A rapid molecular detection protocol for Chikungunya virus directly performed on Aedes albopictus (tiger mosquito)

Abstract

In the last few years tiger mosquitoes (Aedes albopictus), quickly and widely spread in Italy, represent ideal vectors for different Arboviruses, particularly Dengue virus (DenV) and Chikungunya virus (ChikV), who are causing millions of patients in the world per year. For ChikV, appeared for the first time in Italy in 2007, a Surveillance Plan was defined in the Marche Region, a neighbour county of the Italian outbreak site. As a support for this surveillance, we decided to create a new multiplex RT-PCR protocol to detect ChikV directly in tiger mosquitoes. All the mosquitoes were collected with BG-Sentinel® traps (Biogents AG, Regensburg, Germany).Total RNA extraction was carried with Helix RNA plus kit (Diatech srl, Jesi, Italy). For retro-transcription and amplification a Mastercycler® ep gradient S thermal cycler (Eppendorf AG, Hamburg, Germany) was used. From the whole RNA extracted from captured mosquitoes, we developed a new end-point multiplex retro transcriptase polymerase chain reaction (RT-PCR), for both the detection and identification of Aedes spp. and ChikV. This RT-PCR protocol is able to detect ChikV directly from adult insects, during alerts or emergencies.The entomological trapping associated with bio-molecular methods represents an effective strategy to detect ChikV directly from vectors. Moreover, after a specific evaluation, this RT-PCR protocol could be applied also for human blood samples in regions with the certain presence of this virus.

Keywords

• Chikungunya, Aedes albopictus, PCR, multiplex PCR, RT-PCR