Initial evaluation of a mobile SARS-CoV-2 RT-LAMP testing strategy

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) control in the United States remains hampered, in part, by testing limitations. We evaluated a simple, outdoor, mobile, colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay workflow where self-collected saliva is tested for SARS-CoV-2 RNA. From July 16 to November 19, 2020, 4,704 surveillance samples were collected from volunteers and tested for SARS-CoV-2 at 5 sites. A total of 21 samples tested positive for SARS-CoV-2 by RT-LAMP; 12 were confirmed positive by subsequent quantitative reverse-transcription polymerase chain reaction (qRT-PCR) testing, while 8 were negative for SARS-CoV-2 RNA, and 1 could not be confirmed because the donor did not consent to further molecular testing. We estimated the RT-LAMP assay's false-negative rate from July 16 to September 17, 2020 by pooling residual heat-inactivated saliva that was unambiguously negative by RT-LAMP into groups of 6 or less and testing for SARS-CoV-2 RNA by qRT-PCR. We observed a 98.8% concordance between the RT-LAMP and qRT-PCR assays, with only 5 of 421 RT-LAMP negative pools (2,493 samples) testing positive in the more sensitive qRT-PCR assay. Overall, we demonstrate a rapid testing method that can be implemented outside the traditional laboratory setting by individuals with basic molecular biology skills and can effectively identify asymptomatic individuals who would not typically meet the criteria for symptom-based testing modalities.

Figure 1:. Point-of-care RT-LAMP SARS-CoV-2 testing workflow.

Steps 1–5. Saliva sample preparation. Steps 6–7. RT-LAMP reagent preparation. Steps 8–10. RT-LAMP reactions and results interpretation. A reaction color change from pink/orange to yellow after 30 minutes in at least 1 of 2 sample replicates was scored as positive. Figure was created using BioRender.com.

Figure 2:. Detection of SARS-CoV-2 in contrived saliva samples by direct RT-LAMP.

A. Initial limit of detection (LOD) assessment with contrived saliva samples from 3 volunteers (S1, S2, S3). RT-LAMP reactions determined to be negative are pink and those determined to be positive are yellow. Quantitative RT-PCR vRNA loads are presented as copies/μl above the replicates for each sample. B. Bar graph showing an expanded assessment of RT-LAMP LOD for 22 additional contrived saliva samples (S4–S25). Gamma-irradiated SARS-CoV-2 (irSARS-CoV-2) vRNA load is shown as copies/μl on the x-axis, number of samples positive in 2 (black), 1 (dark gray), or 0 (light gray) replicates is shown on the y-axis.

Figure 3:. Detection of SARS-CoV-2 in 38 clinical saliva specimens by direct RT-LAMP.

The vRNA load of each clinical sample is plotted on the x-axis relative to the in-house CDC N1 qRT-PCR assay cycle threshold (Ct) on the y-axis. Black, dark gray, and light gray indicate 2, 1, and 0 positive replicates respectively.