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 significant healthcare costs, along with broader macroeconomic challenges².

In the context of the increasingly overlapping circulation of CHIKV, DENV, and ZIKV viruses, current diagnostic methodologies encounter three primary limitations. First, serological assays are unable to distinguish acute infections due to the prolonged persistence of antibodies following viral clearance¹. Second, viral culture techniques exhibit sensitivity rates low and require several days, thereby impeding timely clinical decision-making. Third, although quantitative PCR assays demonstrate high specificity (exceeding 95%), they cannot detect co-infections within a single reaction, which is a significant drawback given the reported 12.8% CHIKV-DENV co-infection rate in India³. Phylogenetic monitoring indicates a paradigm shift. The ECSA lineage, which was dominant during India's 2019-2022 epidemic, has acquired convergent mutations in its envelope proteins: E1-A226V (enhancing mosquito infectivity) and E2-K252Q (associated with immune evasion). These mutations have resulted in a genotype with documented higher virulence, as reported in recent surveillance studies from Kerala and Maharashtra³. Concurrently, China faces challenges from the importation of the ECSA genotype, as documented in the Yunnan outbreak, and other genotypes, with autochthonous transmission reported in multiple provinces, including Yunnan⁴.

These realities create urgent, resource-limited needs for (i) differential diagnosis despite overlapping clinical manifestations⁵, (ii) diagnostic assays that eliminate the complexity of instrumentation typically required by conventional real-time quantitative PCR (qPCR)⁶, and (iii) comprehensive validation of novel point-of-care technologies, including RT-LAMP and CRISPR-based methods⁵. To address these diagnostic challenges, we propose a rapid triple-PCR assay that uses a fully automated integrated system, enabling simultaneous detection of CHIKV, DENV, and ZIKV in a single reaction. We validated the performance of our triplex qPCR assay through direct comparison with established singleplex assays⁵, which served as the reference standard. The following sections detail the procedures for both the triplex assay and the reference singleplex assays. The comparative analysis of results from both methods is presented in the Representative Results section.

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 lymphadenopathy, and recent travel to chikungunya-endemic areas; or asymptomatic individuals with identical travel history.
3. Exclusion: antimicrobial/antiviral use within 7 days or febrile illness attributable to confirmed HIV or malignancy.

2. Sample collection and processing

1. Check that the outer packaging of the sampling box is not damaged and has not expired, and then remove the internal materials.
2. Position the subject with the arm extended. Identify the median cubital vein, apply a tourniquet, and disinfect the venipuncture site with a 70% alcohol swab.
3. Collect 1~3 mL of blood from the subject's antecubital vein using an anticoagulant-free blood collection tube.
4. Gently invert the tube 5-8 times and allow it to clot at room temperature for 30 min.
5. Centrifuge the blood sample at 4 °C, 1000 x g on a centrifuge, for 10 min. The serum sample obtained after centrifugation can be tested immediately.
6. Carefully aliquot the supernatant serum into sterile microcentrifuge tubes. Use the serum immediately for testing, or store aliquots at -80 °C until further analysis of pathogen nucleic acids via qPCR.

3. Automated Triplex qPCR Detection of CHIKV, DENV, and ZIKV

1. Reagent and tube preparation
   1. Retrieve all required reagents from storage and thaw them at room temperature.
   2. Visually inspect each sealed TD detection tube for damage or seal failure. Discard any compromised tubes.
   3. Briefly vortex the TD nucleic acid extraction buffer for 5 s to ensure homogeneity.
   4. Aseptically dispense 500 µL of the homogenized buffer into a TD extraction reagent tube (containing purple freeze-dried magnetic beads).
   5. Vortex the mixture vigorously for 30 s until all components are fully dissolved and the beads are completely resuspended.
   6. Transfer the entire reconstituted solution into a pre-labeled TD detection tube.
2. Sample loading
   1. Pipette 200 µL of clear, non-hemolyzed, and non-lipemic serum into the prepared TD detection tube.
   2. Seal the tube securely and invert it ten times to mix homogeneously.
      NOTE: During inversion, allow the paraffin layer to float briefly within the tube. This is a normal part of the process.
3. Instrument operation and data acquisition
   1. Insert the sealed detection tubes into the designated slots of the analyzer .
   2. On the touchscreen interface, enter the sample identification information.
   3. Press Start Detection to initiate the fully automated, closed-tube workflow.
      NOTE: The system will automatically perform barcode scanning, nucleic acid extraction via magnetic bead capture, and fluorescence PCR amplification without further user intervention. The standard program includes a denaturation step at 95 °C for 3 min, followed by 40 cycles of amplification.
4. Result interpretation
   1. Upon completion of the run, observe the amplification results for all three targets (CHIKV, DENV, ZIKV) and the internal control on the same screen.
   2. Record the automatically generated readout, "Positive," "Negative," or "Invalid", for each target pathogen in its respective fluorescence channel.
      NOTE: The system's internal software uses a cycle threshold (Ct) of 37.0 for automatic interpretation. No manual data analysis is required.

4. Reference Singleplex qPCR assays for method comparison

1. Preparation of nucleic acid extraction reagents
   1. Take out the pre-packaged 96-well plate from the kit. Gently tap the 96-well plate to make the reagents and magnetic beads all concentrate to the bottom of the 96-well plate. Carefully tear off the aluminum foil sealing film, avoiding vibration of the 96-well plate to prevent liquid splashing out.
   2. In columns 1 and 7 of a 96-well plate, 20 µL of proteinase K is added first, followed by 200~300 µL of the sample to be extracted.
   3. Automated Nucleic Acid Extraction (Magnetic Bead-Based Method)
      1. Prepackaged 96-well plates (with samples) are placed in the corresponding slots of the automated nucleic acid extractor system.
      2. The nucleic acid extraction process is as follows: Adsorb the nucleic acid-bound magnetic beads using a magnetic rod inside the laboratory chamber. Transfer the bead complex into the next designated reagent hole. Activate the stirring device to mix the liquid thoroughly with the magnetic beads through rapid and repeated stirring. Allow the automated nucleic acid extractor to carry out the subsequent steps automatically: i) Cell Lysis: 10 min at 25 °C. ii)Nucleic Acid Adsorption: 15 min at 70 °C, with magnetic adsorption performed 3 times. iii)Washing: Mix for 2 min (repeated 3 times), followed by magnetic adsorption twice (15 s each). iv)Elution: Perform twice (5 min each), with magnetic adsorption carried out 3 times. Eventually, obtain high-purity nucleic acids.
   4. For immediate detection, directly aspirate the nucleic acid-containing eluate from columns 6 and 12. For long-term storage, transfer the remaining nucleic acid samples to -20°C.
2. Preparation of amplification reagents
   NOTE: For a Singleplex PCR reaction, prepare three separate PCR tubes using three different enzymes and three corresponding reaction mixtures. Complete the reagent preparation in three separate steps. Since the preparation steps are similar, we will only demonstrate the preparation steps for the CHIKV detection reagent below.
   1. Remove the nucleic acid amplification reaction solution and CHIKV reaction solution (CHIKV-specific primers/probes) from the kit. After thawing, place them in a 22~25 °C environment, mix thoroughly by vigorous shaking, then perform a brief centrifugation (1000 x g, 15 s, 22~25 °C).
   2. Calculate the total number of reactions as N + 2 (where N = number of test samples, +2 = one negative and one positive quality control). Prepare the Master Mix in a suitably sized microcentrifuge tube by combining the following for each reaction: 17 µL Nucleic Acid Amplification Reaction Solution (containing Buffer, dNTPs, DNA Polymerase, UDG Enzyme), 3 µL CHIKV Reaction Solution (containing Primers, Probes for CHIKV target genes, and IC). Vortex the Master Mix thoroughly to ensure complete mixing. Briefly centrifuge the tube at 10,000 x g for 15 s at room temperature (22~25 °C) to collect the solution at the bottom. Dispense 20 µL of the Master Mix into each PCR reaction tube.
   3. Add nucleic acid and quality control samples: Into the above-prepared PCR reaction tubes (20 µL), respectively add 5 µL of nucleic acid from tested samples, positive control, and negative control, achieving a final volume of 25 µL per tube; tightly cap the tubes and perform instantaneous centrifugation (1000 x g, 15 s, 22 ~25 °C) and then place the tubes into the PCR instrument.
3. PCR amplification
   1. Set the cycling program as follows: Pre-denaturation: 50 °C for 15 min, then at 95 °C for 15 min (both steps together constitute 1 cycle); the following 2 steps for 40 cycles: 94 °C denaturation for 15 s and 55 °C annealing for 45 s.
   2. Set the fluorescence signal detection parameters as follows: FAM fluorescent labeling of CHIKV genes; VIC fluorescent labeling of IC gene used as the kit's internal control. Collect data at 55 °C. Upon completion of the reaction, ensure that the data is saved for future analysis.
   3. Analyze data using qPCR-specific software
      1. Sample information: In the well plate layout diagram of the software, label the sample types (e.g., positive control, negative control, unknown sample, internal reference gene, etc.) corresponding to the sample loading positions.
      2. Placing the reaction tubes: Open the lid of the instrument's heating module, place the sealed PCR plate/tubes steadily inside, ensure they fit closely with the heating module, and close the module lid tightly (to avoid temperature unevenness)
      3. Starting the reaction: After confirming that the program settings in the software are correct, click Start Run and record the experiment ID for future reference.
      4. Stopping the run and removing tubes: Once the program is complete, wait until the heating module temperature drops below 50 °C. Then, open the lid and carefully remove the PCR plate or tubes. Proceed with downstream analysis or sample storage as required.
      5. Data Analysis Before proceeding with analysis, confirm that: i) Amplification curves for all samples and the internal control are characteristically S-shaped; ii) The fluorescence threshold is set within the exponential phase, yielding Ct values ≤ 38 cycles (CV < 5%); iii)Control results are valid, indicating no contamination. If any condition is unmet or if a sample shows abnormal amplification (e.g., an atypical curve or a critical-range Ct), re-check the entire batch or the specific specimen accordingly.
      6. Process the raw data with the software to determine Ct values, using the auto-selected threshold; a sample is considered positive if Ct < 38 and its amplification curve exhibits a sigmoidal shape.

5. Biosafety and waste disposal

1. Transfer all materials to a designated, puncture-resistant biohazard container and decontaminate by autoclaving prior to final disposal through the institutional medical waste system. Immediately transfer these materials to a leak-proof, puncture-resistant biohazard container labeled for high-risk waste. Following institutional guidelines, decontaminate all waste containing amplified products by autoclaving (e.g., 121 °C for 30 min) to inactivate pathogens and degrade nucleic acids. Finally, dispose of the sterilized waste through the designated medical waste management system.
   NOTE: Perform all steps in accordance with Biosafety Level 2 (BSL-2) practices, using appropriate personal protective equipment to minimize exposure risks.

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