支原体检测PCR法与药典方法的对比分析

Comparative analysis of PCR and method in Chinese Pharmacopoeia for mycoplasma examination

目的研究PCR技术在疫苗生产过程支原体检测中应用的可行性。方法选择GP03和MGSO作为引物,与《中国药典》三部(2015)规定的3种支原体阳性株(肺炎支原体、口腔支原体和猪鼻支原体)进行16s r RNA序列比对分析和特异性扩增检测,确定引物适用性。以支原体阳性株为PCR模板,进行浓度梯度试验和不同代次扩增,确认PCR法的检测限和精密度。以口服轮状病毒活疫苗生产过程中20个细胞培养物为样品,分别采用PCR法、液体培养法和固体培养法检测,对比三种方法的检测结果。结果GP03和MGSO适用于支原体检测。PCR法与液体培养法、固体培养法检测结果一致,同时具备操作简单和检出结果快速的特点。结论PCR技术可作为疫苗生产过程中检测支原体的一种手段。

Objective To investigate the feasibility of PCR technique used for mycoplasma examination in the vaccine production process.Methods GP03 and MGSO were selected as primers,of which the sequences were compared with the 16 s r RNA sequences of M.pneumonia,M.orale and M.hyorhinis specified in Chinese Phamacopoeia(VolumeⅢ,2015 edition)for mycloplasma examination.The 16 s r RNA sequences of three mycoplasma positive strains were amplified by PCR using the two primers to determine the primer suitability.Concentration gradient test and amplification test of dif-ferent passages of mycoplasma were carried out using the mycoplasma positive strains as templates to confirm the detection limit and precision of PCR method respectively.Twenty cell cultures in the production process of oral live attenuated ro-tavirus vaccine were detected by PCR method,liquid culture method and solid culture method respectively,and the results were compared.Results GP03 and MGSO were suitable for mycoplasma examination.The detection result by PCR method was consistent with those by liquid and solid culture methods.However,PCR method was rapid and easy to handle.Conclusion PCR may be used as a method for mycoplasma examination in vaccine production.