title: A colorimetric microtiter plate PCR system detects respiratory syncytial virus in nasal aspirates and discriminates subtypes A and B.
abstract: We developed a colorimetric microtiter plate (MTP) PCR system for specific detection of the respiratory syncytial virus (RSV) nucleocapsid gene and differentiation of viral subtypes A and B. Of 47 pediatric nasal aspirate specimens, the sensitivity and specificity were 94.4% (17 of 18) and 100% (15 of 15), respectively, when compared with RSV cell culture isolation in HEp-2 cells. An additional 14 specimens positive for adenoviruses, rhinoviruses, influenza, or parainfluenza viruses did not give positive reactions. PCR testing detected a mean of 0.15 (0.01 to 7.00) plaque-forming units of RSV virions. Inhibition of PCR amplification was detected in 33.3% (6/18) of undiluted specimens and could be avoided by a dilution (1:10) of extracted RNA without decreasing test sensitivity. RSV subtype, as determined by allele-specific probes, was identical to that determined by an immunofluorescence assay. These results indicate that the MTP PCR system provides a sensitive and specific test for clinical laboratory diagnosis and simultaneous subgroup classification of RSV infection.
authors.affiliation: Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee 37232-2605, USA.
authors.forename: Y W
authors.initials: YW
authors.lastname: Tang
authors.forename: P J
authors.initials: PJ
authors.lastname: Heimgartner
authors.forename: S J
authors.initials: SJ
authors.lastname: Tollefson
authors.forename: T J
authors.initials: TJ
authors.lastname: Berg
authors.forename: P N
authors.initials: PN
authors.lastname: Rys
authors.forename: H
authors.initials: H
authors.lastname: Li
authors.forename: T F
authors.initials: TF
authors.lastname: Smith
authors.forename: D H
authors.initials: DH
authors.lastname: Persing
authors.forename: P F
authors.initials: PF
authors.lastname: Wright
mesh_terms: D018929:Cell Culture Techniques / Q000379:methods; D002648:Child; D002675:Child, Preschool; D003124:Colorimetry / Q000379:methods; D004526:Efficiency; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D017817:Nasal Lavage Fluid / Q000821:virology*; D018357:Respiratory Syncytial Virus Infections / Q000175:diagnosis*; D012136:Respiratory Syncytial Viruses / Q000145:classification / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000592:standards; D012680:Sensitivity and Specificity