Portable and accurate diagnostics for COVID-19: Combined use of the miniPCR thermocycler and a well-plate reader for SARS-CoV-2 virus detection
Portable and accurate diagnostics for COVID-19: Combined use of the miniPCR thermocycler and a well-plate reader for SARS-CoV-2 virus detection
复制标题
DOI：
10.1371/journal.pone.0237418
复制
发表时间：
2020-08-13
期刊：
影响因子：
3.7
通讯作者：
Moises Alvarez, Mario
中科院分区：
文献类型：
--
作者：
Gonzalez-Gonzalez, Everardo;Trujillo-de Santiago, Grissel;Moises Alvarez, Mario
2019年冠状病毒病（COVID-19）大流行已经粗略地证明了大规模和快速诊断的必要性。截至7月的第一周，全球已报告了1000多万例COVID-19阳性病例，尽管这一数字可能被大大低估。在发生流行病紧急情况的情况下，第一道反应应以商业上可获得的和经过验证的资源为基础。在这里，我们展示了将最近在市场上销售的小型便携式商用PCR装置miniPCR与商用孔板读取器结合使用，作为检测COVID-19致病因子-严重急性呼吸综合征冠状病毒2 （SARS-CoV-2）遗传物质的诊断系统。我们使用miniPCR检测和扩增SARS-CoV-2 DNA序列，使用世界卫生组织（WHO）推荐的针对编码N蛋白的三个不同区域的启动物组。在扩增之前，将样品与DNA插层试剂（即evgreen Dye）结合。扩增后的样品荧光用商用96孔板读卡器读取。这种简单的方法允许在类似于625到2x10(5)个DNA拷贝的范围内检测和扩增SARS-CoV-2核酸。这种诊断策略的准确性和简单性可为COVID-19大流行检测提供一种具有成本效益和可靠的替代方案，特别是在RT-QPCR仪器可用性可能有限的不发达地区。miniPCR的便携性、易用性和可重复性使其成为大流行期间在医疗点检测SARS-CoV-2工作中部署的可靠替代方案。
The coronavirus disease 2019 (COVID-19) pandemic has crudely demonstrated the need for massive and rapid diagnostics. By the first week of July, more than 10,000,000 positive cases of COVID-19 have been reported worldwide, although this number could be greatly underestimated. In the case of an epidemic emergency, the first line of response should be based on commercially available and validated resources. Here, we demonstrate the use of the miniPCR, a commercial compact and portable PCR device recently available on the market, in combination with a commercial well-plate reader as a diagnostic system for detecting genetic material of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of COVID-19. We used the miniPCR to detect and amplify SARS-CoV-2 DNA sequences using the sets of initiators recommended by the World Health Organization (WHO) for targeting three different regions that encode for the N protein. Prior to amplification, samples were combined with a DNA intercalating reagent (i.e., EvaGreen Dye). Sample fluorescence after amplification was then read using a commercial 96-well plate reader. This straightforward method allows the detection and amplification of SARS-CoV-2 nucleic acids in the range of similar to 625 to 2x10(5)DNA copies. The accuracy and simplicity of this diagnostics strategy may provide a cost-efficient and reliable alternative for COVID-19 pandemic testing, particularly in underdeveloped regions where RT-QPCR instrument availability may be limited. The portability, ease of use, and reproducibility of the miniPCR makes it a reliable alternative for deployment in point-of-care SARS-CoV-2 detection efforts during pandemics.