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

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.

A LAMP assay for the rapid and robust assessment of Wolbachia infection in Aedes aegypti under field and laboratory conditions

“…When compared with the original 5-primer wAlbB wsp LAMP assay (17), the modified 6primer assay was substantially faster (compare the concentration curve for 5-primer LAMP in Figure 1B with the curve for 6-primer (modified) LAMP in Figure S1B). This is expressed by a five-minute difference in intercept values for the respective regressions.…”

“…LAMP primers for the wAlbB wsp sequence were derived from Bhadra et al (17). However, their OSD probe for wsp was replaced by an additional loop primer to increase detection speed (see Supplementary Table 1).…”

“…Three LAMP assays have thus far been published for Wolbachia monitoring (16)(17)(18). Gonçalves et al (2014) targeted Wolbachia's 16S ribosomal protein sequence, and amplified Wolbachia across multiple strains, including wAlbB, wMel, and wMelPop.…”

“…However, an additional assay was also developed specific to wAlbB (ordinarily present within Ae. albopictus) and wPip strain Wolbachia surface proteins (wsp) (17). This assay originally involved one loop primer and a specific detection method with an additional oligomeric probe designed for presence/absence discrimination of fluorescence by eye.…”

Multiplex logic processing isothermal diagnostic assays for an evolving virus

“…12 One-pot LAMP-OSD assays that can work with crude samples are especially appealing for POC use. 13,14 These assays have been coupled to highly sensitive and reliable 'yes/no' output signals such as fluorescence, glucose, or hCG that can be readily read using offthe-shelf cellphones, glucometers, or pregnancy test strips, respectively. 12, 13, 15 16, 17, 18, 19 Toehold switch RNA sensors have also been used to link iNAATs with in vitro reporter protein translation, leading to colorimetric signals.…”