Real-time PCR assays using internal controls for quantitation of HPV-16 and beta-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 beta-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% (10(2) HPV-16 copies), 12% (10(4) HPV-16 copies), 17% (274 beta-globin DNA copies) and 7% (27,400 beta-globin DNA copies). Samples containing 56,800,000, 306,000, 18,000, and 4,070 HPV-16 copies/microg of cellular DNA were tested blindly and estimated to contain 48,800,000, 479,000, 20,300, and 6,620 HPV-16 copies/microg of DNA (mean ratio of measured to expected viral load of 1.27+/-0.32). Inhibition of amplification of HPV-16 and beta-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.
Lefevre
Jonas
J
Laboratoire de Virologie Moléclaire, Centre de Recherche du Centre Hospitalier de l'Université de Montréal, Montréal, Qué., Canada.
Hankins
Catherine
C
Pourreaux
Karina
K
Voyer
Hélène
H
Coutlée
François
F
D002584:Cervix Uteri / Q000821:virology*; D004279:DNA, Viral / Q000032:analysis*; D005260:Female; D005914:Globins / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D027383:Papillomaviridae / Q000235:genetics / Q000302:isolation & purification*; D030361:Papillomavirus Infections / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D015203:Reproducibility of Results; D019914:Taq Polymerase; D002583:Uterine Cervical Neoplasms / Q000821:virology; D014621:Vagina / Q000821:virology*; D044364:Vaginal Douching; D019562:Viral Load
D004279:DNA, Viral; D005914:Globins; D019914:Taq Polymerase