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Jonas Lefevre; Catherine Hankins; Karina Pourreaux; Hélène Voyer; François Coutlée; Janet Conners; Rob Grimshaw; David Haase; Lynn Johnston; Wally Schlech; et al. (Profiled Author: Paula K.A. Braitstein)
Journal of Medical Virology. 2004;72(1):132-137.Abstract
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 β-globin DNA. In the current study, cervicovaginal lavages lysates considered adequate for PCR analysis by a qualitative β-globin PCR test, were screened for the presence of inhibitors using internal controls (IC) for HPV-16 DNA and β-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. © 2004 Wiley-Liss, Inc.
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