Abstract
Detection of the Wolbachia endosymbiont in Aedes aegypti mosquitoes through real-time polymerase chain reaction assays is widely used during and after Wolbachia releases in dengue reduction trials involving the wMel and wAlbB strains. Although several different primer pairs have been applied in current successful Wolbachia releases, they cannot be used in a single assay to distinguish between these strains. Here, we developed a new diagnostic primer pair, wMwA, which can detect the wMel or wAlbB infection in the same assay. We also tested current Wolbachia primers and show that there is variation in their performance when they are used to assess the relative density of Wolbachia. The new wMwA primers provide an accurate and efficient estimate of the presence and density of both Wolbachia infections, with practical implications for Wolbachia estimates in field collected Ae. aegypti where Wolbachia releases have taken place.
Highlights
The bacterium, Wolbachia, is providing an increasingly popular method to inhibit dengue virus transmission in the mosquito, Aedes aegypti
We developed a diagnostic primer pair, wMwA, that can detect and distinguish between the wMel and wAlbB infections in Aedes aegypti (Fig 1), which is important in simplifying current approaches for Wolbachia identification
When template DNA was extracted in Chelex1 100 Resin solution, the efficiencies of all primers ranged from 86.4% to 104.9%, (Table 2 and Fig 2) and the efficiency curves all showed an R2 valued greater than 0.99
Summary
The bacterium, Wolbachia, is providing an increasingly popular method to inhibit dengue virus transmission in the mosquito, Aedes aegypti. Detection of the Wolbachia endosymbiont in Ae. aegypti mosquitoes is a standard requirement for good laboratory practice during Wolbachia mosquito releases in dengue reduction programs and for tracking Wolbachia invasions in the field [4, 5]. In experiments where superinfected lines are used [9], or where mosquitoes carrying different single infections need to be distinguished for experiments or in field collected samples [10], several real-time PCR assays using different primer pairs are currently required.
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