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Table of Contents
Year : 2022  |  Volume : 59  |  Issue : 3  |  Page : 198-205

A validated triplex RT-qPCR protocol to simultaneously detect chikungunya, dengue and Zika viruses in mosquitoes

West Valley Mosquito and Vector Control District, 1295 E. Locust St., Ontario, CA 91761, USA

Date of Submission23-Mar-2020
Date of Acceptance08-Oct-2020
Date of Web Publication08-Dec-2022

Correspondence Address:
Tianyun Su
West Valley Mosquito and Vector Control District, 1295 E. Locust St., Ontario, CA 91761
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0972-9062.316275

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Background & objectives: Recently, the incidences of chikungunya, dengue and Zika infections have increased due to globalization and urbanization. It is vital that reliable detection tools become available to assess the viral prevalence within mosquito populations.
Methods: Based on the previous publications on clinical diagnosis in human infections, for the first time, we described a customized triplex RT-qPCR protocol for simultaneous detection of chikungunya virus (CHIKV), dengue virus serotypes 1-4 (DENV1-4) and Zika virus (ZIKV) in mosquitoes.
Results: In preliminary assessment to determine the specificity and sensitivity of primers and probes, all six targets were detected individually with the following thresholds as indicated by calculated pfu equivalents: 3.96x100 for CHIKV, 3.80x101 for DENV1, 3.20x101 for DENV2, 8.00x104 for DENV3, 1.58x100 for DENV4, and 6.20x100 for ZIKV When tested in a full combination of six targets (CDZ mix), CHIKV, DENV1-4 mix or ZIKV were all detected with the thresholds of 1.32x100 for CHIKV, 3.79x100 for DENV1-4 and 2.06x100 for ZIKV All targets, individually or in full combination were detected in the mixtures of Aedes aegypti (L.) homogenate and viral lysates. A robust evaluation with three replicates in each of three plates for CHIKV, DENV1-4 and ZIKV individually or in full combination was conducted. In individual assays, CHIKV was detected to 3.96x10-1, DENV1-4 to 1.14x100 and ZIKV to 3.20x100. In full combination assays, CHIKV was detected to 1.32x104, DENV1-4 to 3.79x101 and ZIKV to 1.07x100.
Interpretation & conclusion: This triplex RT-qPCR assay appears to consistently detect all six targets and does not cross react with Ae. aegypti homogenate, making it a feasible, practical, and immediately adoptable protocol for use among vector control and other entities, particularly in the endemic areas of CHIKV, DENVs and ZIKV.

Keywords: chikungunya virus; dengue virus; Zika virus; Aedes aegypti; Aedes albopictus; RT-qPCR

How to cite this article:
Lura T, Su T, Thieme J, Brown MQ. A validated triplex RT-qPCR protocol to simultaneously detect chikungunya, dengue and Zika viruses in mosquitoes. J Vector Borne Dis 2022;59:198-205

How to cite this URL:
Lura T, Su T, Thieme J, Brown MQ. A validated triplex RT-qPCR protocol to simultaneously detect chikungunya, dengue and Zika viruses in mosquitoes. J Vector Borne Dis [serial online] 2022 [cited 2023 Mar 30];59:198-205. Available from: http://www.jvbd.org//text.asp?2022/59/3/198/316275

  Introduction Top

With increasing globalization and urbanization, arboviruses are becoming a significant public health burden worldwide, particularly in developing countries in tropical and subtropical regions. Although typically geographically restricted, many arboviruses now have much wider distributions due to frequent human travel and modernized transportation. Furthermore, the spread of vector mosquito species, such as Aedes aegypti (L.) and Aedes albopictus (Skuse), has broadened the distribution of many of the diseases they transmit. It is for these reasons that chikungunya, dengue, yellow fever and Zika have become emerging or resurging public health concerns globally. Chikungunya virus (CHIKV), an Alphavirus within the Togaviridae family, was first described during an outbreak in Tanzania in 1952–1953[1],[2],[3]. Following this discovery, this virus infections were recorded throughout southern Asia with minor outbreaks[4],[5],[6]. But in 2004, a major outbreak occurred on the coast of Kenya[5],[7], ensued by broader spread in Asia, Europe, and North America[5],[8]. Dengue virus (DENV), a Flavivirus within the Flaviviridae family, is further characterized by four distinct viral serotypes, DENV1, DENV2, DENV3, and DENV4. While the clinical manifestation can be moderate mostly, severe cases of dengue, such as dengue hemorrhagic fever, or dengue shock syndrome, can occur even leading to death[9]. Currently, dengue occurrence has been on the up-trend throughout the tropical and subtropical countries[10]. It is calculated that 2.5 to 3.6 billion people live in areas at risk for dengue, with an estimate 50 to 270 million cases of infection worldwide each year[10],[11]. Zika virus (ZIKV), another Flavivirus within the family Flaviviridae, was first documented in Uganda in 1947[12],[13]. The first known major outbreak of documented Zika virus infection occurred in 2007 on the island of Yap in the Federated States of Micronesia[14], followed by that during 2013–2014 in French Polynesia[15],[16]. During this time, Zika virus infection was beginning to be linked with an increase in cases of Guillain-Barré syndrome and other neural complications, such as microcephaly[16]. Recently, ZIKV infection was reported in the Americas, specifically Brazil in 2015, then in Central and North America and the Caribbean by 2016[16].

All three of these viral infections are primarily vectored by Aedes mosquitoes, specifically Ae, aegypti and Ae. albopietus, regardless of conflicting data on the role of Culex quinquefasciatus Say in the transmission of Zika virus[17],[19]. With increased globalization, urbanization and climate changes, the insect vectors and the emerging diseases they transmit have spread globally. Therefore, a dependable testing tool with high sensitivity and specificity for detection of these arboviruses in mosquitoes is desired for use in vector control and other entities. A previously evaluated test kit (Life Technologies, Carlsbad, USA, now Thermo Fisher Scientific, Waltham, USA)[20] has never been commercialized because of high cost and low profit margin. The adoption of this kit is prohibited by proprietary oligonucleotide sequences and manufacturing process. In order to provide a feasible and affordable tool for an average PCR laboratory, based on previously published protocols for clinical diagnosis of human infections[21],[22],[23], a customized, practical and highly adoptable RT-qPCR protocol for simultaneous detection of CHIKV, DENV and ZIKV in mosquito vector species was developed and validated.

  Material & Methods Top

Viral lysates and mosquito homogenate spiked with viral lysates

The viral lysates (C6/36 tissue culture fluid + 20% fetal bovine serum) were prepared with Qiagen AVL buffer and provided by the Diagnostic and Reference Laboratory of the Arboviral Diseases Branch, Centers for Disease Control, Fort Collins, Colorado, USA. Details of all lysates for six viruses are available in [Table 1]. Viral lysate was prepared after plaque assay, stored at -80°C immediately, transported on dry ice from the provider to the user, then stored at the same temperature ever since.
Table 1: Targets, strains and lysate concentration (pfu equivalency/ml)

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To check for cross reactivity with mosquito tissues, Ae. aegypti homogenate was spiked with viral lysates in a 1:1 ratio for RNA extraction. Eggs of Ae. aegypti that were obtained from the Navy Entomology Center of Excellence, CMAVE Detachment (Gainesville, USA), were hatched in an insectary, and the larvae were reared to adults. Homogenate was made from pools of 50 of 3-5 day-old adult females. Mosquitoes were homogenized by Tissue Disruptor Genie (Scientific Industries, Bohemia, NY, USA) in 800 μl of PBS at room temperature for 5 min at 3000 rpm. The homogenate was then centrifuged for 5 min at 4°C (Eppendorf 5424R Centrifuge, Hamburg, Germany). Mosquito homogenate, and PBS alone were also prepared for use as a negative control for RNA extraction.

RNA extraction

The amount of each sample for extraction was 50 μl with various equivalent pfu [Table 2], and all extractions were eluted into 50 μl of elution buffer after lysis and washings. In the preliminary assay to determine specificity and sensitivity of the primers and probes, 50 μl was used for each of six targets, and full combinations of all targets with 1/3 of each CHIKV, DENV1-4 (1/4 for each DENV) and ZIKV. In the later robust evaluation with three replicates in each of three plates, 50 μl was used for each of CHIKV, DENV1-4 (1/4 for each DENV) and ZIKV individually, or 1/3 of each of CHIKV, DENV1-4 (1/4 for each DENV) and ZIKV in full CDZ combination.
Table 2: Estimated pfu equivalency in 50μl of starting concentrations and dilutions for each lysate

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Extractions for RNA were conducted using the Mag-MAX™-96 Viral RNA Isolation Kit (Thermo Fisher Scientific), which extracts total DNA and RNA. RNA extractions were done using the lysates of CHIKV, DENV1-4 (individually and mix), ZIKV, CDZ (CHIKV/DENV1-4/ZIKV) mix, viral lysate plus mosquito homogenate, as well as mosquito homogenate alone. An additional non-template control using PBS (1x) (negative template control 1 - NTC1) was added to the extraction. All extractions were processed on the MagMAX Express 96 machine (Thermo Fisher Scientific) using the manufacturer’s instructions and as previously described[24]. Ten-fold serial dilutions of 100–10-5 were made on elution which were assayed by RT-qPCR.


A preliminary plate was run to ensure that all six viral targets in lysates (CHIKV, each of DENV1, 2, 3, 4, and ZIKV) were amplified individually and all in CDZ mix. All lysates of CHIKV, DENV and ZIKV were analyzed individually and in combinations (CDZ mix), in a 10-fold serial dilution from 100–10-5 of the original viral RNA extractions, using mixed forward primers, reverse primers and probes. As this assay is not to be used to determine which specific DENV serotype is present, all four DENV serotypes were combined into DENV1-4 mix. Furthermore, all six lysates, individually or in CDZ mix, were combined with mosquito homogenate to verify whether mosquito components cross-react with the primers and probes. Additionally, negative controls of mosquito homogenate alone (NC1), NTC1 (PBS buffer), previous extraction of mosquito homogenate (NC2) and NTC2 (nuclease-free water only) were added to the RT-qPCR plate. After verification of lysate amplification for individual targets or CDZ mix, three RT-qPCR plates for CHIKV, DENV1-4 mix, ZIKV individually and in CDZ mix were done in triplicate each to determine the detection threshold as expressed by calculated pfu equivalents.

Each RT-qPCR reaction totaled 25 μl, consisting of 15 μl of RT-qPCR mixture and 10 μl of RNA extraction. For each reaction, the RT-qPCR mixture contains 2.5 μl of nuclease-free water, 6.25 μl of Taqman 1-Step Fast Viral Master Mix, 2.5 μl of forward primer(s) (10K pmol/ml), 2.5 μl of reverse primer(s) (10K pmol/ml), and 1.25 μl of probe(s) (6K pmol/ml). Primers and probes for CHIKV, DENV and ZIKV were described previously[21],[22] with modifications of reporter dyes and quenchers [Table 3]. The following protocol was used for RT-qPCR: reverse transcription was performed at 50°C for 5 min (Holding Stage 1 per Thermo Fisher protocol), followed by initial denaturation at 95°C for 20 sec, then 40 cycles of denaturation at 95°C for 3 sec and annealing/extension (combined for fast PCR) at 60°C for 30 sec[24].
Table 3: Primers, probes and their sequences for CHIKV, DENV1-4, and ZIKV

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A total of 5374 Ae. aegypti and 141 Ae. albopictus were collected from various locations in Chino Hills, Chino, Montclair, Ontario and Upland, California, USA during August 23, 2017 to October 2, 2020. In total, 1076 Ae. aegypti pools and 43 Ae. albopictus pools were made [Table 4] based on collection time and locations with a range of 1–46, and average of 4.93 mosquitoes/pool. Mosquitoes were homogenized, and extraction was done as previously described, and tested for CHIKV, DENV1-4, ZIKV simultaneously, along with positive controls of PC1 and PC2, negative controls of NC1, NC2, NTC1, and NTC2. Viral lysate mixture containing all six targets as described previously was used as PC1 to go through extraction and PCR, while previously extracted CDZ mix was used as PC2 to validate PCR process. The ZIKV lysate (strain H/FP/2013) with titer of 6.2 x 10[6] calculated pfu equivalents [Table 1] was used in the CDZ mix positive controls when testing mosquito samples.
Table 4: Aedes spp. pools tested for chikungunya, dengue and Zika in 2017-2020

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Ethical statement: Not applicable

  Results Top

Verification of primers and probes

To check the specificity and sensitivity of the primers and probes, the viral lysate, individually or in combination, was tested in a serial dilution from 100 to 10-5 from the original elution after extraction. A clear dilution - Ct value correlation was established for all targets. When tested alone, all six targets were detected individually with the following thresholds as indicated by calculated pfu equivalents: 3.96x10° for CHIKV, 3.80x10[1] for DENV1, 3.20x10[1] for DENV2, 8.00x10-1 for DENV3, 1.58x100 for DENV4, and 6.20x100 for ZIKV. When tested in a full combination of six targets (CDZ mix), CHIKV, DENV1-4 mix or ZIKV, all were detected with various thresholds of 1.32x100 for CHIKV, 3.79x100 for DENV1-4 and 2.06x100 for ZIKV. All targets, individually or in full combination were detected in the mixtures of Ae. aegypti homogenate, and viral lysates, with comparable Ct values with 100 lysate dilutions [Table 5]. There were no non-specific amplifications in individual lysates, their mixtures, or combinations with mosquito homogenate.
Table 5: Preliminary plates Ct values for each individual lysate or in combination as in CDZ mix

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Standard curves

The dilution - Ct value curves were generated for CHIKV, DENV1-4 mix, and ZIKV, individually and in CDZ mix. Each assay of these lysates was done in triplicate in each of three plates to check for amplification consistency. In individual assays, CHIKV was detected to 3.96x10-1, DENV1-4 to 1.14x100 and ZIKV to 3.20x100 In full CDZ combination assays, CHIKV was detected to 1.32x10-1, DENV1-4 to 3.79x101 and ZIKV to 1.07x100 calculated pfu equivalents as indicated by Ct values [Table 6].
Table 6: Standard curves for CHIKV, DENV1-4 mix, ZIKV and CDZ mix on three different plates

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Mosquito pools

All 1119 Aedes mosquito pools tested negative for CHIKV, DENV1-4, and ZIKV. The negative controls of NC1, NC2, NTC1, NTC2 all tested negative [Table 4]. Positive control consistently showed amplification curves for all three targets at each assay, CHIKV, DENV1-4, and ZIKV with average Ct values ± SD from all plates as follows: CHIKV with 22.74 ± 0.33, DENV1-4 with 24.83 ± 0.69, and ZIKV with 23.30 ± 0.39.

  Discussion Top

CHIKV, DENVs and ZIKV all share the same mosquito vectors and often present with similar clinical symptoms. Additionally, there have been human cases recorded of concurrent infection with chikungunya, dengue and Zika viruses[25],[26],[27]. Consequently, it can be difficult to differentiate among these diseases. Concurrent infections of more than one virus may also exist in vector mosquitoes, particularly in the areas with high prevalence of these viruses. Control of the spread of these emerging diseases relies heavily on accurate detection of infections in mosquito vectors. Therefore, it is essential that there are rapid, sensitive and specific diagnostic tools available to identify these infections in mosquito populations through molecular technologies at vector control entities. Previously we evaluated a preloaded TaqMan CDZ (CHIKV/DENV/ZIKV) lyophilized 1-step Triplex Kit (Thermo Fisher Scientific) where specified performance was observed[20]. However, this kit was not commercialized probably because of high cost and low profit margin. Furthermore, this kit cannot be adopted by any laboratory because of proprietary oligonucleotide sequences and manufacturing process. In order to provide a feasible, practical, and affordable tool which can be easily adopted by an ordinary laboratory to test CDZ targets simultaneously in mosquito samples, a RT-qPCR protocol was initiated, evaluated and applied for its intention. While working on developing this protocol to test mosquito pools, we tested various primers and probes sets to find what would best work for simultaneous detection of all six targets. Unfortunately, the DENV1-4 primers and probe set from earlier publication on CDZ targets[21] did not produce amplification curves for any of the dengue lysates used, but CHIKV and ZIKV both produced satisfactory results. DENV1-4 primer and probe sets from other previous studies intended for DENVs alone[22],[23] were tested, with the publication[22] being the only set to produce amplification curves for all four serotypes. The primers and probes that were strategically designed by previous studies for human infection diagnosis were chosen and evaluated in the current paper, which are expected to cover common viral strains from various geographical regions worldwide. While the primers and probes described in the current paper were used in RT-qPCR protocol for simultaneous detection of multiple targets, they might be adapted for other PCR procedures for the same targets.

The CDZ triplex RT-qPCR described herein appeared capable of detecting the targets individually or in full CDZ combinations. In preliminary assay for all six lysates, there was a clear and uniform increase in Ct values with serial dilution. This progression was seen consistently in all independent RT-qPCR experiments. Although some lysates turned undetectable quite early among the dilutions, this variability can be explained by the differences in the calculated starting lysate titers. For example, DENV1 was detected down to 10-2 dilution, whereas DENV2 and DENV4 were detected to 10-3 dilution, and DENV3 to 10-4 dilution. However, once the starting concentrations were considered, the sensitivity for DENV1 and DENV2 were lower than that of DENV3 and DENV4. The variation among the DENV1-4 may be due to the differences in copy number of the target sequence, in this case a highly conserved region of 3’ UTR genomic positions 10632–10695 (DENV1, GenBank acc. no. NC_001477)[22]. Additionally, DENV4 needed an additional reverse primer, due to differences of this serotype at the 10670–10695 position compared to DENV1-3[22]. The current protocol negated the need of an additional probe for DENV4 as in the trial CDZ kit (Thermo Fisher Scientific) that was evaluated previously[20]. The necessity of an additional reverse primer in the current paper, or also an additional probe as in previous publication[20] for DENV4 may be due to the slight genetic differences of the DENV4 serotype. DENV4 was one of the first serotypes to diverge in the phylogenetic lineage and is less related to the other three serotypes[28],[29]. Once amplification for each DENV serotype was observed, all four DENV lysates were added together in DENV1-4 mix. As this protocol is primarily to establish presence/absence of these three viruses, differentiation between serotypes was not intended, although any of the four serotypes of DENV was able to be detected. Additional PCR would be needed to differentiate individual DENV. It appeared that there was no interference of mosquito tissues on the RT-qPCR process described herewith as comparable Ct values were observed in the 1:1 mixture of mosquito homogenate versus viral lysate. The possible impact of mosquito bloodmeal on the test protocol was less concerned as the female mosquitoes that render positive PCR results after viral amplification are less likely to have much bloodmeal after the extrinsic incubation period.

Within the CDZ mix in standard curve assays, the DENV 1-4 mix was detected only to dilution 10-2 (3.79x101 calculated pfu equivalents for all DENV) [Table 6], however when amplified separately, the lowest detection threshold was further down to 10-4 dilutions (8.00x10-1 calculated pfu equivalents for DENV3) [Table 5]. The CHIKV lysate on Plate 1 and the ZIKV lysate on Plates 1 and 2 were also detected to one less dilution within the CDZ mix than when amplified separately [Table 6]. The phenomena described above is likely due to competition for reagents from all viral amplifications within the CDZ mix. As all three viral lysates are being amplified concurrently within the CDZ mix, this leaves less reagents for each viral lysate individually. In the wild mosquito populations, however, the concurrent infection of more than one types of virus in a sample is less likely than infection of single virus, therefore the competition of amplification for multiple targets is less concerned. It appeared that standard curve in Plate 3 did show slightly lower detection threshold of all lysates either individually and/or in combination (CHIKV, DENV1-4 mix, ZIKV and CDZ mix), although all three plates had approximately equivalent Ct values at the same dilutions. This is likely due to normal variation in PCR. Slight differences in pipetting, keeping samples cool and/or mixing of reagents may result in slightly different Ct values between plates, particularly for lower concentrations of sample.

The virus lysate supplied by the CDC was prepared in Qiagen AVL lysis buffer to inactivate the live viruses and preserve the RNA. However, our system has already been optimized for the Thermo Fisher MagMAX™-96 Viral RNA Isolation Kit, intended for total DNA and RNA extraction. It is interesting to note that this kit appears to be sufficient enough in extracting the viral RNA as each set of primers and probe amplified their specific target. However, future studies warrant exploration on if the use of the QIAamp® viral RNA mini kit would result in comparable sensitivity in the detection of CHIKV, DENV, and ZIKV.

  Conclusion Top

This CDZ triplex RT-qPCR protocol appears to amplify all three viruses of interest, particularly including all four serotypes for DENV. This test protocol is feasible, practical and affordable with demonstrated performance in specificity and sensitivity for all intended targets. The primers and probe for each target were elaborately designed in prior studies for diagnosis of human infections of various virial strains from different geographical regions. It also simplified the reagent by negating the need of an additional probe for DENV-4 and avoided the high cost of ready-to-use test kit as evaluated previously[20]. Detection of all four dengue serotypes highlighted the significance of this protocol as there has been an increased distribution of all serotypes since 1970[30]. Additionally, it does not appear to cross react with mosquito homogenate of Ae. aegypti or Ae. albopictus, even though at this time we have not come across any positive field samples yet. With increased globalization and human travel, both chikungunya and Zika viruses are being detected in novel locations. Upon the introduction and spread of both Ae. aegypti and Ae. albopictus in California, USA[31],[32], and elsewhere, the risk of spread of these three emerging diseases is imminent when considering the increasing numbers of cases in travelers. Therefore, it is vital that mosquito and vector control entities have efficient assays to detect these diseases as early as possible. Only with early detection and quick action will it be possible to prevent further spread of novel diseases within California and elsewhere. With the full validation of this protocol for its specificity and sensitivity using viral lysates, individually or in combination, in the absence and presence of mosquito homogenate, as well as application to over 1000 field-collected samples of Ae. aegypti and Ae. albopictus, we are confident that this protocol is ready to be adopted in any areas with concerns of the viral pathogens evaluated.

Conflict of interest: None

  Acknowledgements Top

The authors are grateful to the valuable help from Brandy Russell with Diagnostic & Reference Laboratory, Arbovirus Diseases Branch, Centers for Disease Control and Prevention (Fort Collins, CO, USA) for her help and contribution of virus lysates, and to Alden Estep with Navy Entomology Center of Excellence, CMAVE Detachment (Gainesville, FL, USA) for provision of colonized Aedes aegypti.

  References Top

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  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]


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