title: Faster quantitative real-time PCR protocols may lose sensitivity and show increased variability.
abstract: Quantitative real-time PCR has become the method of choice for measuring mRNA transcription. Recently, fast PCR protocols have been developed as a means to increase assay throughput. Yet it is unclear whether more rapid cycling conditions preserve the original assay performance characteristics. We compared 16 primer sets directed against Epstein-Barr virus (EBV) mRNAs using universal and fast PCR cycling conditions. These primers are of clinical relevance, since they can be used to monitor viral oncogene and drug-resistance gene expression in transplant patients and EBV-associated cancers. While none of the primers failed under fast PCR conditions, the fast PCR protocols performed worse than universal cycling conditions. Fast PCR was associated with a loss of sensitivity as well as higher variability, but not with a loss of specificity or with a higher false positive rate.
authors.affiliation: Department of Microbiology and Immunology and Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, NC, USA.
authors.forename: Chelsey
authors.initials: C
authors.lastname: Hilscher
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Vahrson
authors.forename: Dirk P
authors.initials: DP
authors.lastname: Dittmer
mesh_terms: D017931:DNA Primers; D004854:Herpesvirus 4, Human / Q000235:genetics; D025202:Molecular Diagnostic Techniques / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013997:Time Factors; D014158:Transcription, Genetic
chemical_list: D017931:DNA Primers; D012333:RNA, Messenger