Department of Emerging Infectious Diseases, Institute of Tropical Medicine (NEKKEN), Nagasaki University, Nagasaki, Japan.
Centre de Recherches Médicales de Lambaréné, Lambaréné, Gabon.
PLoS Negl Trop Dis. 2022 Dec 1;16(12):e0010964. doi: 10.1371/journal.pntd.0010964. eCollection 2022 Dec.
Despite the development of several methods for diagnosing COVID-19, long-term validation of such methods remains limited. In the early phase of the COVID-19 pandemic, we developed a rapid and sensitive diagnostic method based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) methodology, which is suitable for point-of-care application or for use in resource-limited settings to detect SARS-CoV-2. To assess the applicability of the RT-LAMP assay technique to resource-limited regions, such as rural areas in Africa, and to verify the usability of the method against various SARS-CoV-2 variants, the method was validated using clinical samples collected longitudinally during the pandemic.
METHODOLOGY/PRINCIPAL FINDINGS: First, the sensitivity of the RT-LAMP assay for detecting 10 SARS-CoV-2 variants was evaluated using viral RNA samples extracted from cell culture with a portable battery-supported device, resulting in the successful detection of 20-50 copies of the viral genome within 15 min, regardless of the variant. COVID-19 positive samples collected in Gabon between March 2020 and October 2021 were used to evaluate the sensitivity of the assay and to calculate the copy number of the SARS-CoV-2 genome. More than 292 copies of the viral genome were detected with 100% probability within 15 min in almost all tests.
This long-term validation study clearly demonstrated the applicability of the RT-LAMP assay for the clinical diagnosis of COVID-19 in resource-limited settings of Africa, such as rural areas in Gabon. The results show the potential of the assay as a promising COVID-19 diagnostic method, especially in rural and remote regions located far from the official diagnosis facilities in urban or semi-urban areas.
尽管已经开发出几种用于诊断 COVID-19 的方法，但这些方法的长期验证仍然有限。在 COVID-19 大流行的早期阶段，我们开发了一种基于逆转录环介导等温扩增（RT-LAMP）方法的快速灵敏的诊断方法，该方法适合于即时检测或在资源有限的环境中使用，以检测 SARS-CoV-2。为了评估 RT-LAMP 检测技术在资源有限的地区（如非洲农村地区）的适用性，并验证该方法对各种 SARS-CoV-2 变体的可用性，我们使用在大流行期间纵向收集的临床样本对该方法进行了验证。
方法/主要发现：首先，使用便携式电池支持设备从细胞培养物中提取的病毒 RNA 样本评估了 RT-LAMP 检测方法对 10 种 SARS-CoV-2 变体的敏感性，结果无论变体如何，在 15 分钟内成功检测到 20-50 个病毒基因组拷贝。使用 2020 年 3 月至 2021 年 10 月期间在加蓬收集的 COVID-19 阳性样本评估了该检测方法的敏感性，并计算了 SARS-CoV-2 基因组的拷贝数。在几乎所有测试中，在 15 分钟内以 100%的概率检测到超过 292 个病毒基因组拷贝。
这项长期验证研究清楚地表明，RT-LAMP 检测法适用于资源有限的非洲环境中的 COVID-19 临床诊断，例如加蓬的农村地区。结果表明，该检测法具有作为有前途的 COVID-19 诊断方法的潜力，尤其是在远离城市或半城市地区官方诊断设施的农村和偏远地区。
