Clinical validation of SARS-CoV-2 RT-LAMP in animals

The wide host range, pathogenicity, and zoonotic potential of SARS-CoV-2 infection in animals highlights the need for additional surveillance strategies. Shedding of SARS-CoV-2 RNA within the intestinal tract is prolonged during animal infection, suggesting that surveillance could be accomplished non-invasively through nucleic acid amplification of animal feces. We validated a commercial, pH-based, colorimetric, RT-LAMP assay for the detection of SARS-CoV-2 RNA in animal feces, with comparison to the gold standard assay, rRT-PCR. The limit of detection of the RT-LAMP assay was 72 genome copies per reaction. RT-LAMP was highly specific for SARS-CoV-2 and did not detect other human or animal coronaviruses. RT-LAMP was robust, with valid results generated for incubation lengths of 30 to 45 minutes, incubation temperatures of 60 to 70°C, and reaction volumes of 10 to 25 µL. The diagnostic sensitivity was 100% for clinical fecal samples with high viral loads (Ct ≤25), 97.4% for samples with moderate-to-high viral loads (Ct ≤33), and 62% overall (Ct ≤40). The diagnostic specificity was 97.9% overall. Blinded method testing organized by an independent laboratory confirmed the reproducibility of the assay. SARS-CoV-2 RNA could still be detected by RT-LAMP following storage of fecal suspensions with moderate-to-high or high viral loads at -80°C, -20°C, 4°C, or room temperature for up to 28 days. To our knowledge, this study represents the first clinical evaluation of RT-LAMP for SARS-CoV-2 RNA detection in animal samples. RT-LAMP testing could detect SARS-CoV-2 infection more rapidly and at the point-of-care in animals with moderate-to-high viral loads that are likely to be infectious, allowing for earlier implementation of quarantine and control measures to limit viral spread and therapeutic interventions to reduce animal mortality.

Name thesis:degree_name

M.S.

Level thesis:degree_level

Thesis

Discipline thesis:degree_discipline

VMS-Veterinary Clinical Medcne

Grantor

University of Illinois Urbana-Champaign

Year dc:date

2025

Author dc:creator

• Pepper, Aimee W.

Contributors dc:contributor

• Wang, Leyi
• Samuelson, Jonathan P
• Sander, William E

• SARS-CoV-2
• RT-LAMP
• rRT-PCR
• feces
• animals

Statement dc:rights

• Copyright 2025 Aimee Pepper

Language dc:language

en, eng

Handle dc:identifier

https://hdl.handle.net/2142/129179

OAI record oai:identifier

https://www.ideals.illinois.edu/oai-pmh?verb=GetRecord&metadataPrefix=etdms&identifier=oai%3Awww.ideals.illinois.edu%3A2142%2F129179