Efficient Detection of West Nile Virus in Urine Specimens by a Novel In‐House RT‐qPCR Detection Kit

West Nile Virus (WNV) infection represents a major global public health challenge. Even though most of the patients are asymptomatic, some cases progress to critical condition which may be fatal. Diagnosis traditionally relies on serological methods, but their limitations, including cross-reactivity, highlight the need for alternative approaches. Here, we present the development and validation of a novel RT-qPCR assay for precise and rapid detection of WNV RNA in urine, emerging as a promising specimen due to its noninvasive collection and high viral load. The assay demonstrates high efficiency and sensitivity, with a detection limit comparable to commercially available kits. This study highlights the importance of in-house kit design as a diagnostic tool in regions affected by emerging tropical infections, such as WNV, exemplified Cyprus. It emphasizes the critical role of low-cost, early detection with high sensitivity and specificity in infection control and surveillance efforts.