Fig 1.
Loop-mediated isothermal amplification (LAMP) detection of SARS-CoV-2 RNA captured from saliva using Whatman no. 1 filter.
A. Detection of naked SARS-CoV-2 RNA in saliva. Saliva with spike-in SARS-CoV-2 RNA (tubes 1–3), saliva without RNA spike-in (tubes 4–6), SARS-CoV-2 RNA added directly to LAMP reaction (tube 7), no template control (tube 8). B. Detection of encapsulated SARS-CoV-2 RNA particles in saliva (tubes 1–4); saliva with spike-in encapsulated RNase P RNA particles (tube 5); saliva with spike-in encapsulated SARS-CoV-2 RNA particles with no extraction treatment (tube 6), saliva alone with no spike-in (tube 7), and no saliva (tube 8). LAMP reactions used N2+E1 primers for detection of SARS-CoV-2 RNA. Concentrations are in copies per microliter of saliva.
Table 1.
Compatibility of materials with colorimetric LAMP.
Fig 2.
Overview of WHotLAMP assay and primers.
A. Schematic of the WHotLAMP assay. B. Location of ZI-1 LAMP primers and amplicon relative to mutations (vertical lines) defining SARS-CoV-2 variants.
Fig 3.
Specificity of SARS-CoV-2 LAMP primers.
A. Representative LAMP reactions using ZI-1 LAMP primers with 1x105 copies of SARS-CoV-1 DNA (tubes 1–3), MERS DNA (tubes 4–6), SARS-CoV-2 RNA (tube 7), and no template control (tube 8). B and C, same as A but with CUFC1 or N2+E1 LAMP primers, respectively. D. Representative LAMP reactions with ZI-1 LAMP primers using WHotLAMP detecting different respiratory pathogens (Pools 1–5), no respiratory pathogens (- Ctrl), and with inactivated SARS-CoV-2 virions (+CoV-2 Ctrl). ***P<0.0001 vs. positive CoV-2 control by Fisher’s exact test.
Fig 4.
Detection of RAB7A RNA in saliva.
A. LAMP reactions using RAB7A LAMP primers with purified RNA from healthy saliva (tubes 1–3), or purified RNA treated with RNase A (tubes 4–6). B. LAMP reactions using WHotLAMP detecting RAB7A in saliva (tubes 1–3), or with RNase A treatment (tubes 4–6).
Fig 5.
Colorimetric quantification of LAMP reactions.
A. Illuminated lightbox with automated image acquisition using Raspberry Pi. 1) Raspberry Pi unit; 2) white LED strip; 3) camera unit; 4) test tube rack. B. LAMP reactions using WHotLAMP with ZI-1 primers on saliva samples from different negative nasal-swab qPCR SARS-CoV-2 individuals (top white box) and SARS-CoV-2 positive (nasal swab) samples (bottom white box). C. Processed image showing conversion of colorimetric LAMP results to hues. D. Hue distribution of WHotLAMP saliva results from negative (-) and positive (+) nasal-swab SARS-CoV-2 qPCR donor samples. ***P<0.0001 of negative vs. positive CoV-2 samples by t-test.
Fig 6.
Sensitivity and specificity of WHotLAMP.
A, B Sensitivity and C, D specificity of WHotLAMP using (A, C) ZI-1 or (B, D) CUFC1 primers with qPCR SARS-CoV-2 positive saliva. Yellow circles denote positive (+) LAMP reactions and magenta circles denote negative (-) LAMP reactions. ** P<0.001 and * P<0.01 between WHotLAMP positive and negative samples, by t-test.
Table 2.
Primers used in study.