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

Research output: Contribution to journalArticle

4 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)e0006671
JournalPLoS Neglected Tropical Diseases
Volume12
Issue number8
DOIs
StatePublished - Aug 1 2018

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Wolbachia
Culicidae
Nucleic Acids
Oligonucleotides
Aedes
Electron Transport Complex IV
Membrane Proteins
Hygroscopic Agents
Professional Competence
Immunoassay
Population
Genes
Limit of Detection
Spectrum Analysis
Fluorescence
Viruses
Technology
Bacteria
Sensitivity and Specificity
Polymerase Chain Reaction

ASJC Scopus subject areas

  • Public Health, Environmental and Occupational Health
  • Infectious Diseases

Cite this

Direct nucleic acid analysis of mosquitoes for high fidelity species identification and detection of Wolbachia using a cellphone. / Bhadra, Sanchita; Riedel, Timothy E.; Saldaña, Miguel A.; Hegde, Shivanand; Pederson, Nicole; Hughes, Grant L.; Ellington, Andrew D.

In: PLoS Neglected Tropical Diseases, Vol. 12, No. 8, 01.08.2018, p. e0006671.

Research output: Contribution to journalArticle

Bhadra, Sanchita ; Riedel, Timothy E. ; Saldaña, Miguel A. ; Hegde, Shivanand ; Pederson, Nicole ; Hughes, Grant L. ; Ellington, Andrew D. / Direct nucleic acid analysis of mosquitoes for high fidelity species identification and detection of Wolbachia using a cellphone. In: PLoS Neglected Tropical Diseases. 2018 ; Vol. 12, No. 8. pp. e0006671.
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