Dual-modality loop-mediated isothermal amplification for pretreatment-free detection of Septin9 methylated DNA in colorectal cancer

Abstract

Currently, the determination of DNA methylation is still a challenge due to the limited efficiency of enrichment, bisulfite modification, and detection. In this study, a dual-modality loop-mediated isothermal amplification integrated with magnetic bead isolation is proposed for the determination of methylated Septin9 gene in colorectal cancer. Magnetic beads modified with anti-methyl cytosine antibody were prepared for fast enrichment of methylated DNA through specific immunoaffinity (30 min). One-pot real-time fluorescence and colorimetric loop-mediated isothermal amplification were simultaneously developed for detecting methylated Septin9 gene (60 min). The real-time fluorescence generating by SYTO-9 dye (excitation: 470 nm and emission: 525 nm) and pH indicator (neutral red) was used for quantitative and visualized detection of methylated DNA. This method was demonstrated to detect methylated DNA from HCT 116 cells ranging from 2 to 0.02 ng/μL with a limit of detection of 0.02 ± 0.002 ng/μL (RSD: 9.75%). This method also could discriminate methylated Septin9 in 0.1% HCT 116 cells (RSD: 6.60%), suggesting its high specificity and sensitivity. The feasibility of this assay was further evaluated by clinical plasma samples from 20 colorectal cancer patients and 20 healthy controls, which shows the potential application in simple, low cost, quantitative, and visualized detection of methylated nucleic acids.

Magnetic Nanoparticle-Based Electrochemical Sensing Platform Using Ferrocene-Labelled Peptide Nucleic Acid for the Early Diagnosis of Colorectal Cancer

“…In addition to the inherent magnetic force, superparamagnetic nanoparticles can also improve the analytical signal due to the oxidation/reduction processes, which can decrease the signal-to-noise ratio. Lin et al fabricated a magnetic bead-based optical sensor with low detection limits (0.02 ng/µL) using HCT 116 cells [ 39 ]. Recently, Syedmoradi et al reported an electrochemical system using SPCE electrodes that achieved a good linear range with a detection limit of 0.01 pM [ 40 ].…”

Recent Trends and Innovations in Bead-Based Biosensors for Cancer Detection

“…The core-shell beads' spherical configuration may offer an uncomplicated approach to concentrating and discerning light, thereby possibly further streamlining the optical system's design. Q. Lin et al further highlighted LAMP's adaptability for cancer-biomarker detection [162]. They introduced a dual-modality approach that integrates LAMP with magnetic bead isolation.…”

Recent advancements in nucleic acid detection with microfluidic chip for molecular diagnostics

“…Reproduced with permission from Ref. [ 75 ]. (D) A CRISPR/Cas12a-based SNP detection genotyping method based on the centrifugal microfluidic device.…”

“…The determination of DNA methylation is still a challenge. Researchers have recently proposed a dual-mode LAMP incorporating magnetic bead separation to determine the methylated Septin9 gene in colorectal cancer [ 75 ]. A one-pot real-time fluorescent and colorimetric LAMP was used to detect the methylated Septin9 gene (60 min).…”