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Critical evaluation of HPV16 gene copy number quantification by SYBR green PCR

Roberts, I; Ng, G; Foster, N; Stanley, M; Herdman, MT; Pett, MR; Teschendorff, A; (2008) Critical evaluation of HPV16 gene copy number quantification by SYBR green PCR. BMC Biotechnology , 8 , Article 57. 10.1186/1472-6750-8-57. Green open access

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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/mu l). 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.Conclusion: 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.

Type: Article
Title: Critical evaluation of HPV16 gene copy number quantification by SYBR green PCR
Open access status: An open access version is available from UCL Discovery
DOI: 10.1186/1472-6750-8-57
Publisher version: http://dx.doi.org/10.1186/1472-6750-8-57
Language: English
Additional information: © 2008 Roberts et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Keywords: Human-papillomavirus type-16, Real-time PCR, Carcinoma cell-lines, Cervical-cancer, Viral load, In-situ, Physical status, Uterine cervix, High-risk, DNA oad
UCL classification: UCL
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences
URI: https://discovery.ucl.ac.uk/id/eprint/76077
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