A modified single-tube one-step product-enhanced reverse transcriptase (mSTOS-PERT) assay with heparin as DNA polymerase inhibitor for specific detection of RTase activity.
BACKGROUND
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.


OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Fan
Xiao-Yong
XY
Department of Microbiology, School of Life Sciences, Fudan University, Shanghai 200433, China. xyfan008@hotmail.com
Lü
Guo-Zhen
GZ
Wu
Li-Na
LN
Chen
Jing-Hua
JH
Xu
Wen-Qing
WQ
Zhao
Chun-Nü
CN
Guo
Sheng-Qi
SQ
D002460:Cell Line; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D006493:Heparin / Q000494:pharmacology*; D019384:Nucleic Acid Synthesis Inhibitors; D016133:Polymerase Chain Reaction / Q000379:methods*; D012194:RNA-Directed DNA Polymerase / Q000032:analysis* / Q000378:metabolism; D012190:Retroviridae / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D013698:Templates, Genetic; D014764:Viral Proteins / Q000032:analysis
D019384:Nucleic Acid Synthesis Inhibitors; D014764:Viral Proteins; D006493:Heparin; D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase