prepared with Sabin strain,SIPV)收获液及成品的PCV1和PCV2。结果优化后的退火温度为63℃,建立的PCR方法最低可检测出10^(-1)fg/μL的目的基因,仅对PCV基因组DNA能扩增出特异性条带。用该方法检测生产用细胞KMB_(17)及Vero工作种子批、OPV收获液、f HAV成品、EV71灭活疫苗收获液及成品、SIPV收获液及成品,均未检出PCV1和PCV2。结论成功建立了生产用细胞种子、疫苗收获液及成品中PCV的PCR检测方法,该方法具有较高的灵敏度和较强的特异性,能够用于本所生物制品生产用细胞种子、疫苗收获液及成品中PCV污染的检测,从而提高生物制品的安全性。

Objective To develop and verify a PCR assay for porcine circovirus types 1（PCV1） and 2（PCV2） in vaccines for human use as well as in the cell lines used for production. Methods PCV1 and PCV2 gene sequences were synthesized according to the PCV1（KC447455）and PCV2（AY578327）sequences in Gen Bank and used as the templates for PCR. The temperature for annealing in PCR assay was optimized. The optimized PCR assay was verified for sensitivity and specificity, and used for tests for PCV1 and PCV2 in KMB（17） cells for vaccine production, working cell banks of Vero cells, virus harvests of oral poliomyelitis vaccine（OPV）, final product of freeze-dried hepatitis A vaccine（f HAV）, harvest and final product of enterovirus 71（EV71）vaccine as well as harvest and final product of inactivated poliomyelitis vaccine prepared with Sabin strain（SIPV）. Results The optimal temperature for annealing was 63 ℃. The minimum detection limit of the developed PCR assay was 10-（-1） fg/μL. 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 KMB（17） cells for vaccine production,working cell banks of Vero cells, virus harvests of OPV, final product of f HAV, harvest and final product of EV71 vaccine as well as harvest and final product of SIPV. Conclusion A highly sensitive and specific PCR assay for PCV1 and PCV2 in vaccines for human use and cell lines for production was developed, which might be used for test for PCV1 and PCV2 in the working cell banks, virus harvest and final product and beneficial to the improvement of safety of biological products.