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

Eva Rajh,Žiga Jensterle,Viktorija Tomič,Mojca Benčina,Mojca Milavec,Tina Šket,Katarina Prosenc Trilar,Tina Demšar,Gabriele Turel,Mihaela Zidarn,Polona Kogovšek,Dunja Urbančič,Irena Mlinarič-Raščan,Arne Praznik,Roman Jerala,Tatjana Lejko-Zupanc,Petra Sušjan,Alenka Šmid

doi:10.3390/molecules26216617

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.

Highlights

The COVID-19 pandemic caused by the SARS-CoV-2 virus has, since its beginning in 2020, caused a worldwide humanitarian crisis, with an unprecedented death toll and widespread economic crises
For individuals whose saliva specimens tested positive for SARS-CoV-2, the nasopharyngeal swab (NPS) was collected to confirm the infection
We developed an RNA stabilization buffer that is compatible with nucleic acid amplification tests, meaning that the saliva specimens can be analyzed without RNA extraction before the reverse transcription loop-mediated isothermal amplification (RT-LAMP) and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) tests [16]

Summary

Introduction

The COVID-19 pandemic caused by the SARS-CoV-2 virus has, since its beginning in 2020, caused a worldwide humanitarian crisis, with an unprecedented death toll and widespread economic crises (https://reliefweb.int/topics/covid-19-global, accessed on 10 October 2020). Early detection, isolation, and management of infected individuals all play critical roles in avoiding further escalation of the pandemic. The basic tools for preventing the spread of the virus among the population are disease screening, isolation of infected individuals, identifying and alerting asymptomatic individuals, and contact tracing. A nasopharyngeal swab (NPS) assessed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the recommended diagnosis method for COVID-19 detection [1,2]. For NPS collection, trained personnel are required; this exposes the staff to a higher risk of infection, is costly, and less suitable for mass testing. NPS sampling causes discomfort to individuals and is associated with contraindications, such as coagulopathy, or anticoagulant therapy, and nasal septum deviations

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