title: A real-time PCR approach based on SPF10 primers and the INNO-LiPA HPV Genotyping Extra assay for the detection and typing of human papillomavirus.
abstract: The SPF10 PCR targets a conserved 65bp region of the HPV L1 gene for broad-spectrum amplification. The LiPA assay allows subsequent genotyping of the HPV amplicons. This study aims to develop a SPF10 real-time PCR to achieve simultaneous amplification and detection of the HPV target. That way, LiPA analysis of the HPV-negative samples can be avoided, reducing workload and cost. The real-time PCR shows an analytical sensitivity of 29.7 copies for HPV 6, 16, 18 and 31 and an HPV-specific melting peak. Thirty-one HPV DNA plasmids were genotyped correctly using the SPF10 real-time PCR in combination with the LiPA. Here, the LiPA assay was performed at an increased hybridisation temperature (49.5°C) in combination with a reduced amplicon volume (1μl) to avoid cross-reactivity. In conclusion, the SPF10 real-time PCR proves to be very sensitive and generates amplicons, which are compatible with the LiPA.
authors.affiliation: Applied Molecular Biology Research group, Laboratory of Cell Biology and Histology, University of Antwerp, Groenenborgerlaan 171, Antwerp, Belgium. Isabel.Micalessi@ua.ac.be
authors.forename: M I
authors.initials: MI
authors.lastname: Micalessi
authors.forename: G A
authors.initials: GA
authors.lastname: Boulet
authors.forename: A
authors.initials: A
authors.lastname: Vorsters
authors.forename: K
authors.initials: K
authors.lastname: De Wit
authors.forename: G
authors.initials: G
authors.lastname: Jannes
authors.forename: W
authors.initials: W
authors.lastname: Mijs
authors.forename: M
authors.initials: M
authors.lastname: Ieven
authors.forename: P
authors.initials: P
authors.lastname: Van Damme
authors.forename: J J
authors.initials: JJ
authors.lastname: Bogers
mesh_terms: D036022:Capsid Proteins / Q000235:genetics*; D004279:DNA, Viral / Q000032:analysis / Q000235:genetics; D005838:Genotype; D006801:Humans; D009856:Oncogene Proteins, Viral / Q000235:genetics*; D027383:Papillomaviridae / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D030361:Papillomavirus Infections / Q000821:virology; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D036022:Capsid Proteins; D004279:DNA, Viral; D009856:Oncogene Proteins, Viral; C063317:L1 protein, Human papillomavirus type 16