Department of Chemistry, Chung Yuan Christian University, Taoyuan 320314, Taiwan.
Department of Bioscience Technology, Chung Yuan Christian University, Taoyuan 320314, Taiwan.
J Virol Methods. 2024 Dec;330:115035. doi: 10.1016/j.jviromet.2024.115035. Epub 2024 Sep 17.
The global impact of the COVID-19 pandemic has emphasized the critical need for effective viral diagnostics. Although polymerase chain reaction (PCR) is a well-established nucleotide amplification technique, its limitations, such as the need for expensive equipment and skilled technicians, have led to the exploration of alternative methods, including loop-mediated isothermal amplification (LAMP). Bats, as a crucial natural reservoir of coronaviruses (CoVs), particularly Scotophilus bat coronavirus 512 (Scotophilus bat-CoV 512) prevalent among Taiwan's bat population, are the focus of this study. We aimed to detect Scotophilus bat-CoV 512 from bats in field conditions using loop-mediated isothermal amplification (LAMP) assay for on-site detection. Therefore, our study delves into the specificity of the LAMP reaction, emphasizing the careful design of primers to prevent false positive results. A cross reactivity and primer specificity test involving seven different microorganisms, including closely related bat CoVs and two bacterial species typically found in feces, revealed that the LAMP assay uniquely detected Scotophilus bat-CoV 512. The developed colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was optimized for the primers targeting nucleocapsid (N) gene, and the sensitivity test revealed a detection limit of 2.4 × 10 copies/µL. Our findings indicate the potential of the RT-LAMP assay for on-site detection in the field and subsequent laboratory analysis for comprehensive sampling and further research on bat CoV isolation. The surveillance and monitoring of bat CoVs contribute substantially to mitigating human threats, particularly concerning the emergence of new pandemic variants.
新型冠状病毒肺炎疫情的全球影响凸显了有效病毒诊断的迫切需要。聚合酶链反应（PCR）虽然是一种成熟的核苷酸扩增技术，但由于需要昂贵的设备和熟练的技术人员等限制，促使人们探索替代方法，包括环介导等温扩增（LAMP）。蝙蝠是冠状病毒（CoV）的重要天然宿主，特别是在台湾蝙蝠种群中流行的 Scotophilus bat coronavirus 512（Scotophilus bat-CoV 512），是本研究的重点。我们旨在使用环介导等温扩增（LAMP）检测法在野外条件下从蝙蝠中检测到 Scotophilus bat-CoV 512，以便进行现场检测。因此，我们的研究深入探讨了 LAMP 反应的特异性，强调了引物的精心设计以防止假阳性结果。涉及七种不同微生物的交叉反应性和引物特异性测试，包括密切相关的蝙蝠 CoV 和两种通常在粪便中发现的细菌，结果表明 LAMP 检测法特异性地检测到了 Scotophilus bat-CoV 512。针对核衣壳（N）基因的引物优化了显色逆转录环介导等温扩增（RT-LAMP）检测法，敏感性测试显示检测限为 2.4×10 拷贝/µL。我们的研究结果表明，RT-LAMP 检测法具有在现场进行现场检测的潜力，随后可在实验室中进行全面采样和进一步研究蝙蝠 CoV 分离。对蝙蝠 CoV 的监测和监控对减轻人类威胁具有重要意义，特别是对新的大流行变异体的出现。
