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, 2020, to November 19, 2020, surveillance samples (n = 4704) were collected from volunteers and tested for SARS-CoV-2 at 5 sites. Twenty-one 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, whereas 8 tested 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 false-negative rate of the RT-LAMP assay only from July 16, 2020, to September 17, 2020 by pooling residual heat-inactivated saliva that was unambiguously negative by RT-LAMP into groups of 6 or fewer 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 (2493 total 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 that can effectively identify asymptomatic individuals who would not typically meet the criteria for symptom-based testing modalities.

Keywords: colorimetric LAMP; point-of-care test; saliva-based.

FIGURE 1

Point-of-care reverse-transcription loop-mediated isothermal amplification (RT-LAMP) severe acute respiratory syndrome coronavirus 2 (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 a positive result. Figure was created using BioRender.com (Toronto, ON, Canada).

FIGURE 2

Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in contrived saliva samples by direct reverse-transcription loop-mediated isothermal amplification (RT-LAMP). A) Representative example of a positive sample in 2 of 2 replicates. Sample used negative saliva spiked with irSARS-CoV-2. B) Representative example of a sample positive in 1 of 2 replicates C) Representative negative sample showing no colorimetric change in either replicate. D) Bar graph of results of limit of detection (LOD) assessment with contrived saliva samples from 19 volunteers. γ-Irradiated SARS-CoV-2 (irSARS-CoV-2) vRNA load is shown as copies/μL on the x-axis; the number (No.) of samples positive in 2 (black), 1 (dark gray), or zero (light gray) replicates is shown on the y-axis.

FIGURE 3

Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 38 clinical saliva specimens by direct reverse-transcription loop-mediated isothermal amplification (RT-LAMP). The viral RNA (vRNA) load of each clinical sample is plotted on the x-axis relative to the in-house Centers for Disease Control and Prevention–based N1 quantitative reverse-transcription polymerase chain reaction (qRT-PCR) assay cycle threshold (Ct) on the y-axis. Black, dark gray, and light gray indicate 2, 1, and zero positive replicates, respectively.