Prevalence of selective inhibition of HPV-16 DNA amplification in cervicovaginal lavages.
HPV-16 viral load has been assessed with real-time PCR assays by measuring HPV-16 DNA and a human gene in genital samples. HPV-16 viral load measurements are thus based on the inference that inhibitors contained in samples will equally impede amplification of DNA sequences from HPV-16 and human DNA. We have previously shown that sample lysates can inhibit amplification of HPV-16 but not beta-globin DNA. In the current study, cervicovaginal lavages lysates considered adequate for PCR analysis by a qualitative beta-globin PCR test, were screened for the presence of inhibitors using internal controls (IC) for HPV-16 DNA and beta-globin in real-time PCR assays. Of 150 lysates screened with both ICs, 12 (8%) contained inhibitors. Inhibition of amplification of both ICs was demonstrated in four of these specimens. In eight lysates, amplification of HPV-16 IC only was impeded. Six (50%) of these 12 lysates tested positive for HPV-16 DNA despite the presence of PCR inhibitors. The HPV-16 viral load increased significantly after dilution of 11 of 12 lysates, demonstrating the presence of inhibitors in the undiluted lysate. Nine (90%) of 10 samples with inhibitors that were tested after dilution did not demonstrate inhibitory activity. The use of internal controls in real-time PCR is clearly essential to determine HPV viral loads since the effect of inhibitors on primer-driven genomic amplification is variable.
Lefevre
Jonas
J
Laboratoire de Virologie Moléculaire, Centre de Recherche du Centre Hospitalier de l'Université de Montréal, and Dépatrement de Microbiologie et Immunologie, Université de Montréal, Québec, Canada.
Hankins
Catherine
C
Pourreaux
Karina
K
Voyer
Hélène
H
Coutlée
François
F
D002584:Cervix Uteri / Q000821:virology*; D004247:DNA / Q000032:analysis; D004279:DNA, Viral / Q000032:analysis*; D005260:Female; D005914:Globins / Q000235:genetics; D006801:Humans; D027383:Papillomaviridae / Q000235:genetics / Q000302:isolation & purification*; D030361:Papillomavirus Infections / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods / Q000592:standards; D002583:Uterine Cervical Neoplasms / Q000821:virology; D014621:Vagina / Q000821:virology*; D044364:Vaginal Douching; D019562:Viral Load
D004279:DNA, Viral; D005914:Globins; D004247:DNA