title: Quantitation of enteroviral RNA by competitive polymerase chain reaction.
abstract: The polymerase chain reaction (PCR) is a new diagnostic technique for the detection of enteroviral infection; however, it currently provides only qualitative results. The aim of this study was to adapt PCR for the accurate quantitation of enteroviral RNA in clinical specimens. For this purpose, we designed a standard RNA which was homologous to sequences at the 5' end of the coxsackie B3 enterovirus genome but contained a single-base-pair mutation which created a novel internal restriction site. Serial dilutions of this standard template RNA were mixed with a fixed concentration of coxsackie B3 enterovirus RNA. The viral and standard templates were reversed transcribed to cDNA and coamplified by PCR, and a comparison of the radioactive PCR products was made. Since the templates were both present in a single reaction tube and competed for the same primers, the ratio of products remained proportional throughout the amplification process. By this approach, a fourfold-difference in viral titer was clearly distinguishable. Moreover, we were able to accurately quantitate as few as 15 50% tissue culture infectious doses, which reflects common clinical viral titers. This study lays the foundation for quantitation of enteroviral RNA in clinical specimens and establishes a technique that can readily be applied to the diagnosis of enteroviral infection.
authors.affiliation: Center for Cardiovascular Research, Toronto Hospital, Ontario, Canada.
authors.forename: T A
authors.initials: TA
authors.lastname: Martino
authors.forename: M J
authors.initials: MJ
authors.lastname: Sole
authors.forename: L Z
authors.initials: LZ
authors.lastname: Penn
authors.forename: C C
authors.initials: CC
authors.lastname: Liew
authors.forename: P
authors.initials: P
authors.lastname: Liu
mesh_terms: D001483:Base Sequence; D004770:Enterovirus / Q000235:genetics*; D004769:Enterovirus Infections / Q000175:diagnosis; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000032:analysis*; D012680:Sensitivity and Specificity
chemical_list: D012367:RNA, Viral