Nonisotopic detection of human papillomavirus DNA in clinical specimens using a consensus PCR and a generic probe mix in an enzyme-linked immunosorbent assay format

We assessed the value of a new digoxigenin (DIG)-labeled generic probe mix in a PCR-enzyme-linked immunosorbent assay format to screen for the presenc e of human papillomavirus (RPV) DNA amplified from clinical specimens. Afte r screening with this new generic assay is performed, HPV DNA-positive samp les can be directly genotyped using a reverse blotting method with product from the same PCR amplification. DNA from 287 genital specimens was amplifi ed via PCR using biotin-labeled consensus primers directed to the Ll gene. HPV amplicons were captured on a streptavidin-coated microwell plate (MWP) and detected with a DIG-labeled HPV generic probe mix consisting of nested Ll fragments from types 11, 16, 18, and 51. Coamplification and detection o f human DNA with biotinylated P-globin primers served as a control for both sample adequacy and PCR amplification. All specimens were genotyped using a reverse line blot assay (13). Results for the generic assay using MWPs an d a DIG-labeled HPV generic probe mix (DIG-MWP generic probe assay) were co mpared with results from a previous analysis using dot blots with a radiola beled nested generic probe mix and type-specific probes for genotyping. The DIG-MWP generic probe assay resulted in high intralaboratory concordance i n genotyping results (88% versus 73% agreement using traditional methods). There were 207 HPV-positive results using the DIG-MWP method and 196 positi ves using the radiolabeled generic probe technique, suggesting slightly imp roved sensitivity. Only one sample failed to test positive with the DIG-MWP generic probe assay in spite of a positive genotyping result. Concordance between the two laboratories was nearly 87%. Approximately 6% of samples th at were positive or borderline when tested with the DIG-MWP generic probe a ssay were not detected with the RPV type-specific panel, perhaps representi ng very rare or novel HPV types. This new method is easier to perform than traditional generic probe techniques and uses more objective interpretation criteria, making it useful in studies of HPV natural history.