title: Development of a novel multiplex type-specific quantitative real-time PCR for detection and differentiation of infections with human papillomavirus types HPV2, HPV27, and HPV57.
abstract: BACKGROUND
  The present study describes the development and evaluation of the first multiplex type-specific quantitative real-time PCR (RT-PCR), enabling simple, rapid, sensitive, and specific concurrent detection and differentiation of human papillomavirus (HPV) types HPV2, 27, and 57 in a single PCR reaction.
  
  
  RESULTS
  The HPV2/27/57 multiplex RT-PCR with a dynamic range of seven orders of magnitude (discriminating 10 to 108 viral genome equivalents/reaction) has an analytical sensitivity of at least 10 viral copies of each targeted HPV type/reaction, and no cross-reactivities were observed among the included targets. All three primer/probe combinations were efficient in amplifying 500 copies of targeted DNA in a background of 108, 107, 500, 100, and 10 copies of non-targeted viral DNA/reaction, and the performance of the HPV2/27/57 multiplex RT-PCR was additionally not affected by the presence of background human genomic DNA. When testing DNA isolates obtained from fresh-frozen tissue specimens of various children's warts, the results of the HPV2/27/57 multiplex RT-PCR were completely in line with the results of the conventional Low-risk Alpha-PV PCR.
  
  
  CONCLUSIONS
  The newly developed HPV2/27/57 multiplex RT-PCR is an appropriate test for use in routine clinical laboratory settings and for studies focusing on the molecular epidemiology, pathogenesis, and natural history of HPV2/27/57-related lesions.
authors.affiliation: Institute of Microbiology and Immunology, Faculty of Medicine, Ljubljana, Slovenia.
authors.forename: Lea
authors.initials: L
authors.lastname: Hošnjak
authors.affiliation: Institute of Microbiology and Immunology, Faculty of Medicine, Ljubljana, Slovenia.
authors.forename: Kristina
authors.initials: K
authors.lastname: Fujs Komloš
authors.affiliation: Institute of Microbiology and Immunology, Faculty of Medicine, Ljubljana, Slovenia.
authors.forename: Boštjan J
authors.initials: BJ
authors.lastname: Kocjan
authors.affiliation: Institute of Microbiology and Immunology, Faculty of Medicine, Ljubljana, Slovenia.
authors.forename: Katja
authors.initials: K
authors.lastname: Seme
authors.affiliation: Institute of Microbiology and Immunology, Faculty of Medicine, Ljubljana, Slovenia.
authors.forename: Mario
authors.initials: M
authors.lastname: Poljak
mesh_terms: D002648:Child; D002675:Child, Preschool; D004279:DNA, Viral / Q000302:isolation & purification*; D005260:Female; D006801:Humans; D008297:Male; D060885:Multiplex Polymerase Chain Reaction; D027383:Papillomaviridae / Q000235:genetics*; D030361:Papillomavirus Infections / Q000175:diagnosis* / Q000821:virology*; D060888:Real-Time Polymerase Chain Reaction; D012680:Sensitivity and Specificity
chemical_list: D004279:DNA, Viral
keywords: HPV27; and HPV57; detection; differentiation; human papillomavirus types HPV2; multiplex type-specific quantitative real-time PCR development.