Loop‐mediated isothermal amplification for the detection of SARS‐CoV‐2 in saliva

Abstract

Summary 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.