Multiplex Surveillance Kit Using Sweetened Solid Support to Detect Arboviruses Isothermally in Mosquito Saliva from the Field

Abstract

Recently reported "displaceable probe" loop amplification (DP-LAMP) architecture has shown to amplify viral RNA from SARS-CoV-2 with little sample processing. The architecture allows signals indicating the presence of target nucleic acids to be spatially separated, and independent in sequence, from the complicated concatemer that LAMP processes create as part of their amplification process. This makes DP-LAMP an attractive molecular strategy to integrate with trap and sampling innovations to detect RNA from arboviruses carried by mosquitoes in the field. These innovations include (a) development of organically produced carbon dioxide with ethylene carbonate as a bait deployable in mosquito trap, avoiding the need for dry ice, propane tanks, or inorganic carbonates and (b) a process that induces mosquitoes to lay virus-infected saliva on a quaternary ammonium-functionalized paper (Q-paper) matrix, where (c) the matrix (i) inactivates the deposited viruses, (ii) releases their RNA, and (iii) captures viral RNA in a form that keeps it stable for days at ambient temperatures. We report this integration here, with a surprisingly simple workflow. DP-LAMP with a reverse transcriptase was found to amplify arboviral RNA directly from Q-paper, without requiring a separate elution step. This capture-amplification-detection architecture can be multiplexed, with the entire system integrated into a device that can support a campaign of surveillance, in the wild outdoors, that reports the prevalence of arboviruses from field-captured mosquitoes.

Rapid detection of West Nile and Dengue viruses from mosquito saliva by loop-mediated isothermal amplification and displaced probes

“…All viral RNA samples were stored at −80˚C until required for experiments. Premixed DP-LAMP buffer consisted of 12.5 μL of WarmStart 2X MM (NEB, MA, USA), 2.5 μL of WNV or DENV-I 10 X DP-LAMP primers and probes (1.6 μM of FIP and BIP, 0.4 μM of LB for WNV or 0.5 μM of LB for DENV, 0.5 μM of LF for WNV or 0.4 μM of LF for DENV-I, 0.2 μM of F3, and B3, 0.1 μM of fluorescent FAM-3 0 labeled probe and 0.15 μM of Iowa Black FQ-5 0 labeled LB/LF probe), 0.5 μL of RNase Inhibitor (NEB, MA, USA), 0.5 μL of Antarctic UDG (NEB, MA, USA), 0.25 μL of ET SSB (NEB, MA, USA), and 0.2 μL of dUTP (Promega, WI, USA) [21,24,30]. An aliquot of 7.55 μL of aqueous sucrose was added to the pre-mixed DP-LAMP buffer to achieve specified concentrations, which included 0%, 5%, 10%, 20%, and 40%.…”

“…It is also useful for multiplexing and real-time monitoring, targeting different viruses in a single reaction at a time [21]. Recently, our group successfully detected viral RNA in RT-LAMP with displaced probes from quaternary ammonium functionalized paper by honey-induced mosquito salivation and showed potential fesibility of arbovirus stability and detection [24].…”