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The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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 Furthermore, the large burden of non-specific nucleic acids as well as other molecular and 328 macroscopic components present in a crude mosquito sample did not compromise signal 329 accuracy. We also confirmed the absence of significant inhibition of amplification and signaling 330 by recapitulating the detection limit of synthetic DNA targets in a background of crude non-331 specific mosquito sample. The coi and wsp LAMP-OSD assays could detect 4 and 40 target 332 copies, respectively, even in the presence of 8% reaction volume of crude mosquito sample (S5 333 blood meal could be directly analyzed by visual LAMP-OSD without diminution of signal to noise 342 ratio (Fig 3) . 343 The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/291849 doi: bioRxiv preprint served as controls for monitoring background fluorescence. LAMP-OSD fluorescence was 350 imaged at endpoint using a cellphone. 351 albopictus, and Ochlerotatus species. The mosquitoes were divided into three groups of 30 356 individuals that were stored without desiccant at -20 °C, 4 °C, or 37 °C for 1, 2, or 3 weeks prior 357 to testing. To reduce mosquito processing cost, footprint, and time for this large study, we 358 further simplified sample preparation requirements by optimizing the "in-tube" mosquito 359 preparation method wherein each mosquito was crushed with a micropestle directly in a 360 microcentrifuge tube followed by resuspension in water and introduction in a LAMP-OSD 361