title: Critical evaluation of HPV16 gene copy number quantification by SYBR green PCR.
abstract: BACKGROUND
  Human papilloma virus (HPV) load and physical status are considered useful parameters for clinical evaluation of cervical squamous cell neoplasia. However, the errors implicit in HPV gene quantification by PCR are not well documented. We have undertaken the first rigorous evaluation of the errors that can be expected when using SYBR green qPCR for quantification of HPV type 16 gene copy numbers. We assessed a modified method, in which external calibration curves were generated from a single construct containing HPV16 E2, HPV16 E6 and the host gene hydroxymethylbilane synthase in a 1:1:1 ratio.
  
  
  RESULTS
  When testing dilutions of mixed HPV/host DNA in replicate runs, we observed errors in quantifying E2 and E6 amplicons of 5-40%, with greatest error at the lowest DNA template concentration (3 ng/microl). Errors in determining viral copy numbers per diploid genome were 13-53%. Nevertheless, in cervical keratinocyte cell lines we observed reasonable agreement between viral loads determined by qPCR and Southern blotting. The mean E2/E6 ratio in episome-only cells was 1.04, but with a range of 0.76-1.32. In three integrant-only lines the mean E2/E6 ratios were 0.20, 0.72 and 2.61 (values confirmed by gene-specific Southern blotting). When E2/E6 ratios in fourteen HPV16-positive cervical carcinomas were analysed, conclusions regarding viral physical state could only be made in three cases, where the E2/E6 ratio was < or = 0.06.
  
  
  CONCLUSIONS
  Run-to-run variation in SYBR green qPCR produces unavoidable inaccuracies that should be allowed for when quantifying HPV gene copy number. While E6 copy numbers can be considered to provide a useable indication of viral loads, the E2/E6 ratio is of limited value. Previous studies may have overestimated the frequency of mixed episomal/integrant HPV infections.
authors.affiliation: MRC Cancer Cell Unit, Hutchison/MRC Research Centre, Hills Road, Cambridge, CB2 0XZ, UK. ir210@cam.ac.uk
authors.forename: Ian
authors.initials: I
authors.lastname: Roberts
authors.forename: Grace
authors.initials: G
authors.lastname: Ng
authors.forename: Nicola
authors.initials: N
authors.lastname: Foster
authors.forename: Margaret
authors.initials: M
authors.lastname: Stanley
authors.forename: Michael T
authors.initials: MT
authors.lastname: Herdman
authors.forename: Mark R
authors.initials: MR
authors.lastname: Pett
authors.forename: Andrew
authors.initials: A
authors.lastname: Teschendorff
authors.forename: Nicholas
authors.initials: N
authors.lastname: Coleman
mesh_terms: D001483:Base Sequence; D052160:Benzothiazoles; D002138:Calibration; D002294:Carcinoma, Squamous Cell / Q000821:virology; D045744:Cell Line, Tumor; D004279:DNA, Viral / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics*; D003959:Diamines; D005260:Female; D018628:Gene Dosage; D052162:Human papillomavirus 16 / Q000235:genetics*; D006801:Humans; D011163:Hydroxymethylbilane Synthase / Q000235:genetics; D008969:Molecular Sequence Data; D009856:Oncogene Proteins, Viral / Q000235:genetics*; D009930:Organic Chemicals; D030361:Papillomavirus Infections / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D012097:Repressor Proteins / Q000235:genetics*; D002583:Uterine Cervical Neoplasms / Q000821:virology; D019562:Viral Load; D002578:Uterine Cervical Dysplasia / Q000821:virology
chemical_list: D052160:Benzothiazoles; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D003959:Diamines; C054855:E2 protein, Human papillomavirus type 16; C068651:E6 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; D009930:Organic Chemicals; D011804:Quinolines; D012097:Repressor Proteins; C098022:SYBR Green I; D011163:Hydroxymethylbilane Synthase