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

Summary

This article recommends a fully automated integrated system that employs triple PCR technology to simultaneously diagnose co-infections of Chikungunya, Dengue, and Zika virus using a single sample, thereby optimizing testing efficiency in clinical and primary care laboratories.

Abstract

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.

Introduction

Chikungunya fever (CHIKF) is an increasingly prevalent arboviral disease primarily transmitted by the mosquito vectors Aedes aegypti and Aedes albopictus. Characterized by recurrent outbreaks in tropical regions, this disease clinically manifests as an acute febrile illness, often accompanied by severe joint pain and persistent arthritis¹. As of July 26, the cumulative total of laboratory-confirmed chikungunya cases in Foshan City had reached 4,824, with a remarkable 87.2% of these cases occurring in Shunde District. This pronounced geographic concentration imposes a substantial burden on the regional public health infrastructure and results in....

Protocol

This study adheres to the ethical guidelines established by the Ethics Committee of Guangdong Provincial People's Hospital, Southern Medical University, Guangzhou, China (approval no: KY2025-510-01). Detailed information regarding the materials used in this research (reagents, chemicals, equipment, and software) can be found in the Table of Materials.

1. Participant selection

1. Chooseparticipantsin theagerange of18 to 60years.
2. Choose participantswho present with acute febrile illness (axillary T ≥ 38 °C) within 12 days of symptom onset, plus either rash, arthralgia, or cervical lymphadenop....

Results

To validate the stability and accuracy of the triple PCR detection system, we selected five clinically representative samples from our internal laboratory and ten external EQA blind samples for verification. These five representative clinical samples include both negative and positive cases (S1-S5), including samples positive for CHIKV and DENV, selected to demonstrate the assay's clinical applicability and to support internal validation of test performance (Table 1). The 2025 EQA blinded sample panel fo.......

Discussion

CHIKV is a positive-sense, single-stranded RNA virus classified within the alphavirus genus of the Togaviridae family⁷. Accelerated global warming, intensified international commerce, and expanding tourism networks have collectively heightened the risk of human exposure to arboviral threats⁸. Over the past decade, the rapid evolution of molecular diagnostics has fundamentally transformed the landscape of infectious disease management. Among these technologies, qPCR has gain.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Automated nucleic acid extractor	DAAN	Smart 32	For DNA extraction
BECKMAN X-12	BECKMAN	ALA04H01	For  serum centrifugation
BSC-1500IIA2-X	BIOBASE	SEDA 20143222263	Biosafety cabinet
E-Centrifuge	WEALTEC		Centrifuge the residual liquid off the wall of the tube
Fully automatic medical PCR analyzer	Bioustar‌	UP0102	Fluorescent quantitative PCR amplification
Nucleic acid detection kit for chikungunya virus (PCR-fluorescent probe method)	DAAN	20250804	Detection of  chikungunya virus genes
Nucleic acid detection kit for Dengue virus (PCR-fluorescent probe method)	DAAN	202504003	Detection of  Dengue virusvirus genes
Nucleic acid detection kit for Dengue virus, Zika virus and chikungunya virus (PCR-fluorescent probe method)	Bioustar‌	20250724	Detection of  Dengue virus, Zika virus and chikungunya virus genes
Nucleic acid detection kit for Zika virus (PCR-fluorescent probe method)	DAAN	202508041	Detection of Zika virus  genes
Nucleic acid extraction kit	DAAN		Extract nucleic acid
SLAN Fully automatic medical PCR analysis system	HONGSHI		Data Analysis
SLAN-96S Real-Time PCR machine	HONGSHI	SLAN-96S	Fluorescent quantitative PCR amplification
Ultra-low temperature freezers (DW-YL450)	MELING	SEDA 20172220091	-20 °C for storing reagents
Vortex-5	Kylin-bell		For mixing reagent