Real-time PCR assays using internal controls for quantitation of HPV-16 and β-globin DNA in cervicovaginal lavages

High-risk human papillomavirus 16 (HPV-16) DNA viral load has been measured with real-time PCR assays by amplifying HPV-16 and a human gene. However, these assays have not used internal controls (ICs) to screen for the presence of inhibitors contained in samples. To quantitate HPV-16 DNA and cell content with real-time PCR, ICs for HPV-16 DNA and β-globin were synthesised and used to control for inhibition. The assays were sensitive and linear over 5 logs. Good reproducibility was achieved with inter-run coefficients of variation of 23% (102 HPV-16 copies), 12% (10 4 HPV-16 copies), 17% (274 β-globin DNA copies) and 7% (27400 β-globin DNA copies). Samples containing 56800000, 306000, 18000, and 4070 HPV-16 copies/μg of cellular DNA were tested blindly and estimated to contain 48800000, 479000, 20300, and 6620 HPV-16 copies/μg of DNA (mean ratio of measured to expected viral load of 1.27±0.32). Inhibition of amplification of HPV-16 and β-globin ICs by six samples known to contain PCR inhibitors was variable: four inhibited both ICs while two inhibited only the HPV-16 IC. The use of internal controls with real-time PCR for HPV-16 quantitation allows to screen for the presence of inhibitors that do not affect equally primer-driven genomic amplification. © 2003 Elsevier B.V. All rights reserved.