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

Abstract

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.

Fig. 1

First screening of the 16 published RT‐LAMP primer mixes using 200 copies of the EDX SARS‐CoV‐2 RNA Standard. Yellow colour – positive result – presence of RNA, pink colour – negative result – absence of RNA. NC, negative control.

Fig. 2

Second screening of the selected RT‐LAMP primer mixes using 100 and 50 copies of the EDX SARS‐CoV‐2 RNA Standard. Yellow colour – positive result – presence of RNA, pink colour– negative result – absence of RNA. NC, negative control.

Fig. 3

Sensitivity comparison of the RT‐LAMP primer mixes selected from the second screening using the EDX SARS‐CoV‐2 RNA Standard. Yellow colour – positive result – presence of RNA, pink colour – negative result – absence of RNA. NC, negative control.

Fig. 4

RT‐LAMP with untreated salivary samples with a serial dilution of the EDX SARS‐CoV‐2 RNA Standard as a RNA template using the selected Yan et al. (S‐123) primer mix. Yellow colour – positive result – presence of RNA, pink colour – negative result – absence of RNA. NC, negative control.

Fig. 5

Effect of spiked saliva pretreatment on RT‐LAMP reactions performed with Yan et al. (S‐123) primer mix. Yellow colour – positive result – presence of RNA, pink colour – negative result – absence of RNA. NC, negative control.

Fig. 6

Effect of RNase inhibitor on RT‐LAMP on saliva samples with a serial dilution of the EDX SARS‐CoV‐2 RNA Standard as the RNA template performed with Yan et al. (S‐123) primer mix. Yellow colour – positive result – presence of RNA, pink colour – negative result – absence of RNA. NC, negative control.

Fig. 7

The RT‐LAMP on clinical saliva samples performed with Yan et al. (S‐123) primer mix and RNase inhibitor. Yellow colour – positive result – presence of RNA, pink colour – negative result – absence of RNA. PC, positive control; NC, negative control.

Fig. 8

Sensitivity of real‐time RT‐PCR targeting N1 and N3 gene of the EDX SARS‐CoV‐2 RNA Standard – 100 copies, 10 copies and 1 copy (A). Sensitivity of real‐time RT‐PCR targeting N1 and N3 gene of the SARS‐CoV‐2 Viral RNA – 1000 copies, 100 copies, 10 copies and 1 copy (B). NC – negative control.