A combinatorial probe-coding multiplex RT-qPCR assay for simultaneous detection and differentiation of dengue, Zika, and chikungunya viruses in human and mosquito samples

Abstract Arboviruses such as Zika virus (ZIKV), dengue virus (DENV), and chikungunya virus (CHIKV) remain significant public health threats in tropical and subtropical regions. These viruses are transmitted primarily by Aedes mosquitoes and often co-circulate in the same geographic areas, leading to outbreaks with overlapping clinical presentations and cross-reactivity in serological testing, particularly between ZIKV and DENV. These aspects create diagnostic challenges, and the development of molecular diagnostic methods is critically necessary. Here, we developed a diagnostic kit based on the Multicolor Combinational Probe Coding (MCPC) concept to simultaneously detect six viral targets (ZIKV, DENV1–4, and CHIKV) and an internal control (IC), by encoding targets with four spectrally distinct dyes. Using a panel of 660 characterized human serum or plasma samples, and 9 artificial DENV-4 samples, we obtained diagnostic sensitivity and specificity above 94% and 99%, respectively, for all targets. The assay enables the simultaneous analysis of up to 91 samples, substantially increasing testing throughput compared to commercially available products. The test was also applied to mosquito specimens of Aedes spp., Anopheles spp., Haemagogus spp., and Culex spp., demonstrating its utility as a robust tool not only for emergency response but also for arbovirus surveillance in public health settings.

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Publication Details

Journal
Research Square
Published
2026-11-02
DOI
https://doi.org/10.21203/rs.3.rs-10780424/v1
Primary Topic
Mosquito-borne diseases and control
Type
preprint
Controls
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preprint

A combinatorial probe-coding multiplex RT-qPCR assay for simultaneous detection and differentiation of dengue, Zika, and chikungunya viruses in human and mosquito samples

Thiago Jacomasso, Adriana Yurika Maeda, Irina Nastassja Riediger, Clarice Neuenschwander Lins de Morais et al.
3 links
Research Square
Mosquito-borne diseases and control
preprint

A combinatorial probe-coding multiplex RT-qPCR assay for simultaneous detection and differentiation of dengue, Zika, and chikungunya viruses in human and mosquito samples

Thiago Jacomasso, Adriana Yurika Maeda, Irina Nastassja Riediger, Clarice Neuenschwander Lins de Morais, Victoria Stadler Tasca Ribeiro, Mariana Matos Roll, Cintia Damasceno dos Santos Rodrigues, Aline Andrade Freund, Andrea Cristine Koishi, Lívia Medeiros Neves Casseb, Fabrício Klerynton Marchini, Elisa de Almeida Neves Azevedo, Arabela Leal e Silva de Mello, Tatiane Cinquini Moraes, Lilian Motta Cantanhêde, Felicidade Mota Pereira, Carolina Cardoso Santos, Bruno Tardelli Diniz Nunes, Aline Burda Farias, Ana María Bispo de Filippis, Priscila Souza de Almeida, Nélson Marques, Alessandro Afornali, Felipe Naveca, Ana Cecília Ribeiro Cruza, Fernanda Gatto de Almeida, Marcel Kruchelski Tschá, Nicole Tanuri Romancini, Luciane Keinrath Schier, Adriana Paula de Souza Costa
preprint en

Abstract

Abstract Arboviruses such as Zika virus (ZIKV), dengue virus (DENV), and chikungunya virus (CHIKV) remain significant public health threats in tropical and subtropical regions. These viruses are transmitted primarily by Aedes mosquitoes and often co-circulate in the same geographic areas, leading to outbreaks with overlapping clinical presentations and cross-reactivity in serological testing, particularly between ZIKV and DENV. These aspects create diagnostic challenges, and the development of molecular diagnostic methods is critically necessary. Here, we developed a diagnostic kit based on the Multicolor Combinational Probe Coding (MCPC) concept to simultaneously detect six viral targets (ZIKV, DENV1–4, and CHIKV) and an internal control (IC), by encoding targets with four spectrally distinct dyes. Using a panel of 660 characterized human serum or plasma samples, and 9 artificial DENV-4 samples, we obtained diagnostic sensitivity and specificity above 94% and 99%, respectively, for all targets. The assay enables the simultaneous analysis of up to 91 samples, substantially increasing testing throughput compared to commercially available products. The test was also applied to mosquito specimens of Aedes spp., Anopheles spp., Haemagogus spp., and Culex spp., demonstrating its utility as a robust tool not only for emergency response but also for arbovirus surveillance in public health settings.

Research Square
Instituto Adolfo Lutz (BR), Instituto de Biologia Molecular do Paraná (BR), Ezequiel Dias Foundation (BR), Instituto Evandro Chagas (BR), Fiocruz Amazônia (BR), Instituto Carlos Chagas (BR), Instituto Oswaldo Cruz (BR), Instituto Aggeu Magalhães (BR)
Good health and well-being
Mosquito-borne diseases and control
DOIOpen Access PDFOpenAlex
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