Center for Bio-Medical Engineering Core Facility, Dankook University, Cheonan, 31116, Republic of Korea.
Smart Animal Bio Institute, Dankook University, Cheonan, 31116, Republic of Korea.
Genes Genomics. 2024 Aug;46(8):909-915. doi: 10.1007/s13258-024-01527-5. Epub 2024 Jun 8.
Digital PCR (dPCR) technology allows absolute quantification and detection of disease-associated rare variants, and thus the use of dPCR technology has been increasing in clinical research and diagnostics. The high-resolution melting curve analysis (HRM) of qPCR is widely used to distinguish true positives from false positives and detect rare variants. In particular, qPCR-HRM is commonly used for methylation assessment in research and diagnostics due to its simplicity and high reproducibility. Most dPCR instruments have limited fluorescence channels available and separate heating and imaging systems. Therefore, it is difficult to perform HRM analysis using dPCR instruments.
A new digital real-time PCR instrument (LOAA) has been recently developed to integrate partitioning, thermocycling, and imaging in a single dPCR instrument. In addition, a new technique to perform HRM analysis is utilized in LOAA. The aim of the present study is to evaluate the efficiency and accuracy of LOAA dPCR on HRM analysis for the detection of methylation.
In this study, comprehensive comparison with Bio-Rad qRT-PCR and droplet-based dPCR equipment was performed to verify the HRM analysis-based methylation detection efficiency of the LOAA digital PCR equipment. Here, sodium bisulfite modification method was applied to detect methylated DNA sequences by each PCR method.
Melting curve analysis detected four different Tm values using LOAA and qPCR, and found that LOAA, unlike qPCR, successfully distinguished between different Tm values when the Tm values were very similar. In addition, melting temperatures increased by each methylation were about 0.5℃ for qPCR and about 0.2 ~ 0.6℃ for LOAA. The melting temperature analyses of methylated and unmethylated DNA samples were conducted using LOAA dPCR with TaqMan probes and EvaGreen, and the result found that Tm values of methylated DNA samples are higher than those of unmethylated DNA samples.
The present study shows that LOAA dPCR could detect different melting temperatures according to methylation status of target sequences, indicating that LOAA dPCR would be useful for diagnostic applications that require the accurate quantification and assessment of DNA methylation.
数字 PCR（dPCR）技术允许对与疾病相关的罕见变体进行绝对定量和检测，因此，dPCR 技术在临床研究和诊断中的应用正在增加。qPCR 的高分辨率熔解曲线分析（HRM）广泛用于区分真阳性和假阳性并检测罕见变体。由于其简单性和高重复性，qPCR-HRM 通常用于研究和诊断中的甲基化评估。大多数 dPCR 仪器可用的荧光通道有限，且加热和成像系统独立。因此，使用 dPCR 仪器进行 HRM 分析很困难。
最近开发了一种新型数字实时 PCR 仪器（LOAA），可在单个 dPCR 仪器中集成分区、热循环和成像。此外，在 LOAA 中利用了一种新的 HRM 分析技术。本研究旨在评估 LOAA dPCR 在 HRM 分析中用于检测甲基化的效率和准确性。
在这项研究中，通过与 Bio-Rad qRT-PCR 和液滴式 dPCR 设备进行全面比较，验证了 LOAA 数字 PCR 设备基于 HRM 分析的甲基化检测效率。在此，应用亚硫酸氢钠修饰法通过每种 PCR 方法检测甲基化 DNA 序列。
使用 LOAA 和 qPCR 进行熔解曲线分析检测到四个不同的 Tm 值，发现 LOAA 与 qPCR 不同，当 Tm 值非常相似时，成功区分了不同的 Tm 值。此外，每个甲基化引起的熔解温度升高约为 0.5℃用于 qPCR，而约为 0.2 至 0.6℃用于 LOAA。使用 LOAA dPCR 与 TaqMan 探针和 EvaGreen 对甲基化和未甲基化 DNA 样本进行熔解温度分析，结果发现甲基化 DNA 样本的 Tm 值高于未甲基化 DNA 样本的 Tm 值。
本研究表明，LOAA dPCR 可以根据目标序列的甲基化状态检测到不同的熔解温度，表明 LOAA dPCR 将有助于需要准确定量和评估 DNA 甲基化的诊断应用。
