Site-specific detection of circulating tumor DNA methylation in biological samples utilizing phosphorothioated primer-based loop-mediated isothermal amplification

DNA methylation, a kind of epigenetic alteration, plays a vital role in tumorigenesis and offers a new class of targets for cancer treatment. DNA hypermethylation at the E-Box site (CACGTG, -288 bp) in the SLC22A2 promoter was related to multidrug resistance of renal cell carcinoma (RCC), which can provide the target for both treatment and monitoring. Herein, we developed a novel phosphorothioated primer based loop-mediated isothermal amplification (PS-LAMP) assay to detect circulating tumor DNA (ctDNA) methylation levels in E-Box sites in tumor tissue, urine, and plasma samples from patients with RCC. Bisulfite treatment converted methylated/unmethylated discrepancy to a single base discrepancy (C/U). PS-LAMP amplified the templates to a tremendous amount. One-step strand displacement (OSD) probe provided single base resolution in amplified products and finally realized the specific site methylation detection. Our proposed method provided a linear range from 0% to 100% for methylation levels and was available in samples at low concentrations (10² copies/μL). Visually observable colorimetric detection can be achieved by incorporating the OSD probe with gold nanoparticles (AuNP). Our assay performed better than traditional methods in biological samples with low ctDNA concentration. Further, we found a potential consistency of methylation levels between tumor tissue and plasma sample from the same patient (Spearman's ρ = 0.886, P = 0.019, n = 6). In general, this work provides a PS-LAMP assay combining OSD probes for site-specific methylation detection in various biological samples, offering a method for noninvasive detection.