title: Multiplex nested PCR (MNP) assay for the detection of 15 high risk genotypes of human papillomavirus.
abstract: BACKGROUND
  Human papillomavirus (HPV) is now recognized as the causative agent in cervical cancer. The HPV genotypes that infect the genital region have been classified into high and low risk types according to their oncogenic potential. There is still uncertainty regarding rare HPV genotypes, however the types considered high risk in this study are: HPV-16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68 and 70.
  
  
  OBJECTIVE
  We have set out to develop a multiplex nested PCR (MNP) assay with primers directed at the early region of the HPV genome to detect 15 high risk HPV (HRHPV) genotypes. Since it is known that the late region of HPV is lost on integration into the host cell genome, the primers are directed at the early region of the HPV genome so as to ensure the detection of integrated virus, in the absence of the episomal form of the virus.
  
  
  METHODS
  Primers were designed to detect specifically the high risk HPV in the MNP assay. The MNP assay was compared to a generic mucosal HPV nested PCR and another nested HRHPV PCR assay. DNA sequencing was carried out on the samples tested and matched with the PCR results.
  
  
  RESULTS
  The MNP assay demonstrated that it was able to detect all 15 HRHPV types and was positive for more CIN1, CIN2 and CIN3 cases than the other nested HRHPV PCR. Further to this, the PCR product sizes differ for most of the HRHPV types detected in this system, so it is possible to type most of these HRHPV by the molecular size of the PCR products.
  
  
  CONCLUSIONS
  The MNP assay detects 15 currently recognized HRHPV and could be very useful, in conjunction with the Pap smear, as a screening assay or to help manage Pap smears of uncertain cytology.
authors.affiliation: Division of Microbiology and Infectious Diseases, The Western Australian Centre for Pathology and Medical Research, PathCentre, Locked Bag 2009, Nedlands, WA 6009, Australia. brian.brestovac@health.wa.gov.au
authors.forename: Brian
authors.initials: B
authors.lastname: Brestovac
authors.forename: Gerald B
authors.initials: GB
authors.lastname: Harnett
authors.forename: David W
authors.initials: DW
authors.lastname: Smith
authors.forename: Felicity
authors.initials: F
authors.lastname: Frost
authors.forename: Geoffrey R
authors.initials: GR
authors.lastname: Shellam
mesh_terms: D001315:Australia; D017931:DNA Primers; D004279:DNA, Viral / Q000032:analysis* / Q000737:chemistry / Q000235:genetics*; D004587:Electrophoresis, Agar Gel; D005260:Female; D005838:Genotype; D006801:Humans; D027383:Papillomaviridae / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification* / Q000472:pathogenicity; D030361:Papillomavirus Infections / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA
chemical_list: D017931:DNA Primers; D004279:DNA, Viral