Preliminary optimisation of a simplified sample preparation method to permit direct detection of SARS-CoV-2 within saliva samples using reverse-transcription loop-mediated isothermal amplification (RT-LAMP)

Abstract

We describe the optimisation of a simplified sample preparation method which permits rapid and direct detection of SARS-CoV-2 RNA within saliva, using reverse-transcription loop-mediated isothermal amplification (RT-LAMP). Treatment of saliva samples prior to RT-LAMP by dilution 1:1 in Mucolyse™, followed by dilution in 10 % (w/v) Chelex© 100 Resin and a 98 °C heat step for 2 min enabled detection of SARS-CoV-2 RNA in positive saliva samples. Using RT-LAMP, SARS-CoV-2 RNA was detected in as little as 05:43 min, with no amplification detected in 3097 real-time reverse transcription PCR (rRT-PCR) negative saliva samples from staff tested within a service evaluation study, or for other respiratory pathogens tested (n = 22). Saliva samples can be collected non-invasively, without the need for skilled staff and can be obtained from both healthcare and home settings. Critically, this approach overcomes the requirement for, and validation of, different swabs and the global bottleneck in obtaining access to extraction robots and reagents to enable molecular testing by rRT-PCR. Such testing opens the possibility of public health approaches for effective intervention during the COVID-19 pandemic through regular SARS-CoV-2 testing at a population scale, combined with isolation and contact tracing.

Keywords: COVID-19; Direct detection; Near patient testing; RT-LAMP; Rapid diagnostics; SARS-CoV-2; Saliva.

Fig. 1

Comparison between RT-LAMP and rRT-PCR for 20 saliva samples. (red) 1 in 5 dilution in Chelex® plus 98 °C heat step; (grey) 1 in 10 dilution in Chelex® plus 98 °C heat step; (blue) 1 in 20 dilution in Chelex® plus 98 °C heat step. Half-shaded points represent that of the duplicates, one was positive and the other negative. Both RT-LAMP and rRT-PCR assays were performed at HHFT; the points in the box represent a sample which was also diluted 1:1 in Mucolyse™ prior to analysis by rRT-PCR.