Duplex Real-time PCR assay and SYBR green I melting curve analysis for molecular identification of HPV genotypes 16, 18, 31, 35, 51 and 66

• A Duplex Real-time PCR assay for HPV diagnosis without the use of fluorescent, type specific-probes, reducing the cost and time in HPV genotyping.
• Increased specificity by the use of type-specific primer sets that allowed for the amplification of a part of L1 gene of HPV types 16, 18, 31, 35, 51 and 66.
• The developed Duplex Real-time PCR assay is a simple, specific, sensitive and cost-effective assay that can be easily implemented in medium and small size laboratories.

Long-term infection with high-risk HPV genotypes is the leading cause of cervical cancer. In the present study a Duplex Real-time PCR assay was developed in order to identify HPV types 16, 18, 31, 35, 51 and 66 in three reactions, through SYBR green I melting curve analysis. The method utilizes type-specific primer sets that allowed the amplification of highly conserved regions of L1 gene. Reconstitution experiments were conducted by using HPV DNA plasmids in order to determine the sensitivity of the assay. The newly designed assay has a limit of detection of 10 copies per reaction. The most prevalent HPV genotype in single and in multiple HPV infections was HPV16 followed by HPV18, HPV51, HPV31, HPV35 and HPV66. The proposed method is a simple, specific, sensitive and cost-effective assay that can be easily incorporated in small and medium size laboratories for the rapid identification of the most clinically important HPV genotypes.