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.
Shi
L
L
Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA.
Ho
J
J
Norling
L A
LA
Roy
M
M
Xu
Y
Y
D004279:DNA, Viral / Q000302:isolation & purification; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D011933:Reagent Kits, Diagnostic; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013539:Simian virus 40 / Q000302:isolation & purification*; D019914:Taq Polymerase / Q000378:metabolism
D004279:DNA, Viral; D011933:Reagent Kits, Diagnostic; D019914:Taq Polymerase; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V