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

Pepper, Aimee W.

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

Description

Title

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

Author(s)

Pepper, Aimee W.

Issue Date

2025-03-20

Director of Research (if dissertation) or Advisor (if thesis)

Wang, Leyi

Committee Member(s)

• Samuelson, Jonathan P
• Sander, William E

Department of Study

Vet Clinical Medicine

Discipline

VMS-Veterinary Clinical Medcne

Degree Granting Institution

University of Illinois Urbana-Champaign

Degree Name

M.S.

Degree Level

Thesis

Keyword(s)

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

Abstract

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.

Graduation Semester

2025-05

Type of Resource

Thesis

Handle URL

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

Copyright and License Information

Copyright 2025 Aimee Pepper

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