背景:数字PCR(dPCR)技术允许对疾病相关的罕见变异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越来越多。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异。特别是,qPCR-HRM由于其简单性和高可重复性而通常用于研究和诊断中的甲基化评估。大多数dPCR仪器具有有限的可用荧光通道以及单独的加热和成像系统。因此,使用dPCR仪器执行HRM分析是困难的。
目的:最近开发了一种新的数字实时PCR仪器(LOAA)来集成分区,热循环,并在单个dPCR仪器中成像。此外,LOAA中使用了一种执行HRM分析的新技术。本研究的目的是评估LOAAdPCR在HRM分析中检测甲基化的效率和准确性。
方法:在本研究中,与Bio-RadqRT-PCR和基于液滴的dPCR设备进行综合比较,以验证LOAA数字PCR设备基于HRM分析的甲基化检测效率。这里,应用亚硫酸氢钠修饰方法,通过每种PCR方法检测甲基化的DNA序列。
结果:熔解曲线分析使用LOAA和qPCR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,成功区分不同的Tm值。此外,每次甲基化增加的解链温度qPCR约为0.5℃,LOAA约为0.2~0.6℃。甲基化和未甲基化DNA样品的解链温度分析使用LOAAdPCR与TaqMan探针和EvaGreen,结果发现,甲基化DNA样品的Tm值高于未甲基化DNA样品的Tm值。
结论:本研究表明,LOAAdPCR可以根据靶序列的甲基化状态检测不同的解链温度,这表明LOAAdPCR对于需要准确定量和评估DNA甲基化的诊断应用是有用的。
BACKGROUND: 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.
OBJECTIVE: 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.
METHODS: 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.
RESULTS: 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.
CONCLUSIONS: 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.