Abstract
BackgroundThe global expansion of dengue (DENV), chikungunya (CHIKV), and Zika viruses (ZIKV) is having a serious impact on public health. Because these arboviruses are transmitted by the same mosquito species and co-circulate in the same area, a sensitive diagnostic assay that detects them together, with discrimination, is needed.MethodsWe present here a diagnostics panel based on reverse transcription-PCR amplification of viral RNA and an xMap Luminex architecture involving direct hybridization of PCRamplicons and virus-specific probes. Two DNA innovations (“artificially expanded genetic information systems”, AEGIS, and “self-avoiding molecular recognition systems”, SAMRS) increase the hybridization sensitivity on Luminex microspheres and PCR specificity of the multiplex assay compared to the standard approach (standard nucleotides).ResultsThe diagnostics panel detects, if they are present, these viruses with a resolution of 20 genome equivalents (DENV1), or 10 (DENV3–4, CHIKV) and 80 (DENV2, ZIKV) genome equivalents per assay. It identifies ZIKV, CHIKV and DENV RNAs in a single infected mosquito, in mosquito pools comprised of 5 to 50 individuals, and mosquito saliva (ZIKV, CHIKV, and DENV2). Infected mosquitoes and saliva were also collected on a cationic surface (Q-paper), which binds mosquito and viral nucleic acids electrostatically. All samples from infected mosquitoes displayed only target-specific signals; signals from non-infected samples were at background levels.ConclusionsOur results provide an efficient and multiplex tool that may be used for surveillance of emerging mosquito-borne pathogens which aids targeted mosquito control in areas at high risk for transmission.
Highlights
The global expansion of dengue (DENV), chikungunya (CHIKV), and Zika viruses (ZIKV) is having a serious impact on public health
Development of six-fold multiplexed Reverse transcription polymerase chain reaction (RT-PCR) based Luminex Direct Hybridization Assays (DHAs) platform for detection of four dengue serotypes, chikungunya, and Zika viruses and validation of panel sensitivity Viral RNA stock solutions were purified from viral isolates propagated in Vero cell culture, tittered, and expressed as genome equivalents
All standard primers and probes were tested by monoplexed conventional reverse transcription (RT)-PCR followed by a downstream hybridization on the Luminex platform
Summary
The global expansion of dengue (DENV), chikungunya (CHIKV), and Zika viruses (ZIKV) is having a serious impact on public health. Because these arboviruses are transmitted by the same mosquito species and cocirculate in the same area, a sensitive diagnostic assay that detects them together, with discrimination, is needed. Dengue (DENV), chikungunya (CHIKV), and Zika (ZIKV) viruses are mosquito-borne pathogens that have caused numerous outbreaks in Southeast Asia and more recently undergone geographic expansion in the Americas, causing emerging and serious health problems in humans [1,2,3,4,5]. High viremia levels of these pathogens predisposes epidemics in human populations that are sustained by human-mosquito transmission [4, 16, 17] with ZIKV being the first known arbovirus that could be
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