Detection of human papillomavirus DNA in cervical lavage specimens by a nonisotopic consensus PCR assay

Abstract

A gene amplification method that combines PCR with an enzyme immunoassay (PCR-EIA) for quantitation of amplified DNA was developed for the detection of human papillomavirus (HPV). Samples were amplified with consensus primers MY09 and MY11. Amplified DNA products were reacted in solution with type-specific nested RNA probes labelled with digoxigenin-11-UTP. Hybrids were captured on a microtiter plate coated with an antidigoxigenin antibody. Bound DNA-RNA hybrids were quantitated by the addition of an alkaline phosphatase-labelled monoclonal antibody directed against DNA-RNA hybrids and a fluorogenic substrate. The detection limit of PCR-EIA was six copies of HPV type 18 DNA in the original specimen. The assay was used to assess HPV infection of the uterine cervixes of 65 women referred to a colposcopy clinic. In 66 cervicovaginal lavage specimens, all 23 HPV strains detected by a standard isotopic PCR assay were also detected by the PCR-EIA (sensitivity, 100%; 95% confidence interval, 85.2 to 100%). Forty-two of the 43 samples that did not contain HPV types 6/11, 16, 18, 31, 33, 35, and 45 were also negative by PCR-EIA, for a specificity of 97.7%. Low-level cross-reactivity was encountered between HPV types 18 and 45 as well as between types 33 and 58. PCR-EIA provides a convenient means of objectively measuring PCR-amplified HPV DNA from common genital HPV types.