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Open Access 01-12-2012 | Technical advance

Development of a multiplex methylation-specific PCR as candidate triage test for women with an HPV-positive cervical scrape

Authors: Suzanne Snellenberg, Lise MA De Strooper, Albertus T Hesselink, Chris JLM Meijer, Peter JF Snijders, Daniëlle AM Heideman, Renske DM Steenbergen

Published in: BMC Cancer | Issue 1/2012

Abstract

Background

Quantitative methylation-specific PCR (qMSP) analysis for determining the methylation status of (candidate) tumor suppressor genes has potential as objective and valuable test to triage high-risk human papillomavirus (hrHPV) positive women in cervical screening. Particularly combined methylation analysis of a panel of genes shows most promising clinical performance, with sensitivity levels that equal or exceed that of cytology. However, the wide application of such methylation marker panels is hampered by the lack of effective multiplex assays allowing simultaneous methylation detection of various targets in a single reaction. Here, we designed and analyzed a multiplex qMSP assay for three genes whose methylation was previously found to be informative for cervical (pre)cancer (i.e. CADM1, MAL and hsa-miR-124-2) as well as a reference gene β-actin. Based on our experience, we discuss the optimization of the parameters that provide a practical approach towards multiplex qMSP design.

Methods

Primers and PCR reagents were optimized for multiplex qMSP purposes and the resulting assay was analytically validated on serial dilutions of methylated DNA in unmethylated DNA, and compared with singleplex counterparts on hrHPV-positive cervical scrapings.

Results

Upon optimization, including primer redesign and primer limiting assays, the multiplex qMSP showed the same analytical performance as the singleplex qMSPs. A strong correlation between the obtained normalized ratios of the singleplex and multiplex qMSPs on cervical scrapes was found for all three markers: CADM1 (R²=0.985), MAL (R²=0.986) and hsa-miR-124-2 (R²=0.944).

Conclusion

Multiplex qMSP offers a promising approach for high-throughput diagnostic analysis of the methylation status of multiple genes, which after proper design and validation can be equally specific, sensitive and reproducible as its singleplex versions.

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The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/12/551/prepub

Title: Development of a multiplex methylation-specific PCR as candidate triage test for women with an HPV-positive cervical scrape
Authors: Suzanne Snellenberg
Lise MA De Strooper
Albertus T Hesselink
Chris JLM Meijer
Peter JF Snijders
Daniëlle AM Heideman
Renske DM Steenbergen
Publication date: 01-12-2012
Publisher: BioMed Central
Published in: BMC Cancer / Issue 1/2012
Electronic ISSN: 1471-2407
DOI: https://doi.org/10.1186/1471-2407-12-551

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