Highly Sensitive and Specific Lateral Flow Detection for DNA Methylation Based on GIaI-Mediated Specific-Terminal-Mediated Polymerase Chain Reaction

Ke, Lihui; Zhao, Hang; Shan, Hongbo; Chen, Yicheng; Cai, Yongsheng; Wang, Yang; Wei, Bo; Du, Minghua

doi:10.3390/mi16040387

Open AccessArticle

Highly Sensitive and Specific Lateral Flow Detection for DNA Methylation Based on GIaI-Mediated Specific-Terminal-Mediated Polymerase Chain Reaction

by

Lihui Ke

^1,†,

Hang Zhao

^2,†,

Hongbo Shan

³,

Yicheng Chen

⁴,

Yongsheng Cai

^1,4,

Yang Wang

^4,*,

Bo Wei

^1,* and

Minghua Du

^5,*

¹

Department of Thoracic Surgery, Beijing Tiantan Hospital, Capital Medical University, Beijing 100070, China

²

College of Health Science and Environmental Engineering, Shenzhen Technology University, Shenzhen 518118, China

³

Adicon Clinical Laboratories, Inc., Hangzhou 310023, China

⁴

Key Laboratory of Biomechanics and Mechanobiology, Ministry of Education, Beijing Advanced Innovation Center for Biomedical Engineering, School of Engineering Medicine, Beihang University, Beijing 100191, China

⁵

Department of Emergency, The First Medical Center, Chinese PLA General Hospital, Beijing 100853, China

^*

Authors to whom correspondence should be addressed.

^†

These authors contributed equally to this work.

Micromachines 2025, 16(4), 387; https://doi.org/10.3390/mi16040387

Submission received: 20 February 2025 / Revised: 22 March 2025 / Accepted: 26 March 2025 / Published: 28 March 2025

(This article belongs to the Special Issue The Fabrication of Micro and Nano Bio-Detection Chips, Sensors and Smart Detectors)

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Abstract

Sensitive and specific detection of DNA methylation is crucial for the early diagnosis of various human diseases, particularly cancers. However, conventional methylation detection methods often face challenges in balancing both sensitivity and specificity. In this study, we present a novel approach that integrates the high specificity of methylation-dependent restriction endonuclease (GlaI) digestion with the amplification efficiency of specific terminal-mediated polymerase chain reaction (STEM-PCR). This combination enables selective amplification of methylated DNA, which is then detected through lateral flow detection (LFD), providing a simple, visual readout. As a proof of concept, a STEM-PCR-LFD assay was applied to detect methylated Septin 9, a biomarker for colorectal cancer. The assay demonstrated a sensitivity of approximately 0.1% (10 copies of methylated template per reaction), with no cross-reactivity observed when 10,000 copies of unmethylated DNA were included as background. Furthermore, the assay was validated with ten formalin-fixed paraffin-embedded (FFPE) tissue samples, achieving 100% consistency with standard real-time STEM-PCR. This method offers a highly sensitive, specific, and accessible platform for DNA methylation detection, with potential for early disease diagnosis.

Keywords: DNA methylation detection; STEM-PCR; methylation-dependent restriction endonucleases; Septin 9

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MDPI and ACS Style

Ke, L.; Zhao, H.; Shan, H.; Chen, Y.; Cai, Y.; Wang, Y.; Wei, B.; Du, M. Highly Sensitive and Specific Lateral Flow Detection for DNA Methylation Based on GIaI-Mediated Specific-Terminal-Mediated Polymerase Chain Reaction. Micromachines 2025, 16, 387. https://doi.org/10.3390/mi16040387

AMA Style

Ke L, Zhao H, Shan H, Chen Y, Cai Y, Wang Y, Wei B, Du M. Highly Sensitive and Specific Lateral Flow Detection for DNA Methylation Based on GIaI-Mediated Specific-Terminal-Mediated Polymerase Chain Reaction. Micromachines. 2025; 16(4):387. https://doi.org/10.3390/mi16040387

Chicago/Turabian Style

Ke, Lihui, Hang Zhao, Hongbo Shan, Yicheng Chen, Yongsheng Cai, Yang Wang, Bo Wei, and Minghua Du. 2025. "Highly Sensitive and Specific Lateral Flow Detection for DNA Methylation Based on GIaI-Mediated Specific-Terminal-Mediated Polymerase Chain Reaction" Micromachines 16, no. 4: 387. https://doi.org/10.3390/mi16040387

APA Style

Ke, L., Zhao, H., Shan, H., Chen, Y., Cai, Y., Wang, Y., Wei, B., & Du, M. (2025). Highly Sensitive and Specific Lateral Flow Detection for DNA Methylation Based on GIaI-Mediated Specific-Terminal-Mediated Polymerase Chain Reaction. Micromachines, 16(4), 387. https://doi.org/10.3390/mi16040387

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Highly Sensitive and Specific Lateral Flow Detection for DNA Methylation Based on GIaI-Mediated Specific-Terminal-Mediated Polymerase Chain Reaction

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