title: A quantitative PCR assay for SV40 neutralization adaptable for high-throughput applications.
abstract: 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.
authors.affiliation: Laboratory of DNA Viruses, Division of Viral Products, CBER, FDA, Bethesda, MD 20892, USA. haruhiko.murata@fda.hhs.gov
authors.forename: Haruhiko
authors.initials: H
authors.lastname: Murata
authors.forename: Belete
authors.initials: B
authors.lastname: Teferedegne
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Lewis
authors.forename: Keith
authors.initials: K
authors.lastname: Peden
mesh_terms: D000818:Animals; D057134:Antibodies, Neutralizing / Q000097:blood / Q000276:immunology; D000914:Antibodies, Viral / Q000097:blood / Q000276:immunology; D052160:Benzothiazoles; D002460:Cell Line; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D017931:DNA Primers; D004279:DNA, Viral / Q000235:genetics; D003959:Diamines; D007668:Kidney / Q000166:cytology / Q000821:virology; D009500:Neutralization Tests; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D013539:Simian virus 40 / Q000235:genetics / Q000276:immunology / Q000502:physiology; D013698:Templates, Genetic; D014779:Virus Replication
chemical_list: D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D052160:Benzothiazoles; D017931:DNA Primers; D004279:DNA, Viral; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I