地高辛标记DNA探针杂交法检测Vero细胞DNA残留量的适用性验证及应用

摘要

Objective Objective To detect the residual DNA quantity in Vero cell contained in rabies vaccine( Vero cell) for human use(simplified as rabies vaccine )by Digoxigenin-labeled DNA probe,and the applicability of the method was verified and applied. Methods The Digoxigenin-labeled DNA probe was carried out in verification of the specificity,sensi-tivity,repeatability and stability for detecting the residual DNA quantity in Vero cell contained in rabies vaccine,and the method was applied to detect residual DNA quantity in Vero cell of 3 batches of rabies vaccine. Results The labeling effi-ciency could reach 0. 1 pg. The verification result showed that there was no hybridazation between the labeled probe and heterologous DNA,and the minimal detective limit reached 1. 0 pg and the detective sensitivity was kept in 1. 0 pg after the labeled probe stored at-20℃ for 7 months. The results conformed to the quality control standards published in《Pharmar-copoeia of the People’ s Republic of China》in detecting residual DNA quantity in Vero cell in 3 batches of rabies vaccine. Conclusion The dot hybridization method is specific,sensitive,stable and well repeatable,it is used in detecting residual DNA quantity in Vero cell contained in rabies vaccine. As a result,the method is suitable in production and quantity control of rabies vaccine,it has a helpful effect in preparation of the other viral vaccines by using Vero cell as a substrate.%目的：对地高辛标记DNA探针杂交法检测人用狂犬病疫苗( Vero细胞) DNA残留量进行适用性验证及应用。方法对地高辛标记DNA探针杂交法检测人用狂犬病疫苗( Vero细胞) DNA残留量进行特异性、灵敏度及稳定性验证，并应用该方法检测3批人用狂犬病疫苗( Vero细胞)的DNA残留量。结果地高辛标记探针的标记效率为0.1 pg。验证结果显示探针与非同源DNA无杂交；最低检测限度为1 pg；探针在-20℃放置7个月后，检测灵敏度仍可达到1 pg；3批人用狂犬病疫苗( Vero细胞)中残留DNA含量均符合《中国药典》规定质量控制标准。结论地高辛标记DNA探针杂交法特异性、灵敏度好，结果稳定，适用于人用狂犬病疫苗( Vero细胞)中Vero细胞DNA残留量的检测及疫苗生产过程和其成品的质量控制，对其他以Vero细胞为基质的病毒性疫苗质量控制具有借鉴意义。