Institute of Molecular Biomedicine, Faculty of Medicine, Comenius University, Bratislava, Slovakia.
University Hospital, Bratislava, Slovakia.
Microb Biotechnol. 2021 Jan;14(1):307-316. doi: 10.1111/1751-7915.13737. Epub 2021 Jan 26.
In the fight against the recent COVID-19 pandemics, testing is crucial. Nasopharyngeal swabs and real-time RT-PCR are used for the detection of the viral RNA. The collection of saliva is non-invasive, pain-free and does not require trained personnel. An alternative to RT-PCR is loop-mediated isothermal amplification coupled with reverse transcription (RT-LAMP) that is easy to perform, quick and does not require a thermal cycler. The aim of this study was to test whether SARS-CoV-2 RNA can be detected directly in saliva using RT-LAMP. We have tested 16 primer mixes from the available literature in three rounds of sensitivity assays. The selected RT-LAMP primer mix has a limit of detection of 6 copies of viral RNA per reaction in comparison with RT-PCR with 1 copy per reaction. Whole saliva, as well as saliva collected using Salivette collection tubes, interfered with the RT-LAMP analysis. Neither Chelex-100 nor protease treatment of saliva prevented the inhibitory effect of saliva. With the addition of the ribonuclease inhibitor, the sensitivity of the RT-LAMP assay was 12 copies per reaction of RNA in Salivette® saliva samples and 6 copies per reaction of RNA in whole saliva samples. This study shows that it is possible to combine the use of saliva and RT-LAMP for SARS-CoV-2 RNA detection without RNA extraction which was confirmed on a small set of correctly diagnosed clinical samples. Further studies should prove whether this protocol is suitable for point of care testing in the clinical setting.
在抗击最近的 COVID-19 大流行中，检测至关重要。鼻咽拭子和实时 RT-PCR 用于检测病毒 RNA。采集唾液是非侵入性的，无痛的，不需要经过培训的人员。RT-PCR 的替代方法是环介导等温扩增结合逆转录 (RT-LAMP)，该方法易于操作，快速，并且不需要热循环仪。本研究旨在测试是否可以直接使用 RT-LAMP 在唾液中检测到 SARS-CoV-2 RNA。我们已经在三轮灵敏度检测中测试了来自现有文献的 16 个引物混合物。与每个反应检测 1 个拷贝的 RT-PCR 相比，选定的 RT-LAMP 引物混合物的检测限为每个反应 6 个拷贝的病毒 RNA。全唾液以及使用 Salivette 收集管收集的唾液会干扰 RT-LAMP 分析。Chelex-100 和唾液蛋白酶处理都不能防止唾液的抑制作用。加入核糖核酸酶抑制剂后，RT-LAMP 检测在 Salivette®唾液样本中的 RNA 反应中达到 12 拷贝/反应，在全唾液样本中的 RNA 反应中达到 6 拷贝/反应。本研究表明，有可能在不进行 RNA 提取的情况下将唾液和 RT-LAMP 结合使用来检测 SARS-CoV-2 RNA，这在一小部分正确诊断的临床样本中得到了证实。进一步的研究应证明该方案是否适合临床环境中的即时护理测试。
