Wang Kangning, Li Bin, Guo Yu, Wu Yanqi, Li Yan, Wu Wenming
Institute of Biological and Medical Engineering, Guangdong Academy of Sciences, Guangzhou, China.
Institute of Microbiology Chinese Academy of Sciences, Beijing, China.
Front Bioeng Biotechnol. 2022 Aug 19;10:947895. doi: 10.3389/fbioe.2022.947895. eCollection 2022.
Digital PCR is the most advanced PCR technology. However, due to the high price of the digital PCR analysis instrument, this powerful nucleic acid detection technology is still difficult to be popularized in the general biochemistry laboratory. Moreover, one of the biggest disadvantages of commercial digital PCR systems is the poor versatility of reagents: each instrument can only be used for a few customized kits. Herein, we built a low-cost digital PCR system. The system only relies on low-cost traditional flat-panel PCR equipment to provide temperature conditions for commercial dPCR chips, and the self-made fluorescence detection system is designed and optically optimized to meet a wide range of reagent requirements. More importantly, our system not only has a low cost (<8000 US dollars) but also has a much higher universality for nucleic acid detection reagents than the traditional commercial digital PCR system. In this study, several samples were tested. The genes used in the experiment were plasmids containing UPE-1a fragment, TP53 reference DNA, hepatitis B virus DNA, leukemia sample, SARS-COV-2 DNA, and SARS-COV-2 RNA. Under the condition that DNA can be amplified normally, the function of the dPCR system can be realized with simpler and low-price equipment. Some DNA cannot be detected by using the commercial dPCR system because of the special formula when it is configured as the reaction solution, but these DNA fluorescence signals can be clearly detected by our system, and the concentration can be calculated. Our system is more applicable than the commercial dPCR system to form a new dPCR system that is smaller and more widely applicable than commercially available machinery.
数字PCR是最先进的PCR技术。然而，由于数字PCR分析仪价格高昂，这种强大的核酸检测技术在普通生物化学实验室中仍难以普及。此外，商业数字PCR系统最大的缺点之一是试剂通用性差：每台仪器只能用于少数定制试剂盒。在此，我们构建了一种低成本数字PCR系统。该系统仅依靠低成本的传统平板PCR设备为商业dPCR芯片提供温度条件，并对自制的荧光检测系统进行了设计和光学优化，以满足广泛的试剂需求。更重要的是，我们的系统不仅成本低（<8000美元），而且在核酸检测试剂方面比传统商业数字PCR系统具有更高的通用性。在本研究中，对几个样本进行了测试。实验中使用的基因是含有UPE-1a片段的质粒、TP53参考DNA、乙型肝炎病毒DNA、白血病样本、SARS-CoV-2 DNA和SARS-CoV-2 RNA。在DNA能够正常扩增的情况下，使用更简单、价格更低的设备即可实现dPCR系统的功能。一些DNA在配置为反应溶液时由于特殊配方无法用商业dPCR系统检测，但我们的系统能够清晰检测到这些DNA的荧光信号，并计算出其浓度。我们的系统比商业dPCR系统更适用，形成了一种比市售仪器更小、应用更广泛的新型dPCR系统。
