Regular Article

British Journal of Cancer (1999) 81, 114–121. doi:10.1038/sj.bjc.6690659 www.bjcancer.com
Published online 13 August 1999

A quantitative polymerase chain reaction-enzyme immunoassay for accurate measurements of human papillomavirus type 16 DNA levels in cervical scrapings

M V Jacobs1, J M M Walboomers1, J van Beek1, F J Voorhorst2, R H M Verheijen3, C J L M Meijer1, A J C van den Brule1, ThJ M Helmerhorst3 and P J F Snijders1

  1. 1Department of Pathology Section of Molecular Pathology University Hospital Vrije Universiteit, De Boelelaan 1117, 1081 HV, Amsterdam, The Netherlands
  2. 2Department of Epidemiology and Biostatistics University Hospital Vrije Universiteit, De Boelelaan 1117, 1081 HV, Amsterdam, The Netherlands
  3. 3Department of Obstetrics and Gynecology, University Hospital Vrije Universiteit, De Boelelaan 1117, 1081 HV, Amsterdam, The Netherlands

Received 29 September 1998; Revised 15 February 1999; Accepted 15 March 1999.

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Abstract

A quantitative polymerase chain reaction-enzyme immunoassay (Q-PCR-EIA) was developed to measure the amount of human papillomavirus (HPV) 16 DNA per genome equivalent in cervical scrapings. The quantitative approach was based on a combined competitive PCR for both HPV 16, using the general primer GP5+/6+ PCR, and beta-globin DNA. The two competitive PCRs involve co-amplification of target sequences and exogenously added DNA constructs carrying a rearranged 30 bp sequence in the probe-binding region. The accuracy of quantification by combining the two competitive PCR assays was validated on mixtures of HPV 16 containing cervical cancer cells of CaSki and SiHa cell lines. Comparison of this fully quantitative PCR assay with two semi-quantitative HPV PCR assays on a series of crude cell suspensions from HPV 16 containing cervical scrapings revealed remarkable differences in the calculated relative HPV load between samples. We found evidence that correction for both intertube variations in PCR efficiency and number of input cells/integrity of DNA significantly influence the outcome of studies on viral DNA load in crude cell suspensions of cervical scrapings. Therefore, accurate measurements on viral DNA load in cervical scrapings require corrections for these phenomena, which can be achieved by application of this fully quantitative approach.

Keywords:

HPV, Q-PCR-EIA, cervical scrapings

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