title: Detection of human papillomavirus DNA by PCR/microfluorometry for screening of cervical cancer.
abstract: BACKGROUND
  Cervical cancer screening is conducted by a cytological Papanicolaou (Pap) test. For screening, it is becoming increasingly important to introduce a more objective result, based on human papillomavirus (HPV) DNA test. We describe here a practical method allowing the mass detection of HPV-DNA by PCR followed by fluorogenic DNA intercalation.
  
  
  METHODS
  Samples used were cervical scrapes or biopsy specimens obtained from women who had undergone cytological testing for cervical cancer. Crude DNAs were extracted by a simplified proteinase K-boil method. Common and type-specific primers were newly designed for major types of high-risk HPVs. A fluorogenic DNA intercalator, SYBR Green I was directly added to the specific PCR products. The resultant fluorescence was measured by a conventional fluorometric microplate reader.
  
  
  RESULTS
  The proposed PCR/microfluorometry (MFL) allowed a simple, rapid and economical detection of HPV-DNA without any use of labeling primers or probes. HPV-DNAs were found in 48.2% (123/255) of the cervical scrapes. The detection rate of HPV in cervical cancer biopsy specimen was 92.4% (61/66).
  
  
  CONCLUSIONS
  PCR/MFL detection of HPV-DNA, followed by combined type-specific PCR, is expected to be an extremely useful tool in cervical cancer screening.
authors.affiliation: Sapporo Immunodiagnostic Laboratory, Shinkawa 2-2-12-20, Kita-ku, Sapporo 001-0922, Japan.
authors.forename: Akihiro
authors.initials: A
authors.lastname: Yamaguchi
authors.forename: Naoko
authors.initials: N
authors.lastname: Hashimoto
authors.forename: Wataru
authors.initials: W
authors.lastname: Tsutae
authors.forename: Kuniyoshi
authors.initials: K
authors.lastname: Seino
authors.forename: Yasuhiko
authors.initials: Y
authors.lastname: Ebina
authors.forename: Takashi
authors.initials: T
authors.lastname: Tokino
authors.forename: Noriyuki
authors.initials: N
authors.lastname: Sato
authors.forename: Kokichi
authors.initials: K
authors.lastname: Kikuchi
mesh_terms: D052160:Benzothiazoles; D001706:Biopsy; D017931:DNA Primers; D004279:DNA, Viral / Q000032:analysis* / Q000302:isolation & purification; D003959:Diamines; D005260:Female; D005456:Fluorescent Dyes; D005470:Fluorometry; D006801:Humans; D009930:Organic Chemicals; D027383:Papillomaviridae / Q000737:chemistry*; D011804:Quinolines; D020133:Reverse Transcriptase Polymerase Chain Reaction; D002583:Uterine Cervical Neoplasms / Q000175:diagnosis* / Q000821:virology
chemical_list: D052160:Benzothiazoles; D017931:DNA Primers; D004279:DNA, Viral; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I