Robust Saliva-Based RNA Extraction-Free One-Step Nucleic Acid Amplification Test for Mass SARS-CoV-2 Monitoring

Abstract

Early diagnosis with rapid detection of the virus plays a key role in preventing the spread of infection and in treating patients effectively. In order to address the need for a straightforward detection of SARS-CoV-2 infection and assessment of viral spread, we developed rapid, sensitive, extraction-free one-step reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP) tests for detecting SARS-CoV-2 in saliva. We analyzed over 700 matched pairs of saliva and nasopharyngeal swab (NSB) specimens from asymptomatic and symptomatic individuals. Saliva, as either an oral cavity swab or passive drool, was collected in an RNA stabilization buffer. The stabilized saliva specimens were heat-treated and directly analyzed without RNA extraction. The diagnostic sensitivity of saliva-based RT-qPCR was at least 95% in individuals with subclinical infection and outperformed RT-LAMP, which had at least 70% sensitivity when compared to NSBs analyzed with a clinical RT-qPCR test. The diagnostic sensitivity for passive drool saliva was higher than that of oral cavity swab specimens (95% and 87%, respectively). A rapid, sensitive one-step extraction-free RT-qPCR test for detecting SARS-CoV-2 in passive drool saliva is operationally simple and can be easily implemented using existing testing sites, thus allowing high-throughput, rapid, and repeated testing of large populations. Furthermore, saliva testing is adequate to detect individuals in an asymptomatic screening program and can help improve voluntary screening compliance for those individuals averse to various forms of nasal collections.

Development and implementation of a simple and rapid extraction-free saliva SARS-CoV-2 RT-LAMP workflow for workplace surveillance

“…While several saliva-based approaches have been demonstrated for the detection of SARS-CoV-2 using LAMP [ 8 , 14 , 30 , 31 , 35 , 41 – 45 ], including extraction-free or direct protocols [ 13 , 30 , 35 , 41 , 42 , 44 , 46 , 47 ], no gold standard method exists. We focused on the development of a simple and improved sample preparation protocol compatible with this complex sample and downstream LAMP reactions.…”

“…We also discovered that pooling saliva prior to testing can be beneficial in cases where a particular specimen is problematic due to low pH, color, or viscosity, or has substances that interfere with nucleic acid amplification. Pooling saliva for large-scale surveillance programs has proven to be a highly cost-effective strategy [ 12 , 17 , 42 , 59 , 60 ] and used successfully in schools and universities to routinely identify asymptomatic individuals in pool sizes comprised of 24 samples [ 17 ]. These studies include an RNA purification step and RT-qPCR for detection.…”

Development and Implementation of a Simple and Rapid Extraction-Free Saliva SARS-CoV-2 RT-LAMP Workflow for Workplace Surveillance

“…While several saliva-based approaches have been demonstrated for the detection of SARS-CoV-2 using LAMP [8,14,30,31,35,[37][38][39][40][41], including extraction-free or direct protocols [13,30,35,37,38,40,42,43], no gold standard method exists. We focused on the development of a simple and improved sample preparation protocol compatible with this complex sample and downstream LAMP reactions.…”

“…We also discovered that pooling saliva prior to testing can be beneficial in cases where a particular specimen is problematic due to low pH, color, or viscosity, or has substances that interfere with nucleic acid amplification. Pooling saliva for large-scale surveillance programs has proven to be a highly cost-effective strategy [12, 17, 38, 55, 56] and used successfully in schools and universities to routinely identify asymptomatic individuals in pool sizes comprised of 24 samples [17]. These studies include an RNA purification step and RT-qPCR for detection.…”

“…At 10 days after the initial positive test, we found 13% of positive individuals still testing positive with most of them testing negative for viral RNA by day 14. This may be a general trend for saliva-based COVID diagnostics since in a mass screening program in Slovenia, the viral load in saliva peaked within the first week of infection with most individuals testing negative within 2 weeks after the first positive RT-LAMP test [38].…”