流感疫苗主种子批毒种中外源性禽腺病毒Ⅲ型荧光定量PCR检测方法的建立及初步应用

Development and preliminary application of fluorescent quantitative PCR for determination of extraneous avian adenovirus type Ⅲ in virus seeds from master seed lot of influenza vaccine

目的建立流感疫苗主种子批毒种中外源性禽腺病毒Ⅲ型(egg drop syndrome virus,EDSV)的荧光定量PCR检测方法,并进行验证及初步应用。方法针对EDSV六邻体蛋白保守区分别设计引物和探针,建立荧光定量PCR检测方法,并验证该方法的线性、特异性、精密性、灵敏度及可行性。采用新建立的荧光定量PCR法对16株流感疫苗主种子批毒种进行外源性EDSV检测。结果该方法的最佳线性范围为1×10~4~1×10~9 copies/μl,回归曲线为y=-3.385 x+39.616,R^2>0.99;与其他种属的腺病毒及流感病毒间均无交叉反应;试验内和试验间Ct值的变异系数(CV)均<2%;灵敏度为101 copies/μl;该方法检测P0代样品的最低检测限为10-6掺入体积比例,血清学方法检测P0~P3代样品的最低检测限为10^(-2)~10^(-3)掺入体积比例。该方法检测16株流感毒种中外源性EDSV的结果均为阴性,与血清学方法检测结果一致。结论成功建立了检测EDSV的荧光定量PCR法,提高了检测灵敏度和检测效率,更好地满足了流感疫苗应急检验中快检的要求。

Objective To develop,verify and preliminarily apply a fluorescent quantitative PCR method for determination of extraneous avian adenovirus typeⅢ（egg drop syndrome virus,EDSV）in influenza vaccine.Methods The primers and probes were designed according to the conserved region of EDSV sequence encoding hexon protein,based on which a fluorescent quantitative PCR method was developed and verified for linearity,specificity,precision,sensitivity and feasibility.Sixteen strains from master seed lot of influenza vaccine was tested for extraneous EDSV by the developed method.Results The linear range of the developed method was 1 × 10~4~ 1 × 10~9 copies/μl,while the regression curve was y =-3.385 x ＋ 39.616,with a R^2 value of more than 0.99.No cross reactions with adenovirus and influenza virus of other species were observed.Both the coefficients of variation（CVs）of Ct values in intra-and inter-assays were less than2%,while the sensitivity was 10~1 copies/μl.The detection limit of samples of passage 0（P0） by the developed method was 10-6incorporation volume,while that of P0 ~ P3 was 10^（-2）~ 10^（-3）incorporation volume.All the test results of EDSV in sixteen virus seeds were negative,which were consistent with those by serological method.Conclusion The fluorescent quantitative PCR method for determination of extraneous EDSV in influenza virus was successfully developed,of which the detection sensitivity and efficiency were improved.It met the requirement for emergency inspection on influenza vaccine.