HPV-16 DNA AND MESSENGER-RNA IN CERVICAL BRUSH SAMPLES QUANTIFIED BY PCR AND MICROWELL HYBRIDIZATION

To quantitate HPV 16 DNA and mRNA, biotinylated amplicons from PCR and reverse transcription PCR were captured on streptavidin-coated microt itre plates. The amount of amplicon was determined by colorimetric det ection after hybridization with an alkaline phosphatase-labell ed prob e. Dynamic ranges of between 4 and 6 log(10), sufficient to cover the amounts of viral DNA and mRNA. prepared from cervical samples were ach ieved. The reproducibility of the colorimetric detection step was refl ected in coefficients of variation (C.V.) below 8%, considerably bette r than that of chemiluminescense detection. In a series of 89 HPV 16 D NA positive cervical samples, as compared with a CIN I/normal diagnosi s subgroup, the number of HPV 16 genome copies per assay was significa ntly greater in a CIN II subgroup (P = 0.014), and a high-grade neopla sia 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 concentrati on of E6I mRNA was higher than those of the E6 full-length (P < 0.001 ) and EGII (P < 0.001) transcripts. Despite these differences, no cor relation was found between histological/cytological diagnosis and the amount of viral mRNA relative to the viral load. (C) 1997 Elsevier Sci ence Ireland Ltd.