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 samples may yield important clinical information.


RESULTS
We developed a 5' exonuclease fluorescent probe assay for HPV 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 to quantify a cellular target of known iteration simultaneously with HPV quantitation, which controls for the amount of input DNA. Dilution series of target and heterologous templates were used to verify the assay. The assay was successfully used on fresh and PreservCyt-fixed cell lines, as well as cervical samples. The linear range of the assay is from 10 to 10 million copies. Intraclass correlations for HPV, actin, and globin assays ranged from 0.95 to 0.99, indicating the analytic precision of repeated measures.


CONCLUSIONS
The method is accurate over a large copy number range, reproducible, type specific, normalized for input DNA quantity, and applicable to PreservCyt-fixed material.
Human Papillomavirus Section, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Public Health Service, US Department of Health and Human Services, Atlanta, GA 30333, USA. ratl@cdc.gov
R A
RA
Tucker
E R
ER
Unger
B P
BP
Holloway
D C
DC
Swan
D004268:DNA-Binding Proteins; D005260:Female; D005456:Fluorescent Dyes; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D009856:Oncogene Proteins, Viral / Q000235:genetics; D027383:Papillomaviridae / Q000235:genetics* / Q000302:isolation & purification*; D030361:Papillomavirus Infections / Q000175:diagnosis / Q000235:genetics* / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012097:Repressor Proteins; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured; D014412:Tumor Virus Infections / Q000175:diagnosis / Q000235:genetics* / Q000821:virology; D002583:Uterine Cervical Neoplasms / Q000175:diagnosis / Q000821:virology
D004268:DNA-Binding Proteins; C068651:E6 protein, Human papillomavirus type 16; C118860:E6 protein, Human papillomavirus type 6; C052633:E7 protein, Human papillomavirus type 18; D005456:Fluorescent Dyes; D009856:Oncogene Proteins, Viral; D012097:Repressor Proteins; C062495:oncogene protein E7, Human papillomavirus type 6