Simple, sensitive and cost-effective detection of wAlbB Wolbachia in Aedes mosquitoes, using loop mediated isothermal amplification combined with the electrochemical biosensing method

BackgroundWolbachia is an endosymbiont bacterium generally found in about 40% of insects, including mosquitoes, but it is absent in Aedes aegypti which is an important vector of several arboviral diseases. The evidence that Wolbachia trans-infected Ae. aegypti mosquitoes lost their vectorial competence and became less capable of transmitting arboviruses to human hosts highlights the potential of using Wolbachia- based approaches for prevention and control of arboviral diseases. Recently, release of Wolbachia trans-infected Ae. aegypti has been deployed widely in many countries for the control of mosquito-borne viral diseases. Field surveillance and monitoring of Wolbachia presence in released mosquitoes is important for the success of these control programs. So far, a number of studies have reported the development of loop mediated isothermal amplification (LAMP) assays to detect Wolbachia in mosquitoes, but the methods still have some specificity issues. Methodology/Principal FindingsWe describe here the development of a LAMP combined with the DNA strand displacement-based electrochemical sensor (BIOSENSOR) method to detect wAlbB Wolbachia in trans-infected Ae. aegypti. Our developed LAMP primers were more specific to wAlbB detection than those of the previous published ones if the assays were conducted with low-cost and non-specific detecting dyes. The detection capacity of our LAMP technique was 3.8 nM and the detection limit reduced to 2.16 fM when combined with the BIOSENSOR. Our study demonstrates that the BIOSENSOR can also be applied as a stand-alone method for detecting Wolbachia; and it showed high sensitivity when used with the crude DNA extracts of macerated mosquito samples without DNA purification. Conclusions/SignificanceOur results suggest that both LAMP and BIOSENSOR, either used in combination or stand-alone, are robust and sensitive. The methods have good potential for routine detection of Wolbachia in mosquitoes during field surveillance and monitoring of Wolbachia-based release programs, especially in countries with limited resources. Author SummaryMosquito-borne diseases such as dengue, chikungunya, Zika, and yellow fever are transmitted to humans mainly by the bites of Aedes aegypti mosquitoes. Controlling these diseases relies mostly on the use of insecticides, in which the efficiency has been reduced through development of insecticide resistance in mosquitoes. Wolbachia is the endosymbiotic bacteria that are naturally found in 40% of insects, including mosquitoes. The bacteria could protect their hosts from viral infections and could also cause sterility in host populations, therefore, providing an opportunity to use them for disease control. Application of a Wolbachia-based strategy needs simple, rapid and sensitive methods for detecting the bacteria in released mosquitoes. In this paper, we develop the combined methods of LAMP and BIOSENSORS for detecting wAlbB Wolbachia in mosquitoes. Our positive LAMP reaction can be visualized by color change from violet to blue at a sensitivity of [≥] 60 pg of genomic DNA. When used in combination with the BIOSENSOR method, the sensitivity increased a million fold without losing specificity. Our study indicates that both developed methods, either used in combination or stand-alone, are efficient and cost-effective, hence, it could be applied for routine surveys of Wolbachia in mosquito control programs that use Wolbachia-based approaches.