An MSRE-Assisted Glycerol-Enhanced RPA-CRISPR/Cas12a Method for Methylation Detection
Background: Nasopharyngeal carcinoma (NPC) is a malignant tumor with high prevalence in southern China. Aberrant DNA methylation, as a hallmark of cancer, is extensively present in NPC, the detection of which facilitates early diagnosis and prognostic improvement of NPC. Conventional methylation det...
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2024-12-01
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| author | Zhiquan Lu Zilu Ye Ping Li Yike Jiang Sanyang Han Lan Ma |
| author_facet | Zhiquan Lu Zilu Ye Ping Li Yike Jiang Sanyang Han Lan Ma |
| author_sort | Zhiquan Lu |
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| description | Background: Nasopharyngeal carcinoma (NPC) is a malignant tumor with high prevalence in southern China. Aberrant DNA methylation, as a hallmark of cancer, is extensively present in NPC, the detection of which facilitates early diagnosis and prognostic improvement of NPC. Conventional methylation detection methods relying on bisulfite conversion have limitations such as time-consuming, complex processes and sample degradation; thus, a more rapid and efficient method is needed. Methods: We propose a novel DNA methylation assay based on methylation-sensitive restriction endonuclease (MSRE) HhaI digestion and Glycerol-enhanced recombinase polymerase amplification (RPA)-CRISPR/Cas12a detection (HGRC). MSRE has a fast digestion rate, and HhaI specifically cleaves unmethylated DNA at a specific locus, leaving the methylated target intact to trigger the downstream RPA-Cas12a detection step, generating a fluorescence signal. Moreover, the detection step was supplemented with glycerol for the separation of Cas12a-containing components and RPA- and template-containing components, which avoids over-consumption of the template and, thus, enhances the amplification efficiency and detection sensitivity. Results: The HGRC method exhibits excellent performance in the detection of a CNE2-specific methylation locus with a (limit of detection) LOD of 100 aM and a linear range of 100 aM to 100 fM. It also responds well to different methylation levels and is capable of distinguishing methylation levels as low as 0.1%. Moreover, this method can distinguish NPC cells from normal cells by detecting methylation in cellular genomes. This method provides a rapid and sensitive approach for NPC detection and also holds good application prospects for other cancers and diseases featuring DNA methylation as a biomarker. |
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| institution | Kabale University |
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| spelling | doaj-art-cde9efc9cda74bcfa8c287d1a45f4b752024-12-27T14:14:16ZengMDPI AGBiosensors2079-63742024-12-01141260810.3390/bios14120608An MSRE-Assisted Glycerol-Enhanced RPA-CRISPR/Cas12a Method for Methylation DetectionZhiquan Lu0Zilu Ye1Ping Li2Yike Jiang3Sanyang Han4Lan Ma5Precision Medicine and Healthcare Research Center, Tsinghua-Berkeley Shenzhen Institute (TBSI), Tsinghua Shenzhen International Graduate School, Tsinghua University, University Town of Shenzhen, Nanshan District, Shenzhen 518055, ChinaInstitute of Biopharmaceutical and Health Engineering, Tsinghua Shenzhen International Graduate School, Tsinghua University, University Town of Shenzhen, Nanshan District, Shenzhen 518055, ChinaInstitute of Biomedical Health Technology and Engineering, Shenzhen Bay Laboratory, Shenzhen 518132, ChinaInstitute of Biomedical Health Technology and Engineering, Shenzhen Bay Laboratory, Shenzhen 518132, ChinaInstitute of Biopharmaceutical and Health Engineering, Tsinghua Shenzhen International Graduate School, Tsinghua University, University Town of Shenzhen, Nanshan District, Shenzhen 518055, ChinaPrecision Medicine and Healthcare Research Center, Tsinghua-Berkeley Shenzhen Institute (TBSI), Tsinghua Shenzhen International Graduate School, Tsinghua University, University Town of Shenzhen, Nanshan District, Shenzhen 518055, ChinaBackground: Nasopharyngeal carcinoma (NPC) is a malignant tumor with high prevalence in southern China. Aberrant DNA methylation, as a hallmark of cancer, is extensively present in NPC, the detection of which facilitates early diagnosis and prognostic improvement of NPC. Conventional methylation detection methods relying on bisulfite conversion have limitations such as time-consuming, complex processes and sample degradation; thus, a more rapid and efficient method is needed. Methods: We propose a novel DNA methylation assay based on methylation-sensitive restriction endonuclease (MSRE) HhaI digestion and Glycerol-enhanced recombinase polymerase amplification (RPA)-CRISPR/Cas12a detection (HGRC). MSRE has a fast digestion rate, and HhaI specifically cleaves unmethylated DNA at a specific locus, leaving the methylated target intact to trigger the downstream RPA-Cas12a detection step, generating a fluorescence signal. Moreover, the detection step was supplemented with glycerol for the separation of Cas12a-containing components and RPA- and template-containing components, which avoids over-consumption of the template and, thus, enhances the amplification efficiency and detection sensitivity. Results: The HGRC method exhibits excellent performance in the detection of a CNE2-specific methylation locus with a (limit of detection) LOD of 100 aM and a linear range of 100 aM to 100 fM. It also responds well to different methylation levels and is capable of distinguishing methylation levels as low as 0.1%. Moreover, this method can distinguish NPC cells from normal cells by detecting methylation in cellular genomes. This method provides a rapid and sensitive approach for NPC detection and also holds good application prospects for other cancers and diseases featuring DNA methylation as a biomarker.https://www.mdpi.com/2079-6374/14/12/608nasopharyngeal carcinoma (NPC)MSRERPACRISPR/Cas12afluorescence |
| spellingShingle | Zhiquan Lu Zilu Ye Ping Li Yike Jiang Sanyang Han Lan Ma An MSRE-Assisted Glycerol-Enhanced RPA-CRISPR/Cas12a Method for Methylation Detection Biosensors nasopharyngeal carcinoma (NPC) MSRE RPA CRISPR/Cas12a fluorescence |
| title | An MSRE-Assisted Glycerol-Enhanced RPA-CRISPR/Cas12a Method for Methylation Detection |
| title_full | An MSRE-Assisted Glycerol-Enhanced RPA-CRISPR/Cas12a Method for Methylation Detection |
| title_fullStr | An MSRE-Assisted Glycerol-Enhanced RPA-CRISPR/Cas12a Method for Methylation Detection |
| title_full_unstemmed | An MSRE-Assisted Glycerol-Enhanced RPA-CRISPR/Cas12a Method for Methylation Detection |
| title_short | An MSRE-Assisted Glycerol-Enhanced RPA-CRISPR/Cas12a Method for Methylation Detection |
| title_sort | msre assisted glycerol enhanced rpa crispr cas12a method for methylation detection |
| topic | nasopharyngeal carcinoma (NPC) MSRE RPA CRISPR/Cas12a fluorescence |
| url | https://www.mdpi.com/2079-6374/14/12/608 |
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