title: Defining detection threshold and improving analytical proficiency of HPV testing in clinical specimens.
abstract: OBJECTIVE
  Previous studies have shown that SPF1/GP6+ PCR followed by HPV Blot has high sensitivity and high reproducibility for the detection of human papillomavirus (HPV). However, there have been reports of false-negative results. In the present study we endeavoured to improve the diagnostic performance of HPV DNA testing in cervical swab specimens. We also aimed to identify viral load thresholds.
  
  
  METHODS
  We examined DNA from cervical swabs of 7 women with a cervical intraepithelial neoplasia grade 2 or worse (CIN2+) who were negative by HPV DNA testing. For control purposes, 106 cytology negative/HPV-negative samples were included. PCR was performed with either 1 microL of DNA solution or different amounts of input DNA (25, 50, and 100 ng). We tested two different commercial alkaline phosphatases (ALPs) at two distinct reaction times. The overall conclusions were based on HPV Blot, type-specific PCR, direct sequencing, and real-time quantitative PCR (qPCR).
  
  
  RESULTS
  All seven patients with CIN2+ disease turned HPV-positive when 100 ng of input DNA and E6 type-specific PCR were used. Discrepant and false-negative results were obtained using different amounts of input DNA. Different commercial ALPs showed a significant impact on detection performance. Analysis of viral load indicated that the detection threshold for HPV infection using SPF1/GP6+ PCR plus HPV Blot was approximately 10(2)-10(3) copies.
  
  
  CONCLUSIONS
  Reliable determination of HPV status is crucially dependent on the amount of input genomic DNA.
authors.affiliation: Department of Obstetrics and Gynecology, Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Taoyuan, Taiwan.
authors.forename: Fang-Yu
authors.initials: FY
authors.lastname: Chao
authors.forename: Angel
authors.initials: A
authors.lastname: Chao
authors.forename: Chu-Chuen
authors.initials: CC
authors.lastname: Huang
authors.forename: Swei
authors.initials: S
authors.lastname: Hsueh
authors.forename: Jung-Erh
authors.initials: JE
authors.lastname: Yang
authors.forename: Huei-Jean
authors.initials: HJ
authors.lastname: Huang
authors.forename: Lei-Chiung
authors.initials: LC
authors.lastname: Wang
authors.forename: Cheng-Tao
authors.initials: CT
authors.lastname: Lin
authors.forename: Hung-Hsueh
authors.initials: HH
authors.lastname: Chou
authors.forename: Chyong-Huey
authors.initials: CH
authors.lastname: Lai
mesh_terms: D004279:DNA, Viral / Q000032:analysis; D005260:Female; D005838:Genotype; D006801:Humans; D027383:Papillomaviridae / Q000235:genetics / Q000302:isolation & purification*; D030361:Papillomavirus Infections / Q000473:pathology / Q000821:virology*; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D002583:Uterine Cervical Neoplasms / Q000473:pathology / Q000821:virology*; D019562:Viral Load; D002578:Uterine Cervical Dysplasia / Q000473:pathology / Q000821:virology*
chemical_list: D004279:DNA, Viral