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

Applicability validation and application of Digoxigenin-labeled DNA probe in detection of residual DNA by hybridization in Vero cell

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。

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,sensitivity,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 efficiency 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 《Pharmarcopoeia 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.