Sample-to-answer, extraction-free, real-time RT-LAMP test for SARS-CoV-2 in nasopharyngeal, nasal, and saliva samples: Implications and use for surveillance testing.

Kathryn A Kundrod,Ellen Baker,Sai Paul,Christopher Goh,Megan M Chang,Keyur P Patel,Rebecca Richards-Kortum,Anthony Price,Mary E Natoli,Dereq Ogoe,Chelsey A Smith,Kathleen M Schmeler,Akshaya Santhanaraj,Karen W Eldin,A M Abd El-Aty

doi:10.1371/journal.pone.0264130

Abstract

The global COVID-19 pandemic has highlighted the need for rapid, accurate and accessible nucleic acid tests to enable timely identification of infected individuals. We optimized a sample-to-answer nucleic acid test for SARS-CoV-2 that provides results in <1 hour using inexpensive and readily available reagents. The test workflow includes a simple lysis and viral inactivation protocol followed by direct isothermal amplification of viral RNA using RT-LAMP. The assay was validated using two different instruments, a portable isothermal fluorimeter and a standard thermocycler. Results of the RT-LAMP assay were compared to traditional RT-qPCR for nasopharyngeal swabs, nasal swabs, and saliva collected from a cohort of patients hospitalized due to COVID-19. For all three sample types, positive agreement with RT-LAMP performed using the isothermal fluorimeter was 100% for samples with Ct <30 and 69-91% for samples with Ct <40. Following validation, the test was successfully scaled to test the saliva of up to 400 asymptomatic individuals per day as part of the campus surveillance program at Rice University. Successful development, validation, and scaling of this sample-to-answer, extraction-free real-time RT-LAMP test for SARS-CoV-2 adds a highly adaptable tool to efforts to control the COVID-19 pandemic, and can inform test development strategies for future infectious disease threats.

Highlights

The global COVID-19 pandemic caused by the SARS-CoV-2 virus has demonstrated the need for highly accessible diagnostic and surveillance tests
The oligonucleotide primers for the SARS-CoV-2 RT-loop-mediated isothermal amplification (LAMP) assay were the N2 and E1 primer sets designed by New England Biolabs, Inc. [35] and the Orf1a-HMSe (As1e) primer set designed by Rabe and Cepko [17], which target the N, E, and ORF1a genes
Primers were resuspended in 1X TE buffer at a 1 mM concentration and combined to make a 25X mix consisting of 40 μM FIP and BIP, 5 μM F3 and B3, and 10 μM LF and LB in 1X TE buffer. 2019-nCOV CDC primer and probe mixes were purchased in Emergency Use Authorization (EUA) or RUO kits from Integrated DNA Technologies (IDT), and were used interchangeably in RT-qPCR

Summary

Introduction

The global COVID-19 pandemic caused by the SARS-CoV-2 virus has demonstrated the need for highly accessible diagnostic and surveillance tests. SARS-CoV-2 transmission by individuals who are asymptomatic requires routine population-level surveillance for effective outbreak containment when rates of community transmission are high. RT-qPCR testing requires sample collection into an appropriate medium, RNA extraction, and RT-qPCR reaction assembly prior to running the test on a thermocycler. After RT-qPCR testing became available across the country, supply shortages of sample collection kits, RNA extraction kits, RT-qPCR reagents, automated laboratory liquid handlers, and thermocyclers were common and unpredictable. Traditional RT-qPCR remains the standard-of-care method for SARS-CoV-2 diagnosis, but given the supply chain challenges and infrastructure required, alternative methods for surveillance were desirable

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Results

Conclusion