Introduction. The SARS-CoV-2 pandemic of 2020 has resulted in unparalleled requirements for RNA extraction kits and enzymes required for virus detection, leading to global shortages. This has necessitated the exploration of alternative diagnostic options to alleviate supply chain issues. Aim. To establish and validate a reverse transcription loop-mediated isothermal amplification (RT- LAMP) assay for the detection of SARS-CoV-2 from nasopharyngeal swabs. Methodology. We used a commercial RT-LAMP mastermix from OptiGene in combination with a primer set designed to detect the CDC N1 region of the SARS-CoV-2 nucleocapsid (N) gene. A single-tube, single-step fluorescence assay was implemented whereby 1 µl of universal transport medium (UTM) directly from a nasopharyngeal swab could be used as template, bypassing the requirement for RNA purification. Amplification and detection could be conducted in any thermocycler capable of holding 65 °C for 30 min and measure fluorescence in the FAM channel at 1 min intervals. Results. Assay evaluation by assessment of 157 clinical specimens previously screened by E-gene RT-qPCR revealed assay sensitivity and specificity of 87 and 100%, respectively. Results were fast, with an average time-to-positive (Tp) for 93 clinical samples of 14 min (sd±7 min). Using dilutions of SARS-CoV-2 virus spiked into UTM, we also evaluated assay performance against FDA guidelines for implementation of emergency-use diagnostics and established a limit-of-detection of 54 Tissue Culture Infectious Dose 50 per ml (TCID50 ml−1), with satisfactory assay sensitivity and specificity. A comparison of 20 clinical specimens between four laboratories showed excellent interlaboratory concordance; performing equally well on three different, commonly used thermocyclers, pointing to the robustness of the assay. Conclusion. With a simplified workflow, The N1 gene Single Tube Optigene LAMP assay (N1-STOP-LAMP) is a powerful, scalable option for specific and rapid detection of SARS-CoV-2 and an additional resource in the diagnostic armamentarium against COVID-19.

中文翻译：

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验证用于快速检测 SARS-CoV-2 RNA 的单步、单管逆转录环介导等温扩增测定。


介绍。 2020 年的 SARS-CoV-2 大流行导致对病毒检测所需的 RNA 提取试剂盒和酶的需求空前，导致全球短缺。这就需要探索替代诊断方案来缓解供应链问题。目的。建立并验证逆转录环介导的等温扩增 (RT-LAMP) 检测法，用于检测鼻咽拭子中的 SARS-CoV-2。方法论。我们使用 OptiGene 的商业 RT-LAMP mastermix 与设计用于检测 SARS-CoV-2 核衣壳 (N) 基因的 CDC N1 区域的引物组相结合。实施了单管、单步荧光测定，直接从鼻咽拭子中取 1 µl 通用转运介质 (UTM) 作为模板，绕过了 RNA 纯化的要求。扩增和检测可以在任何能够保持 65°C 30 分钟的热循环仪中进行，并以 1 分钟的间隔测量 FAM 通道中的荧光。结果。通过评估之前通过 E-gene RT-qPCR 筛选的 157 份临床样本进行的检测评估显示，检测灵敏度和特异性分别为 87% 和 100%。结果很快，93 个临床样本的平均阳性时间 (Tp) 为 14 分钟 ( sd ±7 分钟)。使用掺入 UTM 中的 SARS-CoV-2 病毒稀释液，我们还根据 FDA 实施紧急使用诊断指南评估了检测性能，并确定了每毫升 54 组织培养感染剂量 50 的检测限（TCID 50毫升- 1 )、具有令人满意的测定灵敏度和特异性。 对四个实验室之间 20 个临床标本的比较显示出极好的实验室间一致性；在三种不同的常用热循环仪上表现同样良好，表明该测定的稳健性。结论。 N1 基因单管 Optigene LAMP 检测 (N1-STOP-LAMP) 具有简化的工作流程，是一种功能强大、可扩展的选项，可用于特异性、快速检测 SARS-CoV-2，也是针对 COVID-19 的诊断设备中的额外资源。