Department of Virology, Aggeu Magalhães Institute (IAM), Oswaldo Cruz Foundation (Fiocruz), Avenida Professor Moraes Rego, Recife, Pernambuco, 50670-420, Brazil.
Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, ON, M5S 3M2, Canada.
Sci Rep. 2021 Feb 18;11(1):4111. doi: 10.1038/s41598-021-83371-1.
We have previously developed and validated a one-step assay based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of the Zika virus (ZIKV) from mosquito samples. Patient diagnosis of ZIKV is currently carried out in centralized laboratories using the reverse transcription-quantitative polymerase chain reaction (RT-qPCR), which, while the gold standard molecular method, has several drawbacks for use in remote and low-resource settings, such as high cost and the need of specialized equipment. Point-of-care (POC) diagnostic platforms have the potential to overcome these limitations, especially in low-resource countries where ZIKV is endemic. With this in mind, here we optimized and validated our RT-LAMP assay for rapid detection of ZIKV from patient samples. We found that the assay detected ZIKV from diverse sample types (serum, urine, saliva, and semen) in as little as 20 min, without RNA extraction. The RT-LAMP assay was highly specific and up to 100 times more sensitive than RT-qPCR. We then validated the assay using 100 patient serum samples collected from suspected cases of arbovirus infection in the state of Pernambuco, which was at the epicenter of the last Zika epidemic. Analysis of the results, in comparison to RT-qPCR, found that the ZIKV RT-LAMP assay provided sensitivity of 100%, specificity of 93.75%, and an overall accuracy of 95.00%. Taken together, the RT-LAMP assay provides a straightforward and inexpensive alternative for the diagnosis of ZIKV from patients and has the potential to increase diagnostic capacity in ZIKV-affected areas, particularly in low and middle-income countries.
我们之前开发并验证了一种基于逆转录环介导等温扩增（RT-LAMP）的一步法检测，用于从蚊子样本中快速检测寨卡病毒（ZIKV）。目前，对 ZIKV 的患者诊断是在集中式实验室中使用逆转录定量聚合酶链反应（RT-qPCR）进行的，虽然这是金标准的分子方法，但在远程和资源匮乏的环境中使用时存在一些缺点，例如成本高和需要专用设备。即时检测（POC）诊断平台有可能克服这些限制，特别是在寨卡病毒流行的资源匮乏国家。考虑到这一点，我们在这里优化并验证了我们的 RT-LAMP 检测法，用于从患者样本中快速检测 ZIKV。我们发现该检测法无需 RNA 提取，即可在短短 20 分钟内从各种样本类型（血清、尿液、唾液和精液）中检测到 ZIKV。RT-LAMP 检测法具有高度特异性，比 RT-qPCR 高 100 倍。然后，我们使用从巴西伯南布哥州疑似虫媒病毒感染病例中收集的 100 份患者血清样本对该检测法进行了验证，该州是上一次寨卡疫情的中心。与 RT-qPCR 相比，对结果的分析发现，ZIKV RT-LAMP 检测法的敏感性为 100%，特异性为 93.75%，总准确率为 95.00%。总的来说，RT-LAMP 检测法为从患者中诊断 ZIKV 提供了一种简单且廉价的替代方法，并且有可能增加寨卡病毒感染地区的诊断能力，特别是在中低收入国家。
