Direct nucleic acid analysis of mosquitoes for high fidelity species identification and detection of Wolbachia using a cellphone.
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...
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ftdoajarticles:oai:doaj.org/article:5113a60a2cf3435bab15aa018b5c15b2 2023-05-15T15:16:11+02:00 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 2018-08-01T00:00:00Z https://doi.org/10.1371/journal.pntd.0006671 https://doaj.org/article/5113a60a2cf3435bab15aa018b5c15b2 EN eng Public Library of Science (PLoS) http://europepmc.org/articles/PMC6116922?pdf=render https://doaj.org/toc/1935-2727 https://doaj.org/toc/1935-2735 1935-2727 1935-2735 doi:10.1371/journal.pntd.0006671 https://doaj.org/article/5113a60a2cf3435bab15aa018b5c15b2 PLoS Neglected Tropical Diseases, Vol 12, Iss 8, p e0006671 (2018) Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 article 2018 ftdoajarticles https://doi.org/10.1371/journal.pntd.0006671 2022-12-30T22:05:06Z 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. Article in Journal/Newspaper Arctic Directory of Open Access Journals: DOAJ Articles Arctic PLOS Neglected Tropical Diseases 12 8 e0006671 |
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Open Polar |
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Directory of Open Access Journals: DOAJ Articles |
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English |
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Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 |
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Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 Sanchita Bhadra Timothy E Riedel Miguel A Saldaña Shivanand Hegde Nicole Pederson Grant L Hughes Andrew D Ellington Direct nucleic acid analysis of mosquitoes for high fidelity species identification and detection of Wolbachia using a cellphone. |
topic_facet |
Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 |
description |
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. |
format |
Article in Journal/Newspaper |
author |
Sanchita Bhadra Timothy E Riedel Miguel A Saldaña Shivanand Hegde Nicole Pederson Grant L Hughes Andrew D Ellington |
author_facet |
Sanchita Bhadra Timothy E Riedel Miguel A Saldaña Shivanand Hegde Nicole Pederson Grant L Hughes Andrew D Ellington |
author_sort |
Sanchita Bhadra |
title |
Direct nucleic acid analysis of mosquitoes for high fidelity species identification and detection of Wolbachia using a cellphone. |
title_short |
Direct nucleic acid analysis of mosquitoes for high fidelity species identification and detection of Wolbachia using a cellphone. |
title_full |
Direct nucleic acid analysis of mosquitoes for high fidelity species identification and detection of Wolbachia using a cellphone. |
title_fullStr |
Direct nucleic acid analysis of mosquitoes for high fidelity species identification and detection of Wolbachia using a cellphone. |
title_full_unstemmed |
Direct nucleic acid analysis of mosquitoes for high fidelity species identification and detection of Wolbachia using a cellphone. |
title_sort |
direct nucleic acid analysis of mosquitoes for high fidelity species identification and detection of wolbachia using a cellphone. |
publisher |
Public Library of Science (PLoS) |
publishDate |
2018 |
url |
https://doi.org/10.1371/journal.pntd.0006671 https://doaj.org/article/5113a60a2cf3435bab15aa018b5c15b2 |
geographic |
Arctic |
geographic_facet |
Arctic |
genre |
Arctic |
genre_facet |
Arctic |
op_source |
PLoS Neglected Tropical Diseases, Vol 12, Iss 8, p e0006671 (2018) |
op_relation |
http://europepmc.org/articles/PMC6116922?pdf=render https://doaj.org/toc/1935-2727 https://doaj.org/toc/1935-2735 1935-2727 1935-2735 doi:10.1371/journal.pntd.0006671 https://doaj.org/article/5113a60a2cf3435bab15aa018b5c15b2 |
op_doi |
https://doi.org/10.1371/journal.pntd.0006671 |
container_title |
PLOS Neglected Tropical Diseases |
container_volume |
12 |
container_issue |
8 |
container_start_page |
e0006671 |
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1766346475941396480 |