Laboratory for Applied Science and Technology in Health, Carlos Chagas Institute, Oswaldo Cruz Foundation (Fiocruz), Prof. Algacyr Munhoz Mader 3775 Street, Curitiba, 81350-010, Brazil.
Erasto Gaertner Hospital, Dr. Ovande Do Amaral 201 Street, Curitiba, Paraná, 81520-060, Brazil.
Sci Rep. 2024 May 9;14(1):10612. doi: 10.1038/s41598-024-61163-7.
Molecular diagnostics involving nucleic acids (DNA and RNA) are regarded as extremely functional tools. During the 2020 global health crisis, efforts intensified to optimize the production and delivery of molecular diagnostic kits for detecting SARS-CoV-2. During this period, RT-LAMP emerged as a significant focus. However, the thermolability of the reagents used in this technique necessitates special low-temperature infrastructure for transport, storage, and conservation. These requirements limit distribution capacity and necessitate cost-increasing adaptations. Consequently, this report details the development of a lyophilization protocol for reagents in a colorimetric RT-LAMP diagnostic kit to detect SARS-CoV-2, facilitating room-temperature transport and storage. We conducted tests to identify the ideal excipients that maintain the molecular integrity of the reagents and ensure their stability during room-temperature storage and transport. The optimal condition identified involved adding 5% PEG 8000 and 75 mM trehalose to the RT-LAMP reaction, which enabled stability at room temperature for up to 28 days and yielded an analytical and diagnostic sensitivity and specificity of 83.33% and 90%, respectively, for detecting SARS-CoV-2. This study presents the results of a lyophilized colorimetric RT-LAMP COVID-19 detection assay with diagnostic sensitivity and specificity comparable to RT-qPCR, particularly in samples with high viral load.
涉及核酸（DNA 和 RNA）的分子诊断被认为是极具功能性的工具。在 2020 年全球卫生危机期间，人们加紧努力优化用于检测 SARS-CoV-2 的分子诊断试剂盒的生产和交付。在此期间，逆转录环介导等温扩增（RT-LAMP）成为一个重要的关注点。然而，该技术中使用的试剂的热敏性需要特殊的低温基础设施来进行运输、储存和保存。这些要求限制了分配能力，并需要增加成本的适应措施。因此，本报告详细介绍了一种用于检测 SARS-CoV-2 的比色 RT-LAMP 诊断试剂盒中试剂的冻干方案的开发，该方案便于在室温下运输和储存。我们进行了测试，以确定理想的赋形剂，这些赋形剂可以保持试剂的分子完整性，并确保其在室温下储存和运输过程中的稳定性。确定的最佳条件涉及在 RT-LAMP 反应中添加 5% PEG 8000 和 75 mM 海藻糖，这使得试剂在室温下稳定长达 28 天，并且对检测 SARS-CoV-2 的分析和诊断灵敏度和特异性分别为 83.33%和 90%。本研究介绍了一种冻干比色 RT-LAMP COVID-19 检测分析的结果，其诊断灵敏度和特异性可与 RT-qPCR 相媲美，尤其是在病毒载量较高的样本中。
