Cherl-Joon Lee, Wonseok Shin, Minsik Song, Seung-Shick Shin, Yujun Park, Kornsorn Srikulnath, Dong Hee Kim, Kyudong Han
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引用次数: 0
摘要
基于灵敏度、成本、速度、便利性和特异性的考虑,采用定量方法对分子标记进行临床诊断和物种鉴定。然而,典型的聚合酶链反应(PCR)测定是难以量化和有各种局限性。此外,为了使用定量实时PCR (qRT-PCR)设备进行定量分析,必须使用标准曲线或使用内参基因进行归一化。近十年来,已有研究报道第三代PCR技术digital PCR (dPCR)可以克服传统PCR和qRT-PCR的缺点,应用于各个领域。我们选择Stilla Naica System (Stilla Technologies)、Droplet Digital PCR Technology (Bio-Rad)和Lab on an Array Digital real - PCR分析仪系统(OPTOLANE)对目前商业上使用的各种液滴数字PCR平台进行比较分析。我们之前的研究发现了一个分子标记,可以利用韩牛特有的基因组结构变异来区分韩牛。在这里,我们从不同的角度报道了使用该物种鉴定标记的每个dPCR平台操作的利弊。总之,我们希望本研究能够帮助研究者根据其目的和资源选择合适的dPCR平台。
Comparison of digital PCR platforms using the molecular marker.
Assays of clinical diagnosis and species identification using molecular markers are performed according to a quantitative method in consideration of sensitivity, cost, speed, convenience, and specificity. However, typical polymerase chain reaction (PCR) assay is difficult to quantify and have various limitations. In addition, to perform quantitative analysis with the quantitative real-time PCR (qRT-PCR) equipment, a standard curve or normalization using reference genes is essential. Within the last a decade, previous studies have reported that the digital PCR (dPCR) assay, a third-generation PCR, can be applied in various fields by overcoming the shortcomings of typical PCR and qRT-PCR assays. We selected Stilla Naica System (Stilla Technologies), Droplet Digital PCR Technology (Bio-Rad), and Lab on an Array Digital Real-Time PCR analyzer system (OPTOLANE) for comparative analysis among the various droplet digital PCR platforms currently in use commercially. Our previous study discovered a molecular marker that can distinguish Hanwoo species (Korean native cattle) using Hanwoo-specific genomic structural variation. Here, we report the pros and cons of the operation of each dPCR platform from various perspectives using this species identification marker. In conclusion, we hope that this study will help researchers to select suitable dPCR platforms according to their purpose and resources.