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

BMC Cancer

Technical Advance

Peter JF Snijders¹  Renske DM Steenbergen¹  Albertus T Hesselink¹  Chris JLM Meijer¹  Lise MA De Strooper¹  Daniëlle AM Heideman¹  Suzanne Snellenberg¹ 

[1] Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands;

关键词: Cervical cancer;    HPV testing;    DNA methylation;    Promoter;    CADM1;    MAL;    hsa-miR-124-2;    Multiplex qMSP;    Primer/probe design;    7500Fast ABI system;   

DOI  :  10.1186/1471-2407-12-551

 received in 2012-06-12, accepted in 2012-11-08,  发布年份 2012

来源: Springer

【 摘 要 】

BackgroundQuantitative 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.MethodsPrimers 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.ResultsUpon 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).ConclusionMultiplex 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.

【 授权许可】

Unknown   
© Snellenberg et al.; licensee BioMed Central Ltd. 2012. This article is published under license to 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.