DynaLIFE Medical Labs, Edmonton, Alberta, Canada; BioImmuno Designs, Edmonton, Alberta, Canada.
DynaLIFE Medical Labs, Edmonton, Alberta, Canada.
Diagn Microbiol Infect Dis. 2021 Oct;101(2):115458. doi: 10.1016/j.diagmicrobio.2021.115458. Epub 2021 Jun 17.
We describe an extractionless real-time reverse transcriptase-PCR (rRT-PCR) protocol for SARS-CoV-2 nucleic acid detection using heat as an accurate cost-effective high-capacity solution to COVID-19 testing. We present the effect of temperature, transport media, rRT-PCR mastermixes and gene assays on SARS-CoV-2 gene amplification and limits of detection. Utilizing our heated methodology, our limits of detection were 12.5 and 1 genome copy/reaction for singleplex E- and N1-gene assays, respectively, and 1 genome copy/reaction by utilizing an E/N1 or Orf1ab/N1 multiplex assay combination. Using this approach, we detected up to 98% of COVID-19 positive patient samples analyzed in our various cohorts including a significant percentage of weak positives. Importantly, this extractionless approach will allow for >2-fold increase in testing capacity with existing instruments, circumvent the additional need for expensive extraction devices, provide the sensitivity needed for COVID-19 detection and significantly reduce the turn-around time of reporting COVID-19 test results.
我们描述了一种无需提取的实时逆转录聚合酶链反应（rRT-PCR）方案，用于 SARS-CoV-2 核酸检测，该方案使用热作为一种准确、具有成本效益的 COVID-19 检测的高通量解决方案。我们介绍了温度、运输介质、rRT-PCR 主混合物和基因检测对 SARS-CoV-2 基因扩增和检测限的影响。利用我们的加热方法，我们的单重 E 基因和 N 基因检测的检测限分别为 12.5 和 1 基因组拷贝/反应，而利用 E/N1 或 Orf1ab/N1 多重检测组合，则可以达到 1 基因组拷贝/反应。使用这种方法，我们检测到了包括大量弱阳性患者样本在内的各种队列中高达 98%的 COVID-19 阳性患者样本。重要的是，这种无需提取的方法将允许在现有仪器上增加 2 倍以上的检测能力，避免对昂贵的提取设备的额外需求，提供 COVID-19 检测所需的灵敏度，并大大缩短 COVID-19 检测结果报告的周转时间。
