A real time quantitative PCR-based method for the detection and quantification of simian virus 40.

A real time quantitative PCR-based simian virus 40 (SV40) detection and quantification method has been developed. This method takes advantage of the 5' to 3'-exonuclease activity of Taq DNA polymerase and utilizes the PRISM 7700 sequence detection system of PE Applied Biosystems for direct monitoring of PCR product accumulation through a dual-labelled fluorogenic probe. This method provides accurate, precise and reproducible quantification of SV40 DNA over a linear dynamic range of at least 100,000-fold with a minimum detection level of 6.4 copy equivalents/microL of SV40 viral particle in test samples. The sample preparation procedure employed allows for efficient and consistent recovery of SV40 DNA from test samples. High concentrations of protein and cellular DNA presenting in test samples have been demonstrated to have no impact on SV40 quantification. This method offers significant advantages over other PCR methods and cell-based infectivity assays currently available for SV40 detection and quantification. The availability of this method should greatly facilitate the pathogenic investigation of SV40, as well as viral clearance evaluations required for the development of new biological products.

[1]  D. Livingston,et al.  Mammalian Cells in Culture Frequently Release Type C Viruses , 1973, Science.

[2]  J. Lifson,et al.  Plasma SIV RNA viral load determination by real-time quantification of product generation in reverse transcriptase-polymerase chain reaction. , 1998, AIDS research and human retroviruses.

[3]  C. Cicala,et al.  SV40 induces mesotheliomas in hamsters. , 1993, The American journal of pathology.

[4]  S. Kwok,et al.  Quantitation of HIV-1 proviral DNA relative to cellular DNA by the polymerase chain reaction. , 1990, Analytical biochemistry.

[5]  H. Vinters,et al.  High levels of unintegrated HIV-1 DNA in brain tissue of AIDS dementia patients , 1990, Nature.

[6]  Y. Xu,et al.  Real time quantitative PCR as a method to evaluate simian virus 40 removal during pharmaceutical protein purification. , 1999, Biologicals : journal of the International Association of Biological Standardization.

[7]  K. Livak,et al.  Real time quantitative PCR. , 1996, Genome research.

[8]  S. Stenton.  Asbestos, Simian virus 40 and malignant mesothelioma , 1997, Thorax.

[9]  J. Dahlberg,et al.  Structure-specific endonucleolytic cleavage of nucleic acids by eubacterial DNA polymerases. , 1993, Science.

[10]  J. Allain,et al.  High throughput PCR detection of HCV based on semiautomated multisample RNA capture. , 1997, Journal of virological methods.

[11]  K. Livak,et al.  Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. , 1995, PCR methods and applications.

[12]  G. E. Grubbs,et al.  Identification of the oncogenic substance in rhesus monkey kidney cell culture as simian virus 40. , 1962, Virology.

[13]  J. Lifson,et al.  Quantitative competitive polymerase chain reaction for accurate quantitation of HIV DNA and RNA species. , 1993, BioTechniques.

[14]  R. Garcea,et al.  DNA sequences similar to those of simian virus 40 in ependymomas and choroid plexus tumors of childhood. , 1992, The New England journal of medicine.

[15]  H. Pass,et al.  Simian virus 40, poliovaccines and human tumors: a review of recent developments , 1997, Oncogene.

[16]  B. Robertson,et al.  Rapid reverse transcription-PCR detection of hepatitis C virus RNA in serum by using the TaqMan fluorogenic detection system , 1996, Journal of clinical microbiology.

[17]  J. Goedert,et al.  Contamination of poliovirus vaccines with simian virus 40 (1955-1963) and subsequent cancer rates. , 1998, JAMA.

[18]  R. Abramson,et al.  Detection of specific polymerase chain reaction product by utilizing the 5'----3' exonuclease activity of Thermus aquaticus DNA polymerase. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[19]  C. Heid,et al.  A novel method for real time quantitative RT-PCR. , 1996, Genome research.