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

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.

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.

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 for emerging mosquito-borne pathogens comprised 10 inactivated plasma samples (DZC2511-2520). These contained various concentrations and combinations of CHIKV, DENV, and ZIKV. These concentrations and combinations were pre-determined by the NCCL. Analysis of this panel using our triple qPCR platform was entirely consistent with reference results (Table 2), confirming the robustness and clinical applicability of the assay.

The triplex qPCR assay reliably distinguished between negative and positive infections across all validation samples (Figure 1). In the clinical cohort, the assay identified two samples (S2, S3) as positive for CHIKV and one sample (S5) for DENV, while samples S1 and S4 were negative for all three targets (Figure 2, Table 1). The amplification curves for these targets exhibited characteristic sigmoidal kinetics, with clear separation from the baseline, and all internal controls amplified as expected, confirming assay validity.

The singleplex qPCR assay reliably distinguished between negative and positive infections across all validation samples (Figure 3). It is worth noting that the singleplex test results of the same batch of clinical samples are entirely consistent with the singleplex qPCR test results (Figure 4, Table 1). This demonstrates that the multiplexed format does not compromise diagnostic accuracy.

The assay's performance was further confirmed through an external validation using a blinded quality assessment panel. The triplex qPCR results achieved 100% qualitative agreement with the reference outcomes across all 10 samples, accurately identifying CHIKV, DENV, and ZIKV in the respective panels, without any false positives or negatives (Table 2). This confirms the method's robustness and readiness for deployment in routine diagnostic and public health settings.

Figure 1: Quality control results of triplex qPCR detection of negative and positive controls. Negative controls must exhibit an S-shaped growth curve in the CY5-IC detection channel, with a Ct value ≤ 40.00. In contrast, fluorescence signals in the FAM-DENV, HEX-ZIKV, and ROX-CHIKV channels should not demonstrate significant increases, resulting in Ct values > 40.00 or no discernible signal. Positive controls are required to display S-shaped curves across all detection channels, with Ct values ≤ 37.00. Please click here to view a larger version of this figure.

Figure 2: Detection of CHIKV, DENV, and ZIKV nucleic acids in blood samples performed using triplex qPCR. Each fluorescent signal corresponds to a specific gene, with ROX (yellow) indicating CHIKV virus genes, FAM (blue) indicating DENV virus genes, HEX (orange) indicating ZIKV virus genes, and CY5 (green) indicating the internal control gene. Sample phenotypes were as follows: S1 was negative for CHIKV (no signal above threshold); S2 and S3 were positive for CHIKV (ROX signal above threshold); S4 was negative for CHIKV (no signal above threshold); and S5 was positive for DENV (FAM signal above threshold). Please click here to view a larger version of this figure.

Figure 3: Quality control results of Singleplex qPCR detection of negative and positive controls. Negative controls must not exhibit significant increases in fluorescence signals for the FAM and VIC channels, with Ct values exceeding 40.00 or no discernible signal. In contrast, positive controls should display S-shaped curves in all detection channels, with Ct values equal to or less than 32.00. Please click here to view a larger version of this figure.

Figure 4: Detection of CHIKV and dengue virus nucleic acids in blood samples using Singleplex qPCR. Each color denotes a distinct fluorescent signal, with different signals corresponding to specific genes. The fluorescence channels include FAM fluorescent labeling (blue) for CHIKV genes and CY5 fluorescent labeling (green) for the internal control gene. The sample phenotypes are as follows: S1 is a Chikungunya-negative sample (no signal above threshold); S2 is a CHIKV-positive sample (FAM signal above threshold); S3 is also a CHIKV-positive sample (FAM signal above threshold); S4 is a CHIKV-negative sample (no signal above threshold); and S5 is a dengue-positive sample (FAM signal above threshold). Please click here to view a larger version of this figure.

Table1: Results of qPCR for five representative clinical samples. The table provides the qualitative results of detection in samples S2 and S3 indicates that these two patients are infected with the CHIKV virus, while detection in sample S5 signifies infection with the dengue virus. "NA" denotes that no Ct value was detected. Legend: +/- indicates qualitative results; "+" represents a positive result, whereas "-" indicates a negative result.

Table 2: The qualitative outcomes of qPCR analysis from the 2025 EQA panel for emerging and re-emerging mosquito-borne pathogens transmitted through blood. The table provides the qualitative results of samples 2511, 2514, and 2516 tested positive for CHIKV RNA, samples 2512 and 2517 showed positivity for DENV RNA, and samples 2513 and 2519 exhibited positivity for ZIKV RNA. The notation "NA" indicates the absence of a recorded Ct value. In this context, symbols such as "+/-" are used to denote qualitative assessments, with "+" indicating a positive outcome and "-" indicating a negative result.

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 gained particular prominence in clinical laboratories across China due to its rapid turnaround, exceptional analytical sensitivity, high quantitative accuracy, and significantly reduced risk of cross-contamination during routine operations². By integrating a single blood draw with a triplex qPCR platform that simultaneously detects CHIKV, DENV, and ZIKV, we can now screen for both mono- and co-infections within a single analytical window.This streamlined workflow provides clinicians with actionable laboratory evidence that supports early, precise, and decisive public health interventions.

This study advances arboviral diagnostics by integrating triplex qPCR into a fully automated, closed-tube platform optimized for point-of-care use. Unlike conventional multiplex PCR, which requires specialized equipment^9,10,11, our system consolidates extraction and amplification within a single device, delivering simultaneous results for CHIKV, DENV, and ZIKV from one blood sample in 90 min.As shown in Tables 1 and 2, the assay demonstrated perfect agreement with both gold-standard singleplex PCR and EQA samples. This confirms that the streamlined, contamination-resistant workflow enhances operational efficiency in resource-constrained settings without compromising diagnostic accuracy. Illustrated in Methodological Steps 3 and 4, the integrated system maintains diagnostic precision while simplifying procedures, significantly reducing manual handling time, cross-contamination risk, and operator skill requirements. Thus, our system translates laboratory-grade testing into a practical tool for rapid screening and outbreak management in resource-limited environments.

Quality control standards are crucial for qPCR detection, encompassing instrument calibration, verification of detection system performance, evaluation of nucleic acid extraction efficiency, and batch experiment quality control. Sampling instruments undergo biannual calibration, while extraction and amplification equipment are calibrated annually, along with reagent batch calibration.Post-calibration, performance validation is carried out using the SLAN 96S Fluorescence Quantitative PCR system and specific detection kits, extraction reagents, sample tubes, pipettes, and tips. The criteria evaluated include conformity rate, detection limit, anti-interference, cross-reaction, and precision, with unqualified samples necessitating reextraction. Ensuring valid results requires daily adherence to quality control procedures and adherence to established standards for meticulous analysis. Run validity mandates a sigmoidal amplification curve with Ct ≤ 30.00 in the CY5 channel for the negative control.In contrast, no amplification (Ct > 40.00 or undetectable fluorescence) is acceptable in the FAM, HEX, and ROX channels. The positive control should show sigmoidal curves in all four channels, with Ct values ≤ 37.00 in each channel. If one or more pathogen targets are detected, the corresponding internal control is exempt from acceptance criteria if sigmoidal amplification with Ct ≤ 37.00 is observed.

The consistently high performance of qPCR in prior studies underscores its utility in arboviral diagnosis. Direct application to blood samples has proven highly effective in detecting chikungunya and dengue viruses with exceptional sensitivity and specificity^11,12,13. The strong concordance observed across these studies confirms that qPCR serves as a cornerstone methodology for reliable virus monitoring and clinical diagnostics in high-endemic settings. Building on this methodology, our research integrates nucleic acid extraction and amplification into a fully automated system. This innovation minimizes operator-dependent variability and eliminates cross-contamination risks, enabling rapid sample-to-result analysis. The system performs end-to-end processing, from nucleic acid purification to automated report generation, ensuring contact-free operation while maintaining contamination-free conditions. This integrated approach achieves laboratory-grade biosafety throughout the workflow, making it particularly suitable for high-throughput screening scenarios with limited personnel and equipment resources. We rigorously assessed the diagnostic performance of our newly developed triplex qPCR assay using a dual-validation approach.

We initially compared our internal assay to a certified singleplex PCR kit using five clinical samples (S1-S5) from patients with similar symptoms, such as fever and joint pain. The results, depicted in Figures 2，4 and Table 1, showed complete agreement between the two methods, confirming their equal sensitivity and specificity in detecting CHIKV, DENV, and ZIKV. To assess the assay's robustness, we conducted an external validation with 10 blinded plasma samples from the 2025 EQA program. Our Triplex qPCR showed perfect concordance with the reference method across a range of viral concentrations. This dual-verification approach validates the assay's accuracy across multiple targets and its reliability in both laboratory-controlled and external quality-assessment settings. The successful validation of this Triplex qPCR system highlights its significant clinical value. By consolidating the detection of three common arboviruses into a single, rapid test, it streamlines the precise identification of the causative agent, crucial for prompt diagnosis and patient care. This capability is especially vital for distinguishing between infections with similar symptoms, particularly in outbreak scenarios. The method offers a streamlined, efficient, and robust solution for clinical laboratories, with the potential to significantly improve diagnostic throughput and facilitate appropriate public health responses. It is essential to consider pre-analytical variables when assessing the diagnostic accuracy of any blood-based qPCR approach. Factors such as gross lipemia, hemolysis, or the use of heparinized specimens can significantly impact results, potentially leading to false-negative results. Furthermore, RNA arboviruses exhibit rapid evolutionary changes in mosquito vectors, leading to nucleotide divergence over time. While the primers and probes utilized in this study were designed to target conserved genomic regions, it is important to note that rare or emergent mutations occurring outside the specified amplicons may go undetected, potentially affecting the overall completeness of the results¹⁴.

Future research should prioritize developing innovative methodologies to improve mutational surveillance, such as utilizing next-generation sequencing for samples with inconclusive qPCR outcomes to identify undetected viral genomes missed by standard assays, particularly in complex or unusual cases. Simultaneously, public health initiatives need to emphasize comprehensive vector-monitoring schemes and robust mosquito control measures, particularly in high-risk areas, to reduce Aedes mosquito populations and prevent local transmission^15,16. Additionally, enhanced health education programs targeting travelers and communities near borders are crucial for reinforcing personal protective practices and sustaining integrated strategies for reducing arboviral risks.

Despite its proven effectiveness, our triplex qPCR test has limitations that require attention. The primer and probe sequences are not disclosed due to commercial patent constraints. However, the specific genomic regions targeted (e.g., E1 for CHIKV, NS5 for DENV, and ZIKV) are provided to promote scientific transparency. Moreover, as with all molecular tests, pre-analytical factors (e.g., hemolysis, lipemia, heparin use) are critical; deviations can compromise RNA quality and lead to false negatives. Finally, this study did not establish the limit of detection via serial dilution of standardized controls for all targets-a key aspect of analytical validation that remains to be addressed in future work.