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. 2021 Sep 16;16(9):e0257464.
doi: 10.1371/journal.pone.0257464. eCollection 2021.

WHotLAMP: A simple, inexpensive, and sensitive molecular test for the detection of SARS-CoV-2 in saliva

David Ng  1 , Ana Pinharanda  2 , Merly C Vogt  2   3 , Ashok Litwin-Kumar  1 , Kyle Stearns  4 , Urvashi Thopte  1 , Enrico Cannavo  1 , Armen Enikolopov  5 , Felix Fiederling  1 , Stylianos Kosmidis  1 , Barbara Noro  1 , Ines Rodrigues-Vaz  1 , Hani Shayya  1 , Peter Andolfatto  2 , Darcy S Peterka  1 , Tanya Tabachnik  1 , Jeanine D'Armiento  4   6 , Monica Goldklang  4   6 , Andres Bendesky  1   7
Affiliations

WHotLAMP: A simple, inexpensive, and sensitive molecular test for the detection of SARS-CoV-2 in saliva

David Ng et al. PLoS One. .

Abstract

Despite the development of effective vaccines against SARS-CoV-2, epidemiological control of the virus is still challenging due to slow vaccine rollouts, incomplete vaccine protection to current and emerging variants, and unwillingness to get vaccinated. Therefore, frequent testing of individuals to identify early SARS-CoV-2 infections, contact-tracing and isolation strategies remain crucial to mitigate viral spread. Here, we describe WHotLAMP, a rapid molecular test to detect SARS-CoV-2 in saliva. WHotLAMP is simple to use, highly sensitive (~4 viral particles per microliter of saliva) and specific, as well as inexpensive, making it ideal for frequent screening. Moreover, WHotLAMP does not require toxic chemicals or specialized equipment and thus can be performed in point-of-care settings, and may also be adapted for resource-limited environments or home use. While applied here to SARS-CoV-2, WHotLAMP can be modified to detect other pathogens, making it adaptable for other diagnostic assays, including for use in future outbreaks.

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Conflict of interest statement

Patent application 63/088,694 related to this technology was filed by Columbia University, to facilitate that this technology be made widely available. AB became a member of the advisory board of Rapid Diagnostic Systems Limited for which he received options, after the conclusion of experiments and data analyses described here. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1
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.
Fig 2
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
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
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
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
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.
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