Establishment of an efficient multiplex real-time PCR assay for human papillomavirus genotyping in cervical cytology specimens: Comparison with hybrid capture II

Objective: To establish an efficient multiplex real-time PCR assay for 15 human papillomavirus (HPV) genotypes, we designed multiplexing parameters and compared our PCR system with the hybrid capture (HC) II test using cervical cytology samples. Methods: For preventing cross-reactive amplifications, variable HPV genes (E1, E2, E6, E7 and L1) were targeted. The melting temperatures of all primers and probes, and the size of the PCR product were optimized for the multiplex PCR. Our PCR system was compared with the HC II assays in the detection and genotyping of HPV infection using 173 cytology smears. Discordant cases between the two assays were verified by direct HPV DNA sequencing. Results: Of 173 women, 93 (53.8%) were HPV-positive by the HC II assay and/or the multiplex real-time PCR assay. The HPV genotypes were determined in 92 (98.9%) of 93 cases by the multiplex real-time PCR and/or DNA sequencing. The agreement rate between multiplex PCR and HC II methods was 91.9% (kappa=0.84). Although the sample size of this study needs to be increased to have epidemiological significance, multiple infections and HPV 16 were the predominant type. HPV 58, 52 and 18 accounted for 25% of HPV infections. HPV 52, 58 and 31 constituted 30% of CIN 2/3. Conclusion: The multiplex real-time PCR system shows a good and reliable clinical performance. This in house PCR assay is fast and cost-effective for HPV genotyping and the detection of HPV co-infection in the post-HPV vaccination era.