腮腺炎减毒活疫苗感染性滴度荧光定量RT-PCR检测方法的建立及验证

Development and verification of a fluorescent quantitative reverse transcription-polymerase chain reaction assay for infectious titer of live attenuated mumps vaccine

目的建立腮腺炎减毒活疫苗感染性滴度荧光定量RT-PCR检测方法,并进行验证。方法针对腮腺炎减毒活疫苗株S79血凝素(hemagglutinin,H)基因保守区域设计特异性引物和Taq Man荧光探针;以05008批腮腺炎减毒活疫苗S79株成品作为参考品,将参考品或供试品稀释后,接种于长成单层的Vero细胞中,采用低渗合并冻融法将细胞破碎,吸取上清,进行荧光定量RT-PCR。优化病毒感染时间,并对该方法的特异性、精密性及准确性进行验证。结果病毒感染的最佳时间为18 h;建立的荧光定量RT-PCR法只对腮腺炎减毒活疫苗具有特异性扩增,对灭活的腮腺炎减毒活疫苗、水痘病毒、狂犬病病毒、麻疹病毒、风疹病毒和Vero细胞均无扩增曲线出现;该方法检测4个浓度(1、1:5、1:52、1:53)样品6组数据的相对标准偏差(relative standard deviation,RSD)均<5%,不同操作人员于不同日期检测4个浓度(1、1:5、1:52、1:53)样品的标准曲线回归方程R2均大于0.97,RSD均<5%;该方法与细胞病变法测得的11批腮腺炎减毒活疫苗成品的病毒滴度值之差均≤0.2 Lg CCID50/ml,两组数据差异无统计学意义(P>0.05)。结论建立的荧光定量PCR法检测腮腺炎减毒活疫苗感染性滴度特异性较强,精密性和准确性良好,且快速方便,可应用于企业生产过程中的内部质控。

Objective To develop and verify a fluorescent quantitative reverse transcription-polymerase chain reaction（RT-PCR）assay for infectious titer of live attenuated mumps vaccine. Methods Primers and Taq Man fluorescent probe were designed specific to the conserved region of hemagglutinin（H） gene of live attenuated mumps vaccine virus strain S79. The final product prepared with strain S79,with a lot No. of 05008,was served as a reference. Vero cells were inoculated with the reference or test sample,then broken by hypoosmotic method combined with freeze-thawing method,of which the supernatant was collected and determined by fluorescent quantitative RT-PCR. The time for virus infection was optimized,and the developed method was verified for specificity,precision and accuracy. Results The optimal time for virus infection was 18 h. The developed fluorescent quantitative RT-PCR assay was specific to live attenuated mumps vaccine,while no amplification curves were observed in the vaccine after inactivation,varicella virus,rabies virus,measles virus,rebulla virus or Vero cells. The relative standard deviations（RSDs） of six groups of data on samples at four concentrations（1,1 ∶ 5,1 ∶ 52 and 1 ∶ 53） were less than 5%. The R2 values of regression equation of standard curves of samples at four concentrations（1,1 ∶ 5,1 ∶ 52 and 1 ∶ 53） determined by various personnel on various dates were more than 0. 97,with RSDs of less than 5%. The difference between determination results of 11 batches of live attenuated mumps vaccine by the developed method and by CPE method was not more than 0. 2 Lg CCID50/ ml,which showed no significant difference（P 〉 0. 05）. Conclusion The developed fluorescent quantitative RT-PCR assay for infectious titer of live attenuated mumps vaccine showed high specificity,precision and accuracy,which was rapid,simple,and suitable for the in-house quality control during production.