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

BMC Cancer

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

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

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

Others  :  1080060
DOI  :  10.1186/1471-2407-12-551

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

【 摘 要 】

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.

【 授权许可】

   
2012 Snellenberg et al.; licensee BioMed Central Ltd.

【 图 表 】

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