Wolbachia detection in insects through LAMP: loop mediated isothermal amplification

Goncalves, Daniela da Silva; Cassimiro, Anna Paula Alvim; Oliveira, Caroline Dantas de; Rodrigues, Nilton; Moreira, Luciano Andrade

doi:10.1186/1756-3305-7-228

Cited by 28 publications

(31 citation statements)

References 28 publications

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“…The LAMP assay is a sensitive method for detecting low-abundance target DNA in a sample (Notomi et al, 2000). Goncalves Dda et al (2014) developed a LAMP assay for Wolbachia detection in insects (Goncalves Dda, Cassimiro, de Oliveira, Rodrigues, & Moreira, 2014). Recently, Bhadra et al (2018) reported a specific and sensitive assay that combines LAMP and oligonucleotide strand displacement (OSD) for detecting both species identity and Wolbachia .…”

Section: Resultsmentioning

confidence: 99%

Wolbachia pipientisoccurs inAedes aegyptipopulations in New Mexico and Florida, USA

Kulkarni

Jiang

et al. 2018

Preprint

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The mosquitoes Aedes aegypti (L.) and Ae. albopictus Skuse are the major vectors of dengue, Zika, yellow fever and chikungunya viruses worldwide. Wolbachia, an endosymbiotic bacterium present in many insects, is being utilized in novel vector control strategies to manipulate mosquito life history and vector competence to curb virus transmission. Earlier studies have found that Wolbachia is commonly detected in Ae. albopictus but rarely detected in Ae. aegypti. In this study, we used a two-step PCR assay to detect Wolbachia in wild-collected samples of Ae. aegypti. The PCR products were sequenced to validate amplicons and identify Wolbachia strains. A loop-mediated isothermal amplification (LAMP) assay was developed and used for detecting Wolbachia in selected mosquito specimens as well. We found Wolbachia in 85/148 (57.4%) wild Ae. aegypti specimens from various cities in New Mexico and in 2/46 (4.3%) from St. Augustine, Florida. We did not detect Wolbachia in 94 samples of Ae. aegypti from Deer Park, Harris County, Texas. Wolbachia detected in Ae. aegypti from both New Mexico and Florida was the wAlbB strain of Wolbachia pipientis. A Wolbachia positive colony of Ae. aegypti was established from pupae collected in Las Cruces, New Mexico in 2018. The infected females of this strain transmitted Wolbachia to their progeny when crossed with males of Rockefeller strain of Ae. aegypti, which does not carry Wolbachia. In contrast, none of the progeny of progeny of Las Cruces males mated to Rockefeller females were infected with Wolbachia.

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Section: Resultsmentioning

confidence: 99%

Wolbachia pipientisoccurs inAedes aegyptipopulations in New Mexico and Florida, USA

Kulkarni

Jiang

et al. 2018

Preprint

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“…Isothermal nucleic acid amplification assays would facilitate field-based vector monitoring, but most reported approaches rely on nucleic acid purification and non-specific readout, and thus suffer from laborious setup and the risk of false positives [61–63]. Probe-read isothermal methods such as the recombinase polymerase assay (RPA) are more reliable but still require expensive and proprietary reaction formulations and probes, which limits their flexibility and versatility in assay engineering.…”

Section: Discussionmentioning

confidence: 99%

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

Bhadra

Saldaña

Hegde

et al. 2018

Preprint

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20Manipulation of natural mosquito populations using the endosymbiotic bacteria Wolbachia is being 21 investigated as a novel strategy to reduce the burden of mosquito-borne viruses. To evaluate the 22 efficacy of these interventions, it will be critical to determine Wolbachia infection frequencies in 23Aedes aegypti mosquito populations. However, current diagnostic tools are not well-suited to fit this 24 need. Morphological methods cannot identify Wolbachia, immunoassays often suffer from low 25 sensitivity and poor throughput, while PCR and spectroscopy require complex instruments and 26 technical expertise, which restrict their use to centralized laboratories. To address this unmet need, 27we have used loop-mediated isothermal amplification (LAMP) and oligonucleotide strand 28 displacement (OSD) probes to create a one-pot sample-to-answer nucleic acid diagnostic platform 29 for vector and symbiont surveillance. LAMP-OSD assays can directly amplify target nucleic acids 30 from macerated mosquitoes without requiring nucleic acid purification and yield specific single 31 endpoint yes/no fluorescence signals that are observable to eye or by cellphone camera. We 32 demonstrate cellphone-imaged LAMP-OSD tests for two targets, the Aedes aegypti cytochrome 33 oxidase I (coi) gene and the Wolbachia surface protein (wsp) gene, and show a limit of detection 34 of 4 and 40 target DNA copies, respectively. In a blinded test of 90 field-caught mosquitoes, the 35 coi LAMP-OSD assay demonstrated 98% specificity and 97% sensitivity in identifying Ae. aegypti 36 mosquitoes even after 3 weeks of storage without desiccant at 37 °C. Similarly, the wsp LAMP-37 OSD assay readily identified the wAlbB Wolbachia strain in field-collected Aedes albopictus 38 mosquitoes without generating any false positive signals. Modest technology requirements, 39 minimal execution steps, simple binary readout, and robust accuracy make the LAMP-OSD-to-40 cellphone assay platform well suited for field vector surveillance in austere or resource-limited 41 conditions. 42 43 All rights reserved. No reuse allowed without permission. Author summary 44Mosquitoes spread many human pathogens and novel approaches are required to reduce the 45 burden of mosquito-borne disease. One promising approach is transferring Wolbachia into 46Aedes aegypti mosquitoes where it blocks transmission of arboviruses like dengue, Zika and 47Yellow fever viruses and spreads through mosquito populations. For effective evaluation of this 48 approach, regular surveillance of Wolbachia infections in Ae. aegypti is required, but current 49 diagnostic tools are not well suited to support these critical surveillance needs. To fill this need 50we developed a simple, robust and inexpensive assay to identify Ae. aegypti mosquitoes and 51Wolbachia using our unique one-pot assay platform, LAMP-OSD, which uses loop-mediated 52 isothermal amplification to amplify nucleic acid targets at a single temperature. Unlike other 53 LAMP-based tests, our assays assure accuracy by coupling amplification...

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“…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. A study applying this assay to field samples in Malaysia found that it compared favourably with a standard PCR method for Wolbachia detection in Ae.…”

Section: Introductionmentioning

confidence: 99%

“…albopictus mosquitoes, detecting a higher infected rate than observed with PCR (19). However, it should be noted that this study used an experimental design that did not include the loop primers, which Gonçalves et al (2014) consider essential to ensure specificity of the assay.…”

Section: Introductionmentioning

confidence: 99%

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

Jasper

Yang

Ross

et al. 2019

Preprint

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https://orcid.org/0000-0003-4541-3223, Qiong Yang 1 https://orcid.org/0000-0003-3336-5703, Perran A. Ross 1 https://orcid.org/0000-0001-7645-7523, Nancy Endersby-Harshman 1 https://orcid.org/0000-0002-9834-4068, Nicholas Bell 1 https://orcid.org/0000-0002-2420-6682, Ary A. Hoffmann 1 https://orcid.org/0000-0001-9497-7645 AbstractWith Wolbachia-based arbovirus control programs being scaled and operationalised around the world, cost effective and reliable detection of Wolbachia in field samples and laboratory stocks is essential for quality control. Here we validate a modified loop-mediated isothermal amplification (LAMP) assay for routine scoring of Wolbachia in mosquitoes from laboratory cultures and the field, applicable to any setting. We show that this assay is a rapid and robust method for highly sensitive and specific detection of wAlbB Wolbachia infection withinAedes aegypti under a variety of conditions. We test the quantitative nature of the assay by evaluating pooled mixtures of Wolbachia-infected and uninfected mosquitoes and show that it is capable of estimating infection frequencies, potentially circumventing the need to perform large-scale individual analysis for wAlbB infection status in the course of field monitoring. These results indicate that LAMP assays are useful for routine screening particularly under field conditions away from laboratory facilities.Importance. 2Releases of mosquitoes infected with strains of Wolbachia bacteria are expanding around the world because this bacterium that lives inside cells provides an effective tool to suppress mosquito populations and the ability of mosquitoes to transmit viruses. The success of the release programs relies on rapid and effective means of detecting Wolbachia and scoring their frequencies in mosquitoes for quality control and for assessing the success of releases. Here we test a "LAMP" (Loop-mediated isothermal amplification) assay for robust detection of Wolbachia infections in laboratory and field mosquito populations. We show that the assay can detect the bacteria when present at a low density in samples, and with a high degree of reproducibility. The assay uses a simple protocol which requires minimal training. It can readily detect Wolbachia in mosquitoes obtained from traps that are routinely used in field surveys. The assay should be cost effective in a variety of settings.

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Wolbachia detection in insects through LAMP: loop mediated isothermal amplification

Cited by 28 publications

References 28 publications

Wolbachia pipientisoccurs inAedes aegyptipopulations in New Mexico and Florida, USA

Wolbachia pipientisoccurs inAedes aegyptipopulations in New Mexico and Florida, USA

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

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

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