Tomar Priyanka Singh, Patel Sapan, Dash Paban Kumar, Kumar Jyoti S
Division of Virology, Defence Research and Development Establishment, Gwalior, India.
School of Studies in Botany, Jiwaji University, Gwalior, India.
J Appl Microbiol. 2022 Dec;133(6):3512-3522. doi: 10.1111/jam.15783. Epub 2022 Sep 27.
West Nile encephalitis caused by infection with the West Nile virus (WNV) is endemic in many regions of the world and is a global public health threat. The aim of this report was to develop a method using colorimetry-based reverse-transcription loop-mediated isothermal amplification (cRT-LAMP) and RT-LAMP combined with lateral-flow dipstick (LFD) for rapidly detecting WNV in low-infrastructure settings.
The primers for the cRT-LAMP and RT-LAMP-LFD assays were designed based on env gene of the WNV. Primers concentration, temperature and time were optimized for cRT-LAMP and RT-LAMP-LFD. The diagnostic performance of the cRT-LAMP and RT-LAMP-LFD assays was evaluated using human serum samples from 110 patients who were clinically suspected to be infected with WNV. The RT-LAMP was performed in a heating block at 63°C for 40 min. The LAMP amplicons were visible in the lateral-flow dipstick within 5 min. The detection limit of the developed cRT-LAMP and RT-LAMP-LFD assays was 10 copies and this assay showed a high degree of specificity for WNV. Compared with quantitative real-time RT-PCR assay, the kappa value of cRT-LAMP and RT-LAMP-LFD were 0.970.
These results showed that the newly developed WNV-specific cRT-LAMP and RT-LAMP-LFD assays can be employed as an alternative method for screening of WN-suspected human samples. The results revealed that the assay could potentially identify the virus without interference from human serum samples. Collectively, all results revealed that cRT-LAMP and RT-LAMP-LFD assays offer a suitable field-based diagnosis of WNV.
The cRT-LAMP and LAMP-LFD platform for the detection of WNV is rapid, accurate and simple-to-perform. Our present method has not only a short turnaround time but also avoided cross-contamination problem. Moreover, the use of simple lateral flow dipsticks broadens its application potential for the point-of-care use in resource-limited settings during outbreak situations. To the best of our knowledge, this is the first report for the development of cRT-LAMP and LAMP-LFD assays for rapid, simple, specific and sensitive detection of WNV using human clinical samples and EvaGreen dye.
西尼罗河病毒（WNV）感染所致的西尼罗河脑炎在世界许多地区呈地方性流行，是一种全球公共卫生威胁。本报告的目的是开发一种基于比色法的逆转录环介导等温扩增（cRT-LAMP）以及将RT-LAMP与侧向流动试纸条（LFD）相结合的方法，用于在低基础设施环境中快速检测WNV。
基于WNV的env基因设计了cRT-LAMP和RT-LAMP-LFD检测的引物。对cRT-LAMP和RT-LAMP-LFD的引物浓度、温度和时间进行了优化。使用110例临床疑似感染WNV患者的血清样本评估cRT-LAMP和RT-LAMP-LFD检测的诊断性能。RT-LAMP在加热块中于63°C进行40分钟。LAMP扩增产物在5分钟内在侧向流动试纸条上可见。所开发的cRT-LAMP和RT-LAMP-LFD检测的检测限为10个拷贝，且该检测对WNV具有高度特异性。与定量实时RT-PCR检测相比，cRT-LAMP和RT-LAMP-LFD的kappa值为0.970。
这些结果表明，新开发的WNV特异性cRT-LAMP和RT-LAMP-LFD检测可作为筛查疑似WNV感染人类样本的替代方法。结果显示该检测可能不受人类血清样本干扰地鉴定病毒。总体而言，所有结果表明cRT-LAMP和RT-LAMP-LFD检测为WNV提供了合适的现场诊断方法。
用于检测WNV的cRT-LAMP和LAMP-LFD平台快速、准确且操作简单。我们目前的方法不仅周转时间短，而且避免了交叉污染问题。此外，使用简单的侧向流动试纸条拓宽了其在疫情暴发期间资源有限环境中即时检测的应用潜力。据我们所知，这是首次报道使用人类临床样本和EvaGreen染料开发用于快速、简单、特异性和灵敏检测WNV的cRT-LAMP和LAMP-LFD检测方法。
