Fan Xiao-Yong, Lü Guo-Zhen, Wu Li-Na, Chen Jing-Hua, Xu Wen-Qing, Zhao Chun-Nü, Guo Sheng-Qi
Department of Microbiology, School of Life Sciences, Fudan University, Shanghai 200433, China.
J Clin Virol. 2006 Dec;37(4):305-12. doi: 10.1016/j.jcv.2006.08.007. Epub 2006 Sep 12.
Current regulations and recommendations proposed for the production of vaccines in continuous cell lines of any origin demand that these be free of exogenous viruses, particularly retroviruses. Recently, the ultra-sensitive product-enhanced reverse transcriptase (PERT) assay can be used to detect minute of reverse transcriptase (RTase) in single retroviral particle and is 10(6) times more sensitive than the conventional RTase assays. However, coincidental with this increase in sensitivity is an increase in false-positive reactions derived from contaminating cellular DNA polymerases, which are known to have RTase-like activities.
To develop a modified single-tube one-step PERT (mSTOS-PERT) assay with improvements on decreasing significantly the level of false-positive reactions, and to evaluate the mSTOS-PERT assay for sensitivity and specificity.
Ampliwaxtrade mark was used to compartmentalize the reverse transcription (RT) and PCR step in the same micro-tube with more efficiency and reproducibility, while maintaining the high sensitivity. The DNA amplification products were separated by 2% agarose gel electrophoresis, and then analyzed by non-isotopic Southern blot hybridization. A wide variety of cell lines used in biologicals production were detected to validate the improved mSTOS-PERT assay.
The detection limit for the mSTOS-PERT assay was at least 10(-9)U, when using AMV-RTase as a positive control. Furthermore, heparin involvement in the RT step can eliminate completely the false-positive PERT signals which are exhibited by cellular polymerases such as DNA-dependent DNA polymerase alpha, gamma released by cell death. Most mammalian cells (MRC-5, Vero, WISH, 2BS, RK-13, MDCK, etc.) are PERT-negative in cell supernatants. Some PERT-positive signals in cell lysates were found to be introduced by the cellular DNA polymerases and could be inhibited specifically by heparin. Chick cells derived from either chick embryo fibroblasts (CEF) or allantoic fluid from SPF embryonated eggs, murine hybridoma cell SP2/0, etc., contained authentic RTase activities, which could not be inactivated by heparin.
The improved mSTOS-PERT assay described here may distinguish the genuine RTase activity from cellular polymerases with high sensitivity and specificity, and is rapid and easy to perform to screen for the possible contamination of minute retroviruses in the cell substrates used in vaccine production.
目前针对任何来源的连续细胞系生产疫苗所提出的法规和建议要求这些细胞系不含外源病毒，尤其是逆转录病毒。最近，超灵敏的产物增强逆转录酶（PERT）检测法可用于检测单个逆转录病毒颗粒中的微量逆转录酶（RTase），其灵敏度比传统的RTase检测法高10^6倍。然而，随着灵敏度的提高，来自污染的细胞DNA聚合酶的假阳性反应也增加了，已知这些DNA聚合酶具有类似RTase的活性。
开发一种改良的单管一步PERT（mSTOS-PERT）检测法，显著降低假阳性反应水平，并评估mSTOS-PERT检测法的灵敏度和特异性。
使用Ampliwax商标在同一微管中对逆转录（RT）和PCR步骤进行分隔，效率更高且可重复性更好，同时保持高灵敏度。DNA扩增产物通过2%琼脂糖凝胶电泳分离，然后通过非同位素Southern印迹杂交进行分析。检测了生物制品生产中使用的多种细胞系，以验证改进后的mSTOS-PERT检测法。
以禽成髓细胞瘤病毒逆转录酶（AMV-RTase）作为阳性对照时，mSTOS-PERT检测法的检测限至少为10^(-9)U。此外，在RT步骤中加入肝素可以完全消除细胞死亡释放的细胞聚合酶（如依赖DNA的DNA聚合酶α、γ）所呈现的假阳性PERT信号。大多数哺乳动物细胞（MRC-5、Vero、WISH、2BS、RK-13、MDCK等）的细胞上清液PERT检测呈阴性。在细胞裂解物中发现的一些PERT阳性信号是由细胞DNA聚合酶引入的，并且可以被肝素特异性抑制。源自鸡胚成纤维细胞（CEF）或无特定病原体（SPF）胚胎鸡蛋尿囊液的鸡细胞、鼠杂交瘤细胞SP2/0等含有真实的RTase活性，不能被肝素灭活。
本文所述的改进后的mSTOS-PERT检测法可以高灵敏度和特异性区分真正的RTase活性与细胞聚合酶，并且快速简便，可用于筛选疫苗生产中所用细胞底物中可能存在的微量逆转录病毒污染。
