Detection of major HPVs by a new multiplex real-time PCR assay using type-specific primers

• We developed a new SYBR green-based multiplex PCR for the diagnosis of HPV infections.
• New SYBR green-based multiplex PCR is more sensitive than MY09/11 consensus PCR.
• The SYBR green-based multiplex PCR is lower in cost than our new TaqMan-based PCR.
• SYBR green-based multiplex PCR may be useful for other microbiological fields.

In this study, we aimed to develop a cost-effective, practical, and sensitive method to be used for the diagnosis of HPV infections. The presence of HPV-DNA was investigated in cervical smear samples using three different methods: MY09/11 consensus PCR, TaqMan-based type-specific real-time PCR, and SYBR Green-based multiplex PCR. Of the 315 samples, 21.6% (68/315) were HPV-DNA positive by using at least one of the three methods. The relative sensitivities of MY09/11 PCR, type-specific PCR, and multiplex PCR were found to be 86.8% (59/68), 91.2% (62/68), and 91.2% (62/68), respectively. Genotyping analyses were successfully carried out in 62 of 68 HPV-DNA positive samples, and 77 isolates (8 low-risk and 69 high-risk HPV) were identified, while six samples were determined to be positive by consensus PCR only and could not be genotyped. The type distribution of the 69 high-risk HPV strains was as follows: 37.7% HPV 16, 13.0% HPV 52, 11.6% HPV 58, 7.2% HPV 18, 7.2% HPV 31, 7.2% HPV 68, 4.3% HPV 35, 4.3% HPV 39, 4.3% HPV 82, 1.4% HPV 33, and 1.4% HPV 45. Our data suggests that the diagnosis of HPV infections using only consensus PCR may lead to epidemiologically significant data loss, and that our multiplex PCR is more sensitive than consensus PCR and lower in cost than the type-specific PCR. We believe that the SYBR Green-based multiplex PCR may be useful and cost-effective for other microbiological fields. In addition, type-specific screening of HPV-DNA gives more reliable results, but it may also be used in combination with consensus PCR if the type spectrum of the test is not large enough.