Wolbachia detection in insects through LAMP: loop mediated isothermal amplification
© Gonçalves et al.; licensee BioMed Central Ltd. 2014
Received: 25 March 2014
Accepted: 6 May 2014
Published: 19 May 2014
The bacterium Wolbachia is a promising agent for the biological control of vector-borne diseases as some strains have the ability to block the transmission of key human disease-causing pathogens. Fast, accurate and inexpensive methods of differentiating between infected and uninfected insects will be of critical importance as field-based trials of Wolbachia-based bio-control become increasingly common.
We have developed a specific and sensitive method of detecting Wolbachia based on the isothermal DNA amplification. This technique can be performed in an ordinary heat block without the need for gel-based visualisation, and is effective for a wide variety of insect hosts.
Here we present the development of a rapid, highly sensitive and inexpensive method to detect Wolbachia in a variety of insect hosts, including key mosquito disease vectors.
Vector-borne diseases (e.g. malaria, dengue, filariases and Chagas disease) occur in more than 100 countries, primarily within the tropics, with the annual, global death rate in the millions. Despite the existence of a variety of vector control measures disease incidence is generally increasing, and consequently there is an urgent need to develop new and effective control strategies . Ideally these strategies should be able to be used in conjunction with existing control methods, and in this context the bacterium Wolbachia pipientis has proven to be a promising alternative, given its ability to restrict pathogen development in many different mosquito-pathogen combinations [2–5], and recently being applied in the field in Australia, Vietnam, Indonesia and soon in other dengue endemic countries (http://www.eliminatedengue.org).
Wolbachia is a vertically-transmitted bacterial endosymbiont of arthropods that is able to manipulate its host’s reproductive system and consequently spread rapidly through wild populations . Wolbachia was originally identified in the ovaries of the mosquito Culex pipiens, and recent studies have estimated that 40% of terrestrial arthropod species are infected with the bacterium . Critically, infections do not occur in key vector species including the dengue mosquito Aedes aegypti or in the Anopheles genus . Wolbachia is typically detected within the host by PCR against genes such as wsp (Wolbachia surface protein) , ftsZ (cell division protein) , and 16S ribosomal protein , or through fluorescence-based assays . These techniques can be expensive to perform for large numbers of samples, and require the use of laboratory equipment.
The LAMP (loop mediated isothermal amplification) technique utilizes Bst DNA polymerase to create stand-displacement amplification, and operates at a constant temperature . Examples of its use are very broad, ranging from the detection of pathogenic microorganisms to sex determination in embryos . The LAMP product can be detected by the visualisation of turbidity, fluorescence or a metal ion indicator producing a colorimetric change  without the need to run a gel. These characteristics make LAMP a suitable method to detect Wolbachia under circumstances where minimum infrastructure is available. Here we report the use of LAMP to detect Wolbachia in a diverse range of insect species, which will prove beneficial to monitoring the progress of field-based trials utilising the bacterium.
We examined many different species including laboratory-reared mosquito lines of Aedes fluviatilis, naturally infected by the Wolbachia strain w Flu, A. aegypti transinfected with the w MelPop and w Mel strains [17–19], and uninfected Anopheles aquasalis mosquitoes. These lines were reared at FIOCRUZ Minas as previously described . We also examined field-captured mosquitoes including A. aegypti, A. albopictus, and Culex sp. Furthermore, insects belonging to a diverse range of orders, which were known to be Wolbachia-infected (unpublished results) were also included.
Loop-mediated isothermal amplification (LAMP) primers based on Wolbachia 16S sequences, designed using LAMP designer 1.02 software
Each LAMP reaction (Vtotal = 25 μl) contained 1× ThermoPol Reaction Buffer (20 mM Tris–HCl, pH 8.8, 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100), 0.32 mM of each dNTP, 0.64 μM of each internal primer (FIP/BIP), 0.32 μM of LoopF and LoopB, 0.16 μM of each external primer (F3/B3), 0.64 M of Betaine, 6U of Bst DNA polymerase, large fragment (New England Biolabs), and approximately 30 ng of DNA. 1.2 mM of the metal ion indicator Hydroxy Naphthol Blue (HNB) was added to the ThermoPol Reaction Buffer. The mixture was incubated at 63°C for 90 minutes on a thermocycler or heat block, to determine if the reaction could be performed with a simpler setup. To check the sensitivity of the assay we cloned the external primer amplicon (F3/B3) into the pGemT-Easy (Promega) plasmid, and observed the efficacy of the assay on serial dilutions of the product.
Results and discussion
Loop mediated isothermal amplification has been used to detect different organisms, such as Leishmania, Toxoplasma gondii and also bacteria belonging to the same class as Wolbachia (Alphaproteobacteria); Brucella spp , Anaplasma ovis and Ehrlichia ruminantium. We designed a LAMP-based assay to detect Wolbachia in different hosts using the bacterial 16S rRNA sequence . Initially we designed the four basic LAMP primers (F3, B3, FIP and BIP) but due to low specificity we then included the extra Loop primers .
To optimize the LAMP reaction for Wolbachia, samples of A. fluviatilis and w Mel-infected A. aegypti were used to conduct a time course assay where we determined that amplification first occurred after 60 minutes of incubation (at 63°C using a thermocycler – Additional file 1). This was subsequently standardized at 90 minutes in order to account for samples with low bacterial density.
In terms of the cost per reaction, LAMP is approximately half as expensive as conventional PCR. If the cost of equipment is also included, LAMP becomes comparatively even cheaper given that it can be performed using only a heat block. As a further cost-saving measure, or if required in the field, homogenization of specimens could be performed using micro-pestles rather than a beadbeater.
We have demonstrated the reliability and sensitivity of using LAMP to detect Wolbachia, with detection possible for low-density infections, and the results easily visualized by the naked eye. Consequently, this technique could be readily applied in the field, without the need to use expensive laboratory equipment. This is the first report of the use of LAMP to detect Wolbachia infection in different hosts. The technique could potentially be applied to differentiating between multiple strains that could potentially occur within the same host species.
We thank Constancia Ayres for providing mosquito samples. We are in debt to Fernanda Rezende for technical and Alice Guimarães for administrative support, as well as Eric P. Caragata for critical reading. DS Gonçalves and LA Moreira are CNPq fellows. NBR is a CAPES fellow (BEX 11603/13-5).
This work was supported by FAPEMIG, CNPq, the Brazilian Ministry of Health (DECIT/SVS), and a grant to Monash University from the Foundations for the National Institutes of Health through the Vector-Based Transmission of Control: Discovery Research (VCTR) program of the Grand Challenges in Global Health Initiatives of the Bill and Melinda Gates Foundations.
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