Rapid detection of human papillomavirus in cervical scrapes by combined general primer-mediated and type-specific polymerase chain reaction

Abstract

A two-step polymerase chain reaction (PCR) procedure was used as a new screening strategy for the detection of human papillomavirus (HPV) genotypes in cervical scrapes omitting prior DNA extraction. Sample preparation consisted of a freeze-thaw step followed by boiling the cells before the PCR mixture was added. This pretreatment was as efficient and reproducible for HPV DNA amplification as DNA purification. By using crude cell suspensions, a prescreening of the samples with the general primer-mediated PCR method (GP-PCR) was performed to detect a broad spectrum of sequenced and still unsequenced HPV types at the subpicogram level. HPV-containing scrapes by GP-PCR were subjected to HPV 6, 11, 16, 18, 31, and 33 type-specific PCR (TS-PCR) to identify the sequenced HPV types. This direct GP/TS-PCR method was tested on a large group of cervical scrapes (n = 459) from women visiting a gynecologic outpatient clinic. The results were compared with HPV data obtained by a method using modified filter in situ hybridization and TS-PCR in which the PCR was mainly used to confirm HPV positivity. A substantially higher HPV prevalence rate was found by direct GP/TS-PCR strategy. The results indicate that GP/TS-PCR is a rapid, sensitive, and reliable detection method for HPV in cervical scrapes. The easy performance on crude cell suspensions makes this strategy applicable for large HPV-screening programs.