A quantitative PCR assay for SV40 neutralization adaptable for high-throughput applications

A neutralization assay incorporating a quantitative SYBR Green PCR endpoint has been developed for SV40. The present study demonstrates that crude virus samples can serve as suitable amplification templates for quantitative PCR without the need for nucleic acid extraction. The denaturation temperatu...

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Bibliographic Details
Published in:Journal of virological methods 2009-12, Vol.162 (1), p.236-244
Main Authors: Murata, Haruhiko, Teferedegne, Belete, Lewis, Andrew M., Peden, Keith
Format: Article
Language:English
Subjects:
Animals
Antibodies, Neutralizing - blood
Antibodies, Neutralizing - immunology
Antibodies, Viral - blood
Antibodies, Viral - immunology
Antibody
Biological and medical sciences
Cell Line
Cells, Cultured
Cercopithecus aethiops
DNA Primers
DNA, Viral - genetics
Fundamental and applied biological sciences. Psychology
Kidney - cytology
Kidney - virology
Microbiology
Neutralization
Neutralization Tests
Organic Chemicals
Polymerase Chain Reaction - methods
Polyomavirus
Quantitative PCR
Simian virus 40
Simian virus 40 - genetics
Simian virus 40 - immunology
Simian virus 40 - physiology
SV40
SYBR Green
Techniques used in virology
Templates, Genetic
Virology
Virus Replication
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Description
Summary:A neutralization assay incorporating a quantitative SYBR Green PCR endpoint has been developed for SV40. The present study demonstrates that crude virus samples can serve as suitable amplification templates for quantitative PCR without the need for nucleic acid extraction. The denaturation temperature of thermocycling appears to be sufficient to release the encapsidated viral genome and allow its availability as a PCR template. Issues arising from inhibitors of PCR present in crude virus samples can be circumvented easily by a 100-fold dilution step. Using a streamlined procedure that eliminates sample nucleic acid extraction (a hitherto rate-limiting step that diminishes throughput substantially), quantitative PCR was applied in order to assess: (1) the replication kinetics of SV40 and (2) the inhibition of SV40 productive infection by neutralizing antibodies. A similar high-throughput approach might be feasible for related polyomaviruses (e.g., BKV and JCV) as well as for other families of viruses.
ISSN:0166-0934
1879-0984
DOI:10.1016/j.jviromet.2009.08.012