Preliminary optimisation of 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

Abstract We describe the optimization 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 (within the range of 1:5 to 1:40) in 10% (w/v) Chelex © 100 Resin and a 98°C heat step for 2 minutes enabled detection of SARS-CoV-2 RNA in all positive saliva samples tested, with no amplification detected in pooled negative saliva. The time to positivity for which SARS- CoV-2 RNA was detected in these positive saliva samples was proportional to the real-time reverse- transcriptase PCR cycle threshold (C T ), with SARS-CoV-2 RNA detected in as little as 05:43 (C T 21.08), 07:59 (C T 24.47) and 08:35 (C T 25.27) minutes, respectively. The highest C T where direct RT-LAMP detected SARS-CoV-2 RNA was 31.39 corresponding to a 1:40 dilution of a positive saliva sample with a starting C T of 25.27. When RT-LAMP was performed on pools of SARS-CoV-2 negative saliva samples spiked with whole inactivated SARS-CoV-2 virus, RNA was detected at dilutions spanning 1:5 to 1:160 representing C T ’s spanning 22.49-26.43. Here we describe a simple but critical rapid sample preparation method which can be used up front of RT-LAMP to permit direct detection of SARS-CoV- 2 within saliva samples. Saliva is a sample which can be collected non-invasively without the use of highly skilled staff and critically can be obtained from both health care and home settings. Critically, this approach overcomes both the requirement and validation of different swabs and the global bottleneck observed in obtaining RNA extraction robots and reagents to enable molecular testing by PCR. Such testing opens the possibility of public health approaches for effective intervention to control the COVID-19 pandemic through regular SARS-CoV-2 testing at a population scale, combined with isolation and contact tracing for positive cases.