Pepper Aimee, Kayastha Sandipty, Miller Megan, Guag Jake, Tkachenko Andriy, Allender Matthew, Terio Karen, Wang Leyi
Veterinary Diagnostic Laboratory, Department of Veterinary Clinical Medicine, University of Illinois College of Veterinary Medicine, Urbana, IL 61802, USA.
Center for Veterinary Medicine, U.S. Food and Drug Administration, Laurel, MD 20708, USA.
Viruses. 2025 May 29;17(6):783. doi: 10.3390/v17060783.
The wide host range, potential lethality, and zoonotic potential of SARS-CoV-2 infection in animals highlights the need for additional surveillance strategies. We validated a commercial, pH-based, colorimetric RT-LAMP assay for the detection of SARS-CoV-2 RNA in animal feces. The comparator assay was 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 min, 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%. Blinded method testing organized by an independent laboratory confirmed the satisfactory reproducibility of the assay. To our knowledge, this study represents the first validation of RT-LAMP for SARS-CoV-2 detection in animals. 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, allowing for earlier implementation of control measures.
严重急性呼吸综合征冠状病毒2（SARS-CoV-2）感染动物的宿主范围广泛、具有潜在致死性且存在人畜共患病潜力，这凸显了采用更多监测策略的必要性。我们验证了一种基于pH值的商业化比色逆转录环介导等温扩增（RT-LAMP）检测方法，用于检测动物粪便中的SARS-CoV-2 RNA。比较检测方法为逆转录定量聚合酶链反应（rRT-PCR）。RT-LAMP检测方法的检测限为每个反应72个基因组拷贝。RT-LAMP对SARS-CoV-2具有高度特异性，未检测到其他人类或动物冠状病毒。RT-LAMP性能稳定，在30至45分钟的孵育时长、60至70°C的孵育温度以及10至25微升的反应体积条件下均能产生有效结果。对于病毒载量高（Ct≤25）的临床粪便样本，诊断敏感性为100%；对于病毒载量中等至高（Ct≤33）的样本，诊断敏感性为97.4%；总体诊断敏感性为62%（Ct≤40）。诊断特异性为97.9%。由独立实验室组织的盲法测试证实了该检测方法具有令人满意的重复性。据我们所知，本研究是首次对用于动物SARS-CoV-2检测的RT-LAMP进行验证。RT-LAMP检测能够更快速地检测出病毒载量中等至高的动物是否感染SARS-CoV-2，且可在现场进行检测，从而能够更早地实施控制措施。
