A colorimetric microtiter plate PCR system detects respiratory syncytial virus in nasal aspirates and discriminates subtypes A and B

• Published in: Diagnostic microbiology and infectious disease Vol. 34; no. 4; pp. 333 - 337
• Main Authors: Tang, Yi-Wei, Heimgartner, Paul J, Tollefson, Sharon J, Berg, Timothy J, Rys, Paul N, Li, Haijing, Smith, Thomas F, Persing, David H, Wright, Peter F
• Format: Journal Article
• Language: English
• Published: New York, NY Elsevier Inc 01.08.1999; Elsevier
• Subjects: Biological and medical sciences; Cell Culture Techniques - methods; Child; Child, Preschool; Colorimetry - methods; Efficiency; Enzyme-Linked Immunosorbent Assay - methods; Fundamental and applied biological sciences. Psychology; Human viral diseases; Humans; Infant; Infant, Newborn; Infectious diseases; Medical sciences; Microbiology; Nasal Lavage Fluid - virology; Respiratory Syncytial Virus Infections - diagnosis; Respiratory Syncytial Viruses - classification; Respiratory Syncytial Viruses - isolation & purification; Reverse Transcriptase Polymerase Chain Reaction - methods; Reverse Transcriptase Polymerase Chain Reaction - standards; Sensitivity and Specificity; Techniques used in virology; Viral diseases; Viral diseases of the respiratory system and ent viral diseases; Virology; Human; Performance evaluation; Human respiratory syncytial virus; Pneumovirinae; Microtiter plate; Respiratory disease; Secretion; Nucleocapsid; Colorimetry; Method; Differential diagnostic; Paramyxoviridae; Infection; Virus; Polymerase chain reaction; Mononegavirales; Nose; Viral disease; ENT disease; Serotype; Pneumovirus; Detection; Child
• ISSN: 0732-8893; 1879-0070
• DOI: 10.1016/S0732-8893(99)00049-8

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.