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

Abstract

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 presence of human papillomavirus (HPV) DNA amplified from clinical specimens. After screening with this new generic assay is performed, HPV DNA-positive samples can be directly genotyped using a reverse blotting method with product from the same PCR amplification. DNA from 287 genital specimens was amplified via PCR using biotin-labeled consensus primers directed to the L1 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 L1 fragments from types 11, 16, 18, and 51. Coamplification and detection of human DNA with biotinylated ␤-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 and a DIG-labeled HPV generic probe mix (DIG-MWP generic probe assay) were compared with results from a previous analysis using dot blots with a radiolabeled nested generic probe mix and type-specific probes for genotyping. The DIG-MWP generic probe assay resulted in high intralaboratory concordance in genotyping results (88% versus 73% agreement using traditional methods). There were 207 HPV-positive results using the DIG-MWP method and 196 positives using the radiolabeled generic probe technique, suggesting slightly improved 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 that were positive or borderline when tested with the DIG-MWP generic probe assay were not detected with the HPV type-specific panel, perhaps representing 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.Some types of human papillomavirus (HPV) are widely accepted as causative agents for cervical cancer (3,19). There are more than 40 HPV viral types that are commonly found in the genital tract, and approximately one-third of these are associated with cervical cancer and anal neoplasia. The anogenital HPV types are generally categorized as being either "high risk" or "low risk." High-risk types are associated with high-grade precancerous lesions and invasive cancer, while low-risk types are found in asymptomatic or benign conditions such as genital warts. However, the distribution and prevalence of types vary somewhat by geographic region and other demographic factors. Because the significance of the variation in type distribution is still being elucidated, studies of HPV epidemiology need to employ a methodology that can detect the entire spectrum of viral types. One of the most common means to detect and characterize new HPVs has been by PCR using consensus primers...

Inclusion of HPV testing in routine cervical cancer screening for women above 29 years in Germany: results for 8466 patients

“…One contributing factor to the low specificity is the crossreactivity of the HC2 high-risk hybridisation probe with low-risk HPV types or HPV types of undetermined risk as discussed in earlier studies (Peyton et al, 1998;Vernon et al, 2000;Solomon et al, 2001). Here we found a substantial degree of crossreactivity (6.4%) between the HC2 high-risk probe and HPV types not included in the probe cocktail by retesting all HC2 þ samples with PCR followed by direct sequencing and by additional testing with the PGMY09/11-PCR system coupled to a reverse hybridisation line-blot assay (Kornegay et al, 2001) to be able to detect multiple infections. If the problem of crossreactivity theoretically could be eliminated, this would have increased the specificity of the HC2 test up to 97.6% without losing sensitivity (data not shown).…”

Evaluation of the SPF 10 -INNO LiPA Human Papillomavirus (HPV) Genotyping Test and the Roche Linear Array HPV Genotyping Test

“…An advantage of the LiPA is the use of the same amplicon for both detection of 43 different lr-, probable hr-, and hr-HPV genotypes and genotyping of 25 different HPVs. For the LA, a prescreening test with the PGMY primers is available using a generic HPV probe labeled with digoxigenin in a microtiter plate-based assay, as recently described (18). Without the need for further amplification, this amplicon can be directly used for the Linear Array genotyping assay.…”

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Correlates of Human Papillomavirus Viral Load with Infection Site in Asymptomatic Men

“…DNA extracted from swabbed cellular material and semen was used for HPV testing by PCR for amplification of a fragment of the L1 gene using the PGMY system (19). HPV genotyping was conducted using the reverse line blot method (20). This detection method uses the HPV L1 consensus PCR products labeled with biotin to detect 37 HPV types.…”