One-step detection and genotyping of human papillomavirus in cervical samples by reverse hybridization

This study describes a nonisotopic polymerase chain reaction-reverse hybridization-based method (PCR-RH) for the one-step detection and genotyping of anogenital human papillomavirus (HPV) in a microwell format. HPV DNA was amplified and labeled by PCR using GP5+/GP6+ primers. Labeled amplicons were hybridized to 20 HPV type-specific capture probes anchored to the surface of plastic microwells and detected by an immunoenzymatic assay. Assay sensitivity was <50 pg labeled amplicon, and no cross-reactivity was observed, as determined by hybridizing serial dilutions of labeled PCR products to either matched or mismatched capture probes. The assay was tested on 66 clinical samples (23 specimens with normal histology, I fibropapilloma, 26 cervical intraepithelial neoplasia grade 1 [CIN1], 9 CIN2, and 7 CIN3) and compared with a method based on restriction fragment length polymorphism (RFLP) of PCR products. PCR-RH and PCR-RFLP performed equally well on clinical samples. The overall HPV detection rate was similar: 65.1% (43/66) for PCR-RH and 57.6% (38/66) for PCR-RFLP. HPV DNA was found in all CIN2 and CIN3 samples by both methods; however, PCR-RH detected more positives among normal biopsy samples and CINI cases. Overall, there was good agreement between the two genotyping methods, but RH yielded fewer cases with undetermined HPV genotype.