Mirzaei Hadi, Sepahi Neda, Ghasemian Abdolmajid, Ranjbar Razie, Samsami Sahar, Mansoori Yaser, Chenari Maryam, Montaseri Zahra, Namavari Negin, Namavari Sahar, Ghanbariasad Ali
Department of Medical Genetics, School of Medicine, Zabol University of Medical Sciences, Zabol, Iran.
Noncommunicable Diseases Research Center, Fasa University of Medical Sciences, Fasa, Iran.
Can J Infect Dis Med Microbiol. 2025 Apr 5;2025:3343309. doi: 10.1155/cjid/3343309. eCollection 2025.
Coronavirus disease 2019 (COVID-19), an emerging life-threatening viral disease, has rapidly spread worldwide, exerting a detrimental impact on public health. We aimed to devise an innovative platform based on the loop-mediated isothermal amplification (LAMP) method, having priorities over real-time PCR (RT-PCR) in terms of sensitivity, specificity, and low running costs. To develop a novel assay, a new primer set plus four primer sets were designed targeting the N gene of the COVID-19 agent, resulting in the sensitivity reinforcement. The limit of detection (LOD) of the developed approach was determined and compared to those of the standard RT-LAMP and RT-PCR. Two hundred confirmed positive and negative samples initially tested by RT-PCR were recruited to assess the nested-RT-LAMP assay. Furthermore, for the one-step nested-RT-LAMP assay, positive samples were tested directly without the need for RNA extraction. The LOD of nested-RT-LAMP, LAMP, and RT-PCR were 5, 15, and 15 copies/μL, respectively. The findings of the investigation illustrated 100% sensitivity and 98% specificity for both LAMP assays. Moreover, respectively, 94% and 97% sensitivity and specificity were determined regarding the one-step nested-RT-LAMP assay. We offered a novel approach with more sensitivity compared to RT-PCR and common RT-LAMP, not only being a simple, accurate, cost-effective alternative diagnostic tool for RT-PCR but also being able to detect asymptomatic or mildly symptomatic patients more accurately in 2 h by naked eyes.
2019冠状病毒病（COVID-19）是一种新出现的危及生命的病毒性疾病，已在全球迅速传播，对公众健康产生了不利影响。我们旨在设计一种基于环介导等温扩增（LAMP）方法的创新平台，该平台在灵敏度、特异性和低运行成本方面优于实时荧光定量聚合酶链反应（RT-PCR）。为开发一种新型检测方法，针对COVID-19病原体的N基因设计了一组新引物和四组引物，从而提高了灵敏度。确定了所开发方法的检测限（LOD），并与标准RT-LAMP和RT-PCR的检测限进行了比较。招募了200份最初经RT-PCR检测确认为阳性和阴性的样本，以评估巢式RT-LAMP检测方法。此外，对于一步巢式RT-LAMP检测，阳性样本无需RNA提取即可直接检测。巢式RT-LAMP、LAMP和RT-PCR的LOD分别为5、15和15拷贝/μL。调查结果表明，两种LAMP检测方法的灵敏度均为100%，特异性为98%。此外，一步巢式RT-LAMP检测的灵敏度和特异性分别为94%和97%。我们提供了一种比RT-PCR和普通RT-LAMP更灵敏的新方法，它不仅是一种简单、准确、经济高效的RT-PCR替代诊断工具，而且能够在2小时内通过肉眼更准确地检测出无症状或症状轻微的患者。
