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(C) 100 Resin and a 98{degrees}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 (CT), with SARS-CoV-2 RNA detected in as little as 05:43 (CT 21.08), 07:59 (CT 24.47) and 08:35 (CT 25.27) minutes, respectively. The highest CT 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 CT 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 CTs 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.
ABSTRACT
The COVID-19 pandemic has illustrated the importance of rapid, accurate diagnostic testing for the effective triaging and cohorting of patients and timely tracking and tracing of cases. However, a surge in diagnostic testing quickly resulted in worldwide competition for the same sample preparation and real-time RT-PCR diagnostic reagents (rRT-PCR). Consequently, Hampshire Hospitals NHS Foundation Trust, UK sought to diversify their diagnostic portfolio by exploring alternative amplification chemistries including those that permit direct testing without RNA extraction. This study describes the validation of a SARS-CoV-2 RT-LAMP assay, which is an isothermal, autocycling, strand-displacement nucleic acid amplification technique which can be performed on extracted RNA (RNA RT-LAMP) or directly from swab (Direct RT-LAMP). Analytical specificity (ASp) of this new RT-LAMP assay was 100% and analytical sensitivity (ASe) was between 1[x]101 and 1[x]102 copies when using a synthetic DNA target. The overall diagnostic sensitivity (DSe) and specificity (DSp) of RNA RT-LAMP was 97% and 99% respectively, relative to the standard of care (SoC) rRT-PCR. When a CT cut-off of 33 was employed, above which increasingly evidence suggests there is a very low risk of patients shedding infectious virus, the diagnostic sensitivity was 100%. The DSe and DSp of Direct-RT-LAMP was 67% and 97%, respectively. When setting CT cut-offs of [≤]33 and [≤]25, the DSe increased to 75% and 100%, respectively. Time from swab-to-result for a strong positive sample (CT < 25) was < 15 minutes. We propose that RNA RT-LAMP could replace rRT-PCR where there is a need for increase in throughput, whereas Direct RT-LAMP could be used as a screening tool for triaging patients into appropriate hospitals wards, at GP surgeries and in care homes, or for population screening to identify super shedders. Direct RT-LAMP could also be used during times of high prevalence to save critical extraction and rRT-PCR reagents by screening out those strong positives from diagnostic pipelines.
