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Development of dPCR and NGS methods for improved surveillance and detectionof new mosquito densoviruses
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International audience. The use of mosquito densoviruses (MDVs) is considered a promising way to control mosquitoes through insecticide/MDV combinations or in combination with the sterile male technique (boosted SIT). Existing scientific literature mostly focuses on prevalence studies in mosquito populations from limited geographical regions, or on experimental studies evaluating larvicidal efficacy of specific MDV strains, and overall some gaps remain about the diversity, biological as well as geographical distribution of MDV worldwide. To date, only a few MDV candidates have been identified and tested as effective control tools, and methods for monitoring MDV and its spread in wild mosquito populations or persistence in the environment are still lacking. The lack of such tools limits the ability to monitor MDV dynamics and evaluate its efficacy as a control measure in real time. We have developed two complementary methods to detect MDV in wild populations. First, we screened field mosquito populations collected to detect the presence of MDV using an NGS approach based on Illumina sequencing of targeted viral sequences. Second, we developed a pan-MDV monitoring tool using dPCR to detect viruses belonging to the two genera known to infect mosquitoes (Protoambidensovirus and Brevihamaparvovirus). Although dPCR allows accurate virus detection, it currently does not allow sequencing of positive droplets, limiting our ability to identify viruses based on sequence analysis. To overcome this limitation, we investigated the possibility of using double emulsion PCR, which allows droplet recovery, positive sorting and sequencing. I will present the results obtained using both NGS and dPCR approaches on mosquito collections from Gabon, Camargue and Mexico.
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