HPV 16 DNA and mRNA in cervical brush samples quantified by PCR and microwell hybridization

Abstract

To quantitate HPV 16 DNA and mRNA, biotinylated amplicons from PCR and reverse transcription PCR were captured on streptavidin-coated microtitre plates. The amount of amplicon was determined by colorimetric detection after hybridization with an alkaline phosphatase-labelled probe. Dynamic ranges of between 4 and 6 log 10, sufficient to cover the amounts of viral DNA and mRNA prepared from cervical samples were achieved. The reproducibility of the colorimetric detection step was reflected in coefficients of variation (C.V.) below 8%, considerably better than that of chemiluminescense detection. In a series of 89 HPV 16 DNA positive cervical samples, as compared with a CIN I/normal diagnosis subgroup, the number of HPV 16 genome copies per assay was significantly greater in a CIN II subgroup ( P=0.014), and a high-grade neoplasia subgroup ( P=0.040), and the content of HPV 16 mRNA significantly greater in the high-grade neoplasia subgroup ( P=0.0021). The number of mRNA equivalents per copy of viral DNA was higher for E5 than for the other three mRNA species analyzed ( P<0.001), and the concentration of E6*I mRNA was higher than those of the E6 full-length ( P<0.001) and E6*II ( P<0.001) transcripts. Despite these differences, no correlation was found between histological/cytological diagnosis and the amount of viral mRNA relative to the viral load.