Department of Life and Nanopharmaceutical Sciences, Graduate School, Kyung Hee University, Seoul, 02447, South Korea.
R&D Center, Ahram Biosystems Inc, Seoul, 04794, South Korea.
Mol Biol Rep. 2019 Feb;46(1):639-645. doi: 10.1007/s11033-018-4519-7. Epub 2018 Nov 27.
Rapid and on-site DNA-based molecular detection has become increasingly important for sensitive, specific, and timely detection and treatment of various diseases. To prepare and store biomolecule-containing reagents stably, reducing agents are used during protein preparation, and freeze-drying technology has been applied to the protein reagents. Some of the additives used during these processes may affect subsequent processes such as polymerase chain reaction (PCR). In this study, we evaluated the impact of TCEP, a reducing agent, and TBA, a freeze-drying medium, on the performance of convection PCR (cPCR) using a battery-operable PCR device. Singleplex cPCR detection of a 249 bp amplicon from human genomic DNA suggested that approximately 82% of performance was achieved in the presence of 0.1 mM TCEP and 1% TBA. The limit of detection and the minimum number of cycles at which amplicons began to appear was a little lower (~ 82% efficiency) or higher (20 vs 15 cycles), respectively, in the chemical-treated group than in the control group. With larger amplicons of 500 bp, the chemical-treated group revealed approximately 78% of performance and amplicons started to appear at 20 cycles of cPCR in both groups. Similar results were obtained with multiplex cPCR amplification.
快速且现场的基于 DNA 的分子检测对于各种疾病的敏感、特异和及时检测和治疗变得越来越重要。为了稳定地制备和储存含生物分子的试剂，在蛋白质制备过程中使用还原剂，并且将冻干技术应用于蛋白质试剂。这些过程中使用的一些添加剂可能会影响随后的过程，如聚合酶链反应（PCR）。在这项研究中，我们使用可电池供电的 PCR 设备评估了还原剂 TCEP 和冻干介质 TBA 对对流 PCR（cPCR）性能的影响。使用来自人基因组 DNA 的 249 bp 扩增子的单重 cPCR 检测表明，在存在 0.1 mM TCEP 和 1% TBA 的情况下，大约 82%的性能得以实现。在化学处理组中，检测限和扩增子开始出现的最小循环数分别略低（~82%效率）或略高（20 与 15 个循环），而在对照组中则更高。对于更大的 500 bp 扩增子，化学处理组显示出大约 78%的性能，并且在两组中 cPCR 的 20 个循环中扩增子开始出现。多重 cPCR 扩增也得到了类似的结果。
