title: 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.
abstract: 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.
authors.affiliation: University of Thessaly, School of Health Sciences, Department of Biochemistry & Biotechnology, Microbiology-Virology Laboratory, Larissa, Greece.
authors.forename: D
authors.initials: D
authors.lastname: Tsakogiannis
authors.affiliation: University of Thessaly, School of Health Sciences, Department of Biochemistry & Biotechnology, Microbiology-Virology Laboratory, Larissa, Greece.
authors.forename: M
authors.initials: M
authors.lastname: Papacharalampous
authors.affiliation: University of Thessaly, School of Health Sciences, Department of Biochemistry & Biotechnology, Microbiology-Virology Laboratory, Larissa, Greece.
authors.forename: E
authors.initials: E
authors.lastname: Toska
authors.affiliation: University of Thessaly, School of Health Sciences, Department of Biochemistry & Biotechnology, Microbiology-Virology Laboratory, Larissa, Greece.
authors.forename: Z
authors.initials: Z
authors.lastname: Kyriakopoulou
authors.affiliation: University of Thessaly, School of Health Sciences, Department of Biochemistry & Biotechnology, Microbiology-Virology Laboratory, Larissa, Greece.
authors.forename: T G
authors.initials: TG
authors.lastname: Dimitriou
authors.affiliation: University of Thessaly, School of Health Sciences, Department of Biochemistry & Biotechnology, Microbiology-Virology Laboratory, Larissa, Greece.
authors.forename: I G A
authors.initials: IG
authors.lastname: Ruether
authors.affiliation: University of Thessaly, School of Health Sciences, Department of Biochemistry & Biotechnology, Microbiology-Virology Laboratory, Larissa, Greece.
authors.forename: D
authors.initials: D
authors.lastname: Komiotis
authors.affiliation: University of Thessaly, School of Health Sciences, Department of Biochemistry & Biotechnology, Microbiology-Virology Laboratory, Larissa, Greece. Electronic address: markoulatos@bio.uth.gr.
authors.forename: P
authors.initials: P
authors.lastname: Markoulatos
mesh_terms: D004279:DNA, Viral / Q000032:analysis; D005260:Female; D005838:Genotype; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D027383:Papillomaviridae / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D030361:Papillomavirus Infections / Q000175:diagnosis* / Q000821:virology; D012680:Sensitivity and Specificity; D044366:Transition Temperature
chemical_list: D004279:DNA, Viral
keywords: Duplex Real-time PCR; HPV genotyping; L1 gene; Melting curve analysis