A Point-of-Care Platform for Simultaneous Triplex PCR Detection of Chikungunya, Dengue, and Zika Virus Infections

Chikungunya fever (CHIKF), an acute viral illness transmitted by mosquitoes and first discovered in Tanzania in 1952, has shown intermittent spread in China, with increasing local outbreaks. By July 2025, Guangdong Province, currently the outbreak's center, had reported 4,824 local cases, including nearly 3,000 new infections in just one week. This rapid surge creates an urgent need for advanced diagnostic technologies. Such tools are critical to support containment efforts on the front lines. We propose a solution for the rapid simultaneous detection of dengue (DENV), chikungunya (CHIKV), and Zika virus (ZIKV) infections to enhance early epidemic prevention and control capabilities. This approach involves developing customized testing protocols for border checkpoints and primary healthcare facilities. Traditional diagnostic methods for CHIKV and DENV often lack sensitivity/specificity in coinfection cases. Since CHIKV, DENV, and ZIKV share similar transmission patterns and symptoms, simultaneous monitoring is crucial. This protocol presents a triplex qPCR method using TaqMan probes to detect these arboviruses in a single tube within 90 min. Compared to viral culture and serological tests, this method improves efficiency by identifying active infections and coinfections with high specificity while simplifying workflow and eliminating specialized equipment requirements. Validation with clinical and External Quality Assessment (EQA) samples confirmed 100% concordance with gold-standard singleplex assays. This method serves as an effective tool for monitoring febrile illnesses in endemic regions, particularly in resource-limited settings where rapid outbreak containment is critical.