中国生产用细胞基质污染支原体类型的分析及猪鼻支原体特异性检测方法的初步建立

Analysis of mycoplasma species responsible for contamination of cell substrates used for production of biologicals in China and primary development of specific detection method for Mycoplasma hyorhinis

目的分析我国生物制品生产用细胞基质中污染的支原体的类型,并建立猪鼻支原体感染特异性检测方法。方法对半巢式PCR法的退火温度及引物进行优化,利用优化后方法对70份经培养法及染色法确证为支原体阳性的生产用细胞基质培养上清进行PCR检测及序列分析;用支原体特异的抗生素Plasmocin TM处理猪鼻支原体阳性细胞株NCI-H1703 1~3周后,收集上清,进行优化后方法的特异性验证。使用猪鼻支原体特异抗体PD-4对感染不同类型支原体及不同稀释度的猪鼻支原体的Vero细胞进行检测验证。结果 70份样本中60份PCR检测阳性,其中猪鼻支原体（44份）占62.9%,其他分别为口腔支原体（6份）、精氨酸支原体（3份）、肺支原体（2份）、人型支原体（2份）、滑液囊支原体（1份）、发酵支原体（1份）、牛鼻支原体（1份）;优化后的方法检测支原体为阴性,且阴性结果随药物持续作用而维持不变,可特异地监控支原体增殖活性。猪鼻支原体特异抗体PD-4可特异地检测猪鼻支原体污染。结论猪鼻支原体是中国生物制品生产用细胞基质的主要支原体污染源,初步建立的猪鼻支原体感染的有效特异检测方法,可明显提高与支原体污染相关的质量控制能力。

Objective To analyze the major mycoplasma species responsible for contamination of cell substrates used for production of biologicals in China and develop a method for specific detection of Mycoplasma hyorhinis. Methods The annealing temperature and primers of semi-nested PCR-based method were optimized, and 70 mycoplasma-positive culture supernatant of cell substrates confirmed by culture method and staining method were detected by the optimized PCR method combined with DNA sequencing. M. hyorhinis-positive NCI-H1703 cell strain was treated with mycoplasmaspecific PlasmocinTMfor 1 ~ 3 weeks, of which the supernatant was collected for verification of specificity of the optimized method. The Vero cells infected with various mycoplasma species and M. pulmonis at various dilutions were detected by M. pulmonis-specific antibody PD-4. Results Sixty of the 70 samples were tested as positive for contamination with eight mycoplasma species by PCR, among which 44（62. 9%） were infected with M. pulmonis, while 6 with M. orale, 3 with M. arginini, 2 with M. pulmonis, 2 with human mycoplasma, one with M. synoviae, one with M. fermentans, and one with M. bovirhinis. The negative result proved by the optimized method showed no change after continuous treatment with drug,indicating that the proliferative activity of mycoplasma might be monitored specifically. M. pulmonis contamination was specifically detected by antibody PD-4. Conclusion M. pulmonis is the most prevalent mycoplasma species responsible for the contamination of cell substrates used for production of biologicals in China. An effective and specific detection method for M. pulmonis was developed preliminarily, which greatly improved the quality control capability for mycoplasma contamination in China.