Real-time PCR-based fluorescent assay for quantitation of human papillomavirus types 6, 11, 16, and 18

Background: Quantitation of human papillomavirus (HPV) DNA in clinical samp les may yield important clinical information. Methods and Results: We developed a 5' exonuclease fluorescent probe assay for I-IPV quantitation that uses real-time PCR. The assay was optimized for HPV types 6 (HPV-6), -11, -16, and -18. A multiplex format was developed t o quantify a cellular target of known iteration simultaneously with HPV qua ntitation, which controls for the amount of input DNA. Dilution series of t arget and heterologous templates were used to verify the assay. The assay w as successfully used on fresh and PreservCyt-fixed cell lines, as well as c ervical samples. The linear range of the assay is from 10 to 10 million cop ies. Intraclass correlations for HPV, actin, and globin assays ranged from 0.95 to 0.99, indicating the analytic precision of repeated measures. Conclusion: The method is accurate over a large copy number range, reproduc ible, type specific, normalized for input DNA quantity, and applicable to P reservCyt-fixed material.