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Development and evaluation of a quantitative real time reverse transcriptase polymerase chain reaction assay for respiratory syncytial virus



Development and evaluation of a quantitative real time reverse transcriptase polymerase chain reaction assay for respiratory syncytial virus



Abstracts of the Interscience Conference on Antimicrobial Agents & Chemotherapy 43: 498



Background: Evaluation of RSV antivirals and understanding pathogenesis requires quantitative RSV testing. Plaque assay is currently used. However, RTrtPCR offers possible greater sensitivity, stability after freeze/thaw, and lower cost, thus facilitating multi center studies. Methods: N and F gene primers/probes were developed from conserved regions (GeneBank). An extraction technique for resp secretions was followed by RT and separate PCR using an ABI 7900HT. RSV-A patient aliquots frozen from 2-30 months at -80degreeC, but quantified fresh using plaque assay were analyzed. Standards were prepared by parallel RNA extraction of RSV-A long culture and electron microscopic standards. Results: RSV quantity measured by RTrtPCR (N-gene) in spiked nasal washings correlated with predicted values (R2=0.96). The lower limit of detection of F was inferior to N gene (101.15 PFU/mL). RSV quantity in cultured patient isolates (A) and in frozen resp fluid (B) by RTrtPCR correlated with fresh plaque assay. Assay reproducibility on resp fluid was high (C) and was unaffected by freeze thaw. Conclusions: RSV-A quantification by RTrtPCR of N-gene is reproducible, has a broad range of detection, and correlates well with fresh plaque assay in cultured patient isolates. This correlation is less robust in resp fluid, likely due to non-infectious particles.

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