Abstract：
Japanese encephalitis (JE), a mosquito-borne zoonotic disease caused by the Japanese encephalitis virus (JEV), poses a serious threat to global public health. The low viremia levels typical in JEV infections make RNA detection challenging, necessitating early and rapid diagnostic methods for effective control and prevention. This study introduces a novel one-pot detection method that combines recombinant enzyme polymerase isothermal amplification (RPA) with CRISPR/EsCas13d targeting, providing visual fluorescence and lateral flow assay (LFA) results. Our portable one-pot RPA-EsCas13d platform can detect as few as two copies of JEV nucleic acid within 1 h, without cross-reactivity with other pathogens. Validation against clinical samples showed 100 % concordance with real-time PCR results, underscoring the method\'s simplicity, sensitivity, and specificity. This efficacy confirms the platform\'s suitability as a novel point-of-care testing (POCT) solution for detecting and monitoring the JE virus in clinical and vector samples, especially valuable in remote and resource-limited settings.
摘要：
日本脑炎(JE),由日本脑炎病毒（JEV）引起的蚊子传播的人畜共患疾病，对全球公共卫生构成严重威胁。JEV感染中典型的低病毒血症水平使RNA检测具有挑战性，需要早期和快速的诊断方法来有效控制和预防。本研究介绍了一种新型的一锅法检测方法，该方法将重组酶聚合酶等温扩增（RPA）与CRISPR/EsCas13d靶向相结合，提供视觉荧光和侧流测定(LFA)结果。我们的便携式一罐RPA-EsCas13d平台可以在1小时内检测到少至两个拷贝的JEV核酸，与其他病原体没有交叉反应性。对临床样本的验证显示与实时PCR结果100%一致，强调方法的简单性，灵敏度,和特异性。这种功效证实了该平台作为一种新型的即时检测（POCT）解决方案的适用性，用于检测和监测临床和载体样品中的JE病毒，在远程和资源有限的设置中尤其有价值。
