Direct nucleic acid analysis of mosquitoes for high fidelity species identification and detection of Wolbachia using a cellphone

Sanchita Bhadra; Timothy E. Riedel; Miguel A. Saldaña; Shivanand Hegde; Nicole Pederson; Grant L. Hughes; Andrew D. Ellington

The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/291849 doi: bioRxiv preprint 40 copies of its target sequence. In the absence of target DNA, neither assay generated 281 spurious signal. 282 Our next goal was to demonstrate the ability of these LAMP-OSD assays to detect naturally 290 occurring target sequences in mosquitoes. At the same time, we wanted to ensure that 291 minimally processed samples would be compatible with our detection platform in order to 292 facilitate rapid in-field vector testing with fewest instruments and user-required steps. 293 Therefore, as an initial approach, we developed the 'in-syringe' method for rapid sample 294 preparation wherein individual mosquitoes were crushed inside 1 cc syringes using the syringe 295 plunger as a pestle. A small chromatography column frit placed inside the syringe served as a 296 pedestal that aided maceration and removed larger particulates when the macerated sample 297 was re-suspended in water and recovered. Small portions (up to 8% of a LAMP-OSD reaction) 298 of these macerated samples were added directly to LAMP-OSD reactions, which were then 299 incubated for 90 min at 65 °C to initiate and sustain amplification. 300