Abstract
BackgroundHuman 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.ResultsWhen 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/μl). 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 ≤ 0.06.ConclusionRun-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.
Highlights
Human papilloma virus (HPV) load and physical status are considered useful parameters for clinical evaluation of cervical squamous cell neoplasia
The E2 primers, amplifying bp 3,361–3,442 of the HPV16 genome (GenBank accession AF125673), were located in the part of the E2 open reading frame (ORF) that is most often deleted on HPV16 integration [5]
Following four replicate quantitative PCR (qPCR) runs, seven point external calibration curves were generated for E2, E6 and hydroxymethylbilane synthase (HMBS)
Summary
Human papilloma virus (HPV) load and physical status are considered useful parameters for clinical evaluation of cervical squamous cell neoplasia. 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. Most cases are squamous cell carcinomas (SCCs), which develop from precursor squamous intraepithelial lesions (SILs). At present it is not possible to discriminate progressive from non progressive SIL, leading to over-treatment of large numbers of women, with attendant physical and psychological morbidity. Persistent infection by high risk human papillomavirus (HR-HPV) represents the most significant risk factor in development of cervical carcinoma [2,3], with HPV16 being the virus type most commonly seen in SCC. In cervical malignancy HPV is usually integrated into the host genome. Integration is characterised by retention of the viral oncogenes E6 and E7, and by disruption or loss of expression of the viral transcriptional repressor E2, leading to deregulated production of E6 and E7 [4]
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.