Abstract
Manipulation of natural mosquito populations using the endosymbiotic bacteria Wolbachia is being investigated as a novel strategy to reduce the burden of mosquito-borne viruses. To evaluate the efficacy of these interventions, it will be critical to determine Wolbachia infection frequencies in Aedes aegypti mosquito populations. However, current diagnostic tools are not well-suited to fit this need. Morphological methods cannot identify Wolbachia, immunoassays often suffer from low sensitivity and poor throughput, while PCR and spectroscopy require complex instruments and technical expertise, which restrict their use to centralized laboratories. To address this unmet need, we have used loop-mediated isothermal amplification (LAMP) and oligonucleotide strand displacement (OSD) probes to create a one-pot sample-to-answer nucleic acid diagnostic platform for vector and symbiont surveillance. LAMP-OSD assays can directly amplify target nucleic acids from macerated mosquitoes without requiring nucleic acid purification and yield specific single endpoint yes/no fluorescence signals that are observable to eye or by cellphone camera. We demonstrate cellphone-imaged LAMP-OSD tests for two targets, the Aedes aegypti cytochrome oxidase I (coi) gene and the Wolbachia surface protein (wsp) gene, and show a limit of detection of 4 and 40 target DNA copies, respectively. In a blinded test of 90 field-caught mosquitoes, the coi LAMP-OSD assay demonstrated 98% specificity and 97% sensitivity in identifying Ae. aegypti mosquitoes even after 3 weeks of storage without desiccant at 37°C. Similarly, the wsp LAMP-OSD assay readily identified the wAlbB Wolbachia strain in field-collected Aedes albopictus mosquitoes without generating any false positive signals. Modest technology requirements, minimal execution steps, simple binary readout, and robust accuracy make the LAMP-OSD-to-cellphone assay platform well suited for field vector surveillance in austere or resource-limited conditions.
Highlights
Mosquitoes are vectors that can transmit an array of pathogens that often cause devastating human diseases [1]
For effective evaluation of this approach, regular surveillance of Wolbachia infections in Ae. aegypti is required. Current diagnostic tools, such as real time polymerase chain reaction, are not well suited to support these critical surveillance needs in resource poor settings due to their dependence on expensive instruments and technical expertise. To fill this need we developed a simple, robust and inexpensive assay to identify Ae. aegypti mosquitoes and Wolbachia using our unique one-pot assay platform, LAMP-oligonucleotide strand displacement (OSD), which uses loop-mediated isothermal amplification to amplify nucleic acid targets at a single temperature
Unlike other LAMP-based tests, our assays assure accuracy by coupling amplification with novel nucleic acid strand displacement (OSD) probes that hybridize to specific sequences in LAMP amplification products and thereby generate simple yes/no readout of fluorescence readable by human eye and by offthe-shelf cellphones
Summary
Mosquitoes are vectors that can transmit an array of pathogens that often cause devastating human diseases [1]. Wolbachia is a maternally-transmitted endosymbiont that can rapidly become established in the natural mosquito populations and can inhibit a variety of pathogens, including arboviruses, malaria parasites, and filarial nematodes [10,11,12,13,14,15]. Wolbachia control strategies are currently being deployed into the field to alter the capacity of Aedes aegypti to transmit arboviruses or to suppress mosquito populations [16,17,18]. In addition to screening for Wolbachia infection, it would be desirable to identify the host mosquito species using these assays since different mosquito species differ in their ability to transmit pathogens [20]. Prevalence and stability of Wolbachia is essential for effective vector control and pre-emption of disease outbreaks with public health measures [21]
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