Article

Loop‐mediated isothermal amplification (LAMP) for the identification of invasive Aedes mosquito species

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Abstract

Invasive Aedes mosquito species (Diptera: Culicidae) are of public health concern in Europe because they are either recognized or potential vectors of pathogens. Loop‐mediated isothermal amplification (LAMP) is a rapid and simple method for amplifying DNA with high specificity and efficiency, with the technique having potential for application in the field, including in high‐throughput format. Specific LAMP assays based on rDNA internal transcribed spacers 1 or 2 sequences, considering intraspecies variability at these loci, were developed for Aedes aegypti, Aedes albopictus, Aedes japonicus, Aedes koreicus and the indigenous Aedes geniculatus. No such assays could be developed for Aedes atropalpus and Aedes triseriatus because both loci were too short to serve as target. The assays rely on the clearly visible colour change from violet to sky blue after successful amplification. Sensitivity of egg detection was confirmed with ratios of up to one mosquito egg in 99 other eggs. Simple sample preparation of adults or eggs by mechanical homogenization in water required an additional heat treatment or centrifugation step to avoid non‐specific colour changes. Thus, further technical improvements are needed to render these assays truly field‐applicable, which would greatly facilitate surveillance of these invasive mosquito species and allow for prompt implementation of control measures. Loop‐mediated isothermal amplification assays for the invasive container‐breeding mosquitoes Aedes aegypti, Aedes albopictus, Aedes atropalpus, Aedes japonicus, Aedes koreicus, Aedes triseriatus and the indigenous Aedes geniculatus were developed. Intra‐individual and interspecies sequence variability were detected and considered for robust primer design. The sensitivity of the assays was tested, showing that one egg of a species could be detected in 99 other eggs.

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... In this study, we demonstrate proof-of-principle of a rapid colorimetric loop-mediated isothermal amplification (LAMP) based field test, which requires only minimal sample preparation. The Ae. aegypti assay was adapted from [16], whilst we developed the Ae. albopictus assay as part of the current study. ...
... Primers for the detection of Ae. aegypti (Table 1) were targeted to the ITS1 ribosomal spacer region [16]. Primers for the detection of Ae albopictus were designed targeting the ITS1 region using previously described LAMP assay parameters [18]. ...
... ITS1 has sufficient sequence variation that it enabled the design of species-specific primers. The primers used to detect Ae. albopictus were designed for this work and the primers used to detect Ae. aegypti were published previously [16] (Table 1). ...
Article
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Background Yellow fever, dengue, chikungunya and Zika viruses are responsible for considerable morbidity and mortality in humans. Aedes aegypti and Aedes albopictus are the most important mosquito vectors involved in their transmission. Accurate identification of these species is essential for the implementation of control programs to limit arbovirus transmission, during suspected detections at ports of first entry, to delimit incursions or during presence/absence surveillance programs in regions vulnerable to invasion. We developed and evaluated simple and rapid colorimetric isothermal tests to detect these two mosquito species based on loop-mediated isothermal amplification (LAMP) targeting the ribosomal RNA internal transcribed spacer 1 (ITS1). Methodology/Principal findings Samples were prepared by homogenizing and heating at 99 oC for 10 min before an aliquot was added to the LAMP reaction. After 40 min incubation at 65 oC, a colour change indicated a positive result. The tests were 100% sensitive and species-specific, and demonstrated a limit of detection comparable with PCR-based detection (TaqMan chemistry). The LAMP assays were able to detect target species for various life stages tested (adult, 1st instar larva, 4th instar larva and pupa), and body components, such as legs, wings and pupal exuviae. Importantly, the LAMP assays could detect Ae. aegypti DNA in mosquitoes stored in Biogents Sentinel traps deployed in the field for 14 d. A single 1st instar Ae. aegypti larva could also be detected in a pool of 1,000 non-target 1st instar Aedes notoscriptus, thus expediting processing of ovitrap collections obtained during presence/absence surveys. A simple syringe-sponge protocol facilitated the concentration and collection of larvae from the ovitrap water post-hatch. Conclusions/Significance We describe the development of LAMP assays for species identification and demonstrate their direct application for surveillance in different field contexts. The LAMP assays described herein are useful adjuncts to laboratory diagnostic testing or could be employed as standalone tests. Their speed, ease-of-use, low cost and need for minimal equipment and training make the LAMP assays ideal for adoption in low-resource settings without the need to access diagnostic laboratory services.
... LAMP has been widely used for the detection of pathogens, genetically modified crops, and tumors (Mori and Notomi, 2009;Tsukane et al., 2013;Zhou et al., 2016;Feng et al., 2019). Recently, this technique has been suggested for the detection and identification of various agricultural and medical pests, such as Spodoptera frugiperda (Kim et al., 2021), small hive beetle (Ponting et al., 2020), Aedes mosquito species (Schenkel et al., 2019), non-adult tephritid fruit fly (Kitano and Takakura, 2020), etc. Agarose gel electrophoresis analysis and visual detection are two methods usually used to present results for LAMP identification. For visual identification, hydroxy naphthol blue (HNB) is commonly used as a metal ion indicator, and the identification results could be judged more directly and conveniently just from the color change of the amplification mixture after heating for less than 1 h (Huang and Weng, 2015). ...
... According to the A-T base bias of the Liposcelis COI gene, the Tm value was appropriately lowered. The specificity of the primers was preliminarily evaluated using the BLAST function in Genbank (Altschul et al., 1990;Schenkel et al., 2019), revealing high consistency and similarity between the primers and L. entomophila. The regions of the primers in L. entomophila COI genes and primer sequences are given in Fig. 1 and Table 1, respectively. ...
... Qualified PCR products were selected using 2% agarose gel electrophoresis in 1 × TAE buffer and sequenced by Sangon Biotech (Shanghai, China). The obtained COI gene sequences of each booklouse were aligned using BLAST in GenBank (Atschul et al., 1990;Schenkel et al., 2019). A phylogenetic tree was also reconstructed for these newly collected species using the neighbor-joining (NJ) method and K2P (Kimura-2-parameter) model in Mega X to ensure their identification (Zhang et al., 2019). ...
Article
Liposcelis entomophila (Psocodea: Liposcelididae) is one of the most economically important booklice in the world. This species damages stored grain and represents an allergenic health risk to humans. Because of the small size of booklice, they are difficult to identify using morphological characteristics. Traditional molecular identification, such as DNA barcoding and qRT-PCR, requires specialized equipment and takes a long time, which is not appropriate for practical usage by plant health inspectors at ports. Therefore, for storage psocid identification, we suggested a simple, rapid and relatively new technique known as loop-mediated isothermal amplification (LAMP). In our study, a new rapid molecular identification method using the LAMP technique based on the mitochondrial gene cytochrome c oxidase I (COI) was developed for L. entomophila with high specificity and efficiency. Evident amplification results could be presented if the DNA concentration was 0.001 ng/μL. Visual detection was also possible with our LAMP primers using hydroxy naphthol blue (HNB), relying on the clear change in color from violet to sky blue after amplification. This method successfully determined unknown species that were newly collected from grain stores and may facilitate L. entomophila identification in plant quarantine and stored product protection.
... Accordingly, the aims of this research were to (a) adapt and extend the assay of Bhadra et al. [17] using a wAlbB wsp primer set as well as an Ae. aegypti ITS1 primer set [24] for efficient and specific detection of wAlbB-infected Ae. aegypti mosquitoes in the context of control efforts including those in sub-optimal conditions, (b) compare this method to established qPCR monitoring methods on samples taken from field locations, and (c) develop a quantitative form of the assay for use on pooled mosquito extractions to determine relative wAlbB frequencies. The results are expected to be applicable to a variety of projects involving Wolbachia within health and agricultural contexts. ...
... LAMP primers for the Ae. aegypti ITS1 gene were taken from Schenkel et al. [24]. Primers were manufactured according to our specifications by Integrated DNA Technologies Inc. (Coralville, IA, USA) under the standard desalting purification process. ...
... The Ae. aegypti ITS1 primers from Schenkel et al. [24] exhibited a stable annealing point of 92.7˚C (S.D. 0.11˚C). With fresh reagents (i.e. ...
Article
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With 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 within Aedes 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.
... LAMP is an efficient and cost-effective assay (Lee, 2017) most often focused on pointof-care detection of pathogens of domestic animals and humans in a medical setting (Wong et al., 2018;Mori and Notomi, 2020). Increasingly, this technology is now being harnessed to detect DNA of invasive species in environmental samples where results can be used to take immediate actions to avoid spreading or introducing the species (Sillo et al., 2018;Schenkel et al., 2019). ...
... A sensitive and specific field applicable tool needs to be simple, quick, and accurate (Lee, 2017;Williams et al., 2017;Schenkel et al., 2019). The assay described herein detects AFT within 20 min, making it a potential tool to test baitfish or free-living fish to prevent the dissemination of AFT via translocation activities. ...
Article
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The Asian fish tapeworm (Schyzocotyle acheilognathi syn. Bothriocephalus acheilognathi) (AFT) is an invasive parasite that can infect many species of fish, although most hosts are primarily members of Cyprinidae. Pathogenicity has most often been reported in aquaculture settings in fry and fingerling stages of carp (Cyprinus spp.). More recently, it has been shown to cause growth retardation in the endangered bonytail chub (Gila elegans) and found to be widespread in populations of endangered humpback chub (Gila cypha) in the Colorado River, Grand Canyon, Arizona. AFT spreads most often through the transport of infected fish, particularly baitfish. Despite its harmful potential, there is no efficient or accurate ante mortem test to detect AFT in water or fish samples before transport. Herein, we report on the development of a sensitive and specific loop-mediated isothermal amplification (LAMP) assay to detect the parasite in under 30 min from laboratory prepared samples. Six LAMP primers were designed to amplify a variable region of the 18S ribosomal RNA gene in AFT with the detection and quantification of DNA on a real-time fluorometer. The limit of detection was 1 × 101 copies/μl of DNA extracted from as few as 2 AFT eggs. Future application of our assay would be a low-cost test to rapidly and accurately detect AFT DNA from environmental samples on-site so that preventive actions can be taken to halt the spread of the AFT through the movement of infected fish.
... More recently, LAMP assays combined with a microfluidic system for the identification of four Asian Anopheles species (Anopheles dirus Peyton & Harrison, 1979, Anopheles lesteri Baisas & Hu, 1936, Anopheles minimus Theobald, 1901and Anopheles sinensis Wiedemann, 1828 reported published (Mao et al., 2018) but the primer sequences are not disclosed. LAMP assays for Aedes and Culex species have been developed by our group (Schenkel et al., 2019;T. Kamber & A. Mathis, unpublished work), combining simple sample preparation (heating to 80 ∘ C) of mosquito stages (eggs, larvae, pupae, adults) and by calorimetric detection of amplicons using the naked eye. ...
... one Aedes aegypti Linnaeus, 1762 adult mosquito among 49 Ae. japonicus Theobald, 1901(Schenkel et al., 2019] after heat-treatment of the mosquitoes in 1 mL of water and using 2 μL in the assay. LAMP assays typically are incubated for a default time of 1 h but often evaluated for shorter incubation times, and the reactions are heat-inactivated or stored cooled if not analysed immediately (Britton et al., 2015;Hardinge & Murray, 2019;Yu et al., 2019). ...
Article
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Species of the genus Anopheles vary with regard to their vector capacity for Plasmodium spp., the causative agent of malaria, and their accurate identification is often required. Loop-mediated isothermal amplification (LAMP) is a rapid, simple and low-cost method for specific DNA amplification. Primers for LAMP assays specific for the Anopheles funestus group and Anopheles gambiae complex species as well as for the species Anopheles arabiensis, An. funestus, An. gambiae s.s/Anopheles coluzzii (major vectors) and Anopheles rivulorum (minor vector) were designed targeting specific genome or rDNA internal transcribed spacer regions. Reaction conditions (buffer composition, primer concentrations, incubation time) were evaluated and the specificities of the assays confirmed with DNA from non-target Anopheles species. DNA release from the mosquitoes is achieved simply by heating them for 5 min in water. An aliquot of the DNA solutions is transferred to the reaction tube using disposable inoculation loops. The outcome of the LAMP amplifications after 1 h incubation at 65 °C can easily be visualized by a colour change visible to the naked eye. The assays are operable under field conditions requiring only basic equipment (portable heat block programmable at 65 and 80 °C, cooler for master mixes).
... The major vector of these arboviruses is Aedes aegypti. There is also a risk of other arboviruses being introduced in New Caledonia such as West Nile virus, Rift Valley fever virus, Japanese encephalitis virus or Ross River virus, as suitable vector species are present: Culex quinquefasciatus; Culex annulirostris; Culex sitiens; Aedes vigilax; Aedes vexans; and Aedes notoscriptus [6][7][8][9][10]. In addition, the risk for introduction of exotic mosquito species is also important because other vector species are documented in neighboring countries [11][12][13]. ...
... Aedes mosquitoes are the most abundant mosquito species, and pose a health hazard for humans worldwide as well-known potential vectors of disease pathogens [35,43]. During the last few decades, various mosquito species have spread expeditiously from their area of origin and colonised temperate zones [35]. ...
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The natural distribution range of Aedes koreicus is Korea, China, Japan, and the Russian Far East. Since 2008, this species has been recorded as an invasive species in some European countries (Belgium, European Russia, Germany, Hungary, Italy, Slovenia, and Switzerland). The invasive mosquito species Ae. koreicus is reported from the Republic of Kazakhstan for the first time. Its morphological identification was confirmed by molecular-genetic analyses of ND4 sequences using specific primers. Aedes koreicus larvae were found in an artificial water reservoir together with the larvae of Culiseta longiareolata and Culex pipiens s.l. Aedes koreicus successfully overwintered in Almaty at low winter temperatures in 2018–2019. This suggests that the Ae. koreicus acclimation capacity is greater than it has been considered until now. We assume that Ae. koreicus will spread over the west and south of the Republic of Kazakhstan and territories of Kyrgyzstan and Uzbekistan Republics bordering the Almaty region.
... For instance, the multiplex allele-specific PCR technique was used to diagnose similar Aedes mosquitoes from the Stegomyia subgenus [11] and mosquitoes from the Anopheles gambiae and Anopheles barbirostris complexes [12,13]. In another area of molecular biology, loop-mediated isothermal amplification (LAMP) assays were created with possible outcomes in field surveillance of invasive species [14]. Finally, proteomic approaches have recently flourished in entomological identification. ...
Article
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Background: In the context of the increasing circulation of arboviruses, a simple, fast and reliable identification method for mosquitoes is needed. Geometric morphometrics have proven useful for mosquito classification and have been used around the world on known vectors such as Aedes albopictus. Morphometrics applied on French indigenous mosquitoes would prove useful in the case of autochthonous outbreaks of arboviral diseases. Methods: We applied geometric morphometric analysis on six indigenous and invasive species of the Aedes genus in order to evaluate its efficiency for mosquito classification. Results: Six species of Aedes mosquitoes (Ae. albopictus, Ae. cantans, Ae. cinereus, Ae. sticticus, Ae. japonicus and Ae. rusticus) were successfully differentiated with Canonical Variate Analysis of the Procrustes dataset of superimposed coordinates of 18 wing landmarks. Conclusions: Geometric morphometrics are effective tools for the rapid, inexpensive and reliable classification of at least six species of the Aedes genus in France.
... In situ detection based on colour change proves vital in alien species management as it decreases the need for expensive equipment as well as reduces the time taken to positively identify a non-native species. The benefits of LAMP assays for species detection have been proven by studies carried out on invasive flies [21,22] and Aedes mosquito [23]. In terms of identification of fish, several studies have used LAMP to detect freshwater fish [24,25]. ...
Article
Invasive alien fish species have become a silent treat towards the ecosystem especially the native fish population in Malaysia. There has been a need to develop rapid identification methods that can aid management teams in identifying fish species that are not native to our ecosystem. Current visual identification methods are highly tedious and require time, delaying action towards curbing the invasion. The LAMP assay successfully identified six popular invasive fish species in Malaysia. None of the LAMP assays showed false positives and the Limit of Detection of the LAMP primers were highly sensitive and could detect DNA samples up to 1 × 10 − 15 ng/μl. The LAMP primers designed were highly specific to the target species and did not amplify non target species. DNA sequencing was done to ensure the accuracy of LAMP assay results. This study demonstrates that LAMP is a suitable tool in species identification efforts of invasive fish species in Malaysia.
... Alors que la morphologie a été la méthode unique d'identification jusqu'aux années 1970, l'émergence des méthodes moléculaires a apporté une nouvelle gamme d'outils (électrophorèse des protéines sur gel, précédée par l'étude cytogénétique des chromosomes) [7,8]. Dans les années 1990, la PCR (Polymerase Chain Reaction) et le séquençage de gènes ont été développés [9,10], et plus récemment l'amplification isotherme (Loop-Mediated Amplification -LAMP) [11,12]. Depuis quelques années, la spectrométrie de masse a également été appliquée à l'identification des moustiques, avec des résultats prometteurs [13,14]. ...
Article
Résumé Parmi les nombreuses espèces de moustiques décrites, seule une petite proportion est responsable de transmissions d’agents pathogènes à l’homme. Les espèces invasives comme le moustique tigre comptent parmi les vecteurs les plus efficaces (dengue, chikungunya, Zika). L’identification précise des espèces, indispensable, n’est pas aisée. La méthode d’identification morphologique reste le standard mais plusieurs méthodes moléculaires ont été développées, telles que la PCR, le séquençage et le barcoding, l’ADN environnemental, l’amplification isotherme LAMP et la spectrométrie de masse Maldi-TOF. Dans un premier temps développées pour identifier des espèces membres d’un complexe taxonomique, les techniques les plus efficaces sont maintenant étendues à de larges spectres d’espèces. Chaque méthode d’identification a ses forces et ses faiblesses. L’existence d’une variabilité intra spécifique morphologique et génétique ne simplifie pas le contexte. Dans tous les cas, il est souhaitable de ne pas se cantonner à une méthode unique, mais d’avoir recours à plus d’une méthode sur la base de la complémentarité des méthodes et techniques, et dans la perspective d’une taxonomie intégrée. Pour ce qui concerne les moustiques invasifs Aedes, à identifier en routine dans le cadre de surveillances entomologiques, nous préconisons l’identification morphologique pour les larves et les adultes, et l’identification par Maldi-TOF pour les œufs.
... LAMP primers for the Ae. aegypti ITS1 gene were taken from Schenkel et al. (24). Primers were manufactured according to our specifications by Reactions were incubated at 65°C for 20-30 minutes. ...
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With 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 w AlbB Wolbachia infection within Aedes 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 w AlbB 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 Releases 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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Insecticide resistance is a large and growing problem for the control of mosquito disease vectors. The World Health Organization (WHO) established the Global Plan for Insecticide Resistance Management (GPIRM) in 2012. In that context, both classical and molecular tools, as well as entomological databases and decision support platforms have been developed and used for IRM. Despite major advances in the molecular elucidation of resistance mechanisms and the development of diagnostic tools, their impact on disease control programs has been limited. In most cases diagnostic tools provide a retrospective examination of changes imposed by insecticides rather than a prospective analysis to guide vector control strategies. The uncertainty of the predictive value of markers, the assay robustness and the common misconceptions in resistance diagnosis terminology are continuing challenges in monitoring vector resistance. Furthermore, an often logistics, as opposed to systematic scientific evidence, based approach to decision for the use of the very few alternative chemicals in vector control, has reduced the value of resistance monitoring in practice. The current deployment of new insecticidal active ingredients should necessitate the application of companion diagnostics (CDx) and the development of modern ways for interpretation and management of the data by trained programme managers. This will establish their real value for use in decision-making, in line with evidence based choice of chemicals in agriculture and medical applications.
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Background The mosquito-borne filarial nematodes Dirofilaria immitis and Dirofilaria repens primarily affect dogs but also cats, causing heartworm disease or subcutaneous dirofilariosis, respectively, and both may also cause zoonotic diseases in humans. Several mosquito species have been reported as competent vectors for these nematodes, but no data are available for the invasive mosquito species Aedes japonicus (Theobald, 1901). The objective of this study was to describe the development of both D. immitis and D. repens under standardised experimental laboratory conditions in mosquitoes. Methods For this purpose, both a laboratory strain and field-collected individuals of the invasive mosquito species Ae. japonicus and, for comparative purposes, a laboratory strain of Aedes geniculatus, a rare indigenous species sharing habitats with Ae. japonicus, and of the tropical species Aedes aegypti were used. Anticoagulated microfilariaemic blood was fed at a density of 3000 mf/ml to mosquitoes with a hemotek system. Blood-fed mosquitoes were incubated at 27 °C and 85% relative humidity, and specimens were dissected under the microscope at pre-set time points to observe developmental stages of both Dirofilaria species. Additionally, real-time PCRs were carried out in some microscopically negative samples to determine the infection rates. ResultsIn field-collected Ae. japonicus infectious L3 larvae of both D. immitis and D. repens developed, rendering this mosquito species an efficient vector for both filarial species. Additionally, Ae. geniculatus was shown to be an equally efficient vector for both filarial species. Aedes japonicus mosquitoes from a laboratory colony were refractory to D. immitis but susceptible to D. repens, whereas Ae. aegypti was refractory to both filarial species. Conclusions To our knowledge, Aedes japonicus was for the first time shown to be an efficient vector for both D. immitis and D. repens, indicating that this invasive and locally highly abundant species may contribute to a transmission of filarial worms. The data emphasize the necessity to perform vector competence studies with local mosquito populations as basis for risk assessments. We further demonstrated that detection of filarial DNA in a mosquito species alone does not allow to draw reliable conclusions with regard to its vector competence.
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It would not be an exaggeration to say that among molecular technologies, it is PCR (polymerase chain reaction) that underpins the discipline of molecular ecology as we know it today. With PCR, it has been possible to target the amplification of particular fragments of DNA, which can then be analysed in a multitude of ways. The capability of PCR to amplify DNA from a mere handful of copies further means that conservationists and ecologists are able to sample DNA unobtrusively and with minimal disturbance to the environment and the organisms of interest. However, a key disadvantage of PCR-based methods has been the necessity for a generally non-portable, laboratory setting to undertake the time-consuming thermocycling protocols. LAMP (loop-mediated isothermal amplification) offers a logistically simpler protocol: a relatively rapid DNA amplification reaction occurs at one temperature, and the products are visualized with a colour change within the reaction tubes. In the first field application of LAMP for an ecological study, Centeno-Cuadros et al. (2016) demonstrates how LAMP can be used to determine the sex of three raptor species. By enabling DNA amplification in situ and in ‘real-time’, LAMP promises to revolutionize how molecular ecology is practised in the field.
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Accurate detection of filarial parasites in humans is essential for the implementation and evaluation of mass drug administration programs to control onchocerciasis and lymphatic filariasis. Determining the infection levels in vector populations is also important for assessing transmission, deciding when drug treatments may be terminated and for monitoring recrudescence. Immunological methods to detect infection in humans are available, however, cross-reactivity issues have been reported. Nucleic acid-based molecular assays offer high levels of specificity and sensitivity, and can be used to detect infection in both humans and vectors. In this study we developed loop-mediated isothermal amplification (LAMP) tests to detect three different filarial DNAs in human and insect samples using pH sensitive dyes for enhanced visual detection of amplification. Furthermore, reactions were performed in a portable, non-instrumented nucleic acid amplification (NINA) device that provides a stable heat source for LAMP. The efficacy of several strand displacing DNA polymerases were evaluated in combination with neutral red or phenol red dyes. Colorimetric NINA-LAMP assays targeting Brugia Hha I repeat, Onchocerca volvulus GST1a and Wuchereria bancrofti LDR each exhibit species-specificity and are also highly sensitive, detecting DNA equivalent to 1/10-1/5000th of one microfilaria. Reaction times varied depending on whether a single copy gene (70 minutes, O. volvulus) or repetitive DNA (40 min, B. malayi and W. bancrofti) was employed as a biomarker. The NINA heater can be used to detect multiple infections simultaneously. The accuracy, simplicity and versatility of the technology suggests that colorimetric NINA-LAMP assays are ideally suited for monitoring the success of filariasis control programs.
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Loop-mediated isothermal amplification (LAMP) is a simple, powerful state-of-the-art gene amplification technique used for the rapid diagnosis and early detection of microbial diseases. Many LAMP assays have been developed and validated for important epizootic diseases of livestock. We review the LAMP assays that have been developed for the detection of 18 viruses deemed notifiable of ruminants, swine and poultry by the World Organization for Animal Health (OIE). LAMP provides a fast (the assay often takes less than an hour), low cost, highly sensitive, highly specific and less laborious alternative to detect infectious disease agents. The LAMP procedure can be completed under isothermal conditions so thermocyclers are not needed. The ease of use of the LAMP assay allows adaptability to field conditions and works well in developing countries with resource-limited laboratories. However, this technology is still underutilized in the field of veterinary diagnostics despite its huge capabilities.
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Background The dramatic worldwide expansion of Aedes albopictus (the Asian tiger mosquito) and its vector competence for numerous arboviruses represent a growing threat to public health security. Molecular markers are crucially needed for tracking the rapid spread of this mosquito and to obtain a deeper knowledge of population structure. This is a fundamental requirement for the development of strict monitoring protocols and for the improvement of sustainable control measures. Methods Wild population samples from putative source areas and from newly colonised regions were analysed for variability at the ribosomal DNA internal transcribed spacer 2 (ITS2). Moreover, a new set of 23 microsatellite markers (SSR) was developed. Sixteen of these SSRs were tested in an ancestral (Thailand) and two adventive Italian populations. Results Seventy-six ITS2 sequences representing 52 unique haplotypes were identified, and AMOVA indicated that most of their variation occurred within individuals (74.36%), while only about 8% was detected among populations. Spatial analyses of molecular variance revealed that haplotype genetic similarity was not related to the geographic proximity of populations and the haplotype phylogeny clearly indicated that highly related sequences were distributed across populations from different geographical regions. The SSR markers displayed a high level of polymorphism both in the ancestral and in adventive populations, and FST estimates suggested the absence of great differentiation. The ancestral nature of the Thai population was corroborated by its higher level of variability. Conclusions The two types of genetic markers here implemented revealed the distribution of genetic diversity within and between populations and provide clues on the dispersion dynamics of this species. It appears that the diffusion of this mosquito does not conform to a progressive expansion from the native Asian source area, but to a relatively recent and chaotic propagule distribution mediated by human activities. Under this scenario, multiple introductions and admixture events probably play an important role in maintaining the genetic diversity and in avoiding bottleneck effects. The polymorphic SSR markers here implemented will provide an important tool for reconstructing the routes of invasion followed by this mosquito.
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The need for simple and effective assays for detecting nucleic acids by isothermal amplification reactions has led to a great variety of end point and real-time monitoring methods. Here we tested direct and indirect methods to visualize the amplification of potato spindle tuber viroid (PSTVd) by loop-mediated isothermal amplification (LAMP) and compared features important for one-pot in-field applications. We compared the performance of magnesium pyrophosphate, hydroxynaphthol blue (HNB), calcein, SYBR Green I, EvaGreen, and berberine. All assays could be used to distinguish between positive and negative samples in visible or UV light. Precipitation of magnesium-pyrophosphate resulted in a turbid reaction solution. The use of HNB resulted in a color change from violet to blue, whereas calcein induced a change from orange to yellow-green. We also investigated berberine as a nucleic acid-specific dye that emits a fluorescence signal under UV light after a positive LAMP reaction. It has a comparable sensitivity to SYBR Green I and EvaGreen. Based on our results, an optimal detection method can be chosen easily for isothermal real-time or end point screening applications.
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Background: Malaria is a major public health problem in sub-Saharan African countries including Ethiopia. Early and accurate diagnosis followed by prompt and effective treatment is among the various tools available for prevention, control and elimination of malaria. This study aimed to evaluate the performance of non-instrumented nucleic acid amplification loop-mediated isothermal amplification (NINA-LAMP) compared to standard thick and thin film microscopy and nested PCR as gold standard for the sensitive diagnosis of malaria in Northwest Ethiopia. Methods: A cross-sectional study was conducted in North Gondar, Ethiopia from March to July 2014. Eighty-two blood samples were collected from malaria suspected patients visiting Kola Diba Health Centre and analysed for Plasmodium parasites by microscopy, NINA-LAMP and nested PCR. The NINA-LAMP method was performed using the Loopamp Malaria Pan/Pf detection kits for detecting DNA of the genus Plasmodium and more specifically Plasmodium falciparum using an electricity-free heater. Diagnostic accuracy outcome measures (analytical sensitivity, specificity, predictive values, and Kappa scores) of NINA-LAMP and microscopy were compared to nested PCR. Results: A total of 82 samples were tested in the primary analysis. Using nested PCR as reference, the sensitivity and specificity of the primary NINA-LAMP assay were 96.8% (95% confidence interval (CI), 83.2% - 99.5%) and 84.3% (95% CI, 71.4% - 92.9%), respectively for detection of Plasmodium genus, and 100% (95% CI, 75.1% - 100%) and 81.2% (95% CI, 69.9% - 89.6%), respectively for detection of P. falciparum parasite. Microscopy demonstrated sensitivity and specificity of 93.6% (95% CI, 78.5% - 99.0%) and 98.0% (95% CI, 89.5% - 99.7%), respectively for the detection of Plasmodium parasites. Post-hoc repeat NINA-LAMP analysis showed improvement in diagnostic accuracy, which was comparable to nested PCR performance and superior to microscopy for detection at both the Plasmodium genus level and P. falciparum parasites. Conclusion: NINA-LAMP is highly sensitive for the diagnosis of malaria and detection of Plasmodium parasite infection at both the genus and species level when compared to nested PCR. NINA-LAMP is more sensitive than microscopy for the detection of P. falciparum and differentiation from non-falciparum species and may be a critical diagnostic modality in efforts to eradicate malaria from areas of low endemicity.
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AliView is an alignment viewer and editor designed to meet the requirements of next-generation sequencing era phylogenetic datasets. AliView handles alignments of unlimited size in the formats most commonly used, i.e. FASTA, Phylip, Nexus, Clustal and MSF. The intuitive graphical interface makes it easy to inspect, sort, delete, merge and realign sequences as part of the manual filtering process of large datasets. AliView also works as an easy-to-use alignment editor for small as well as large datasets. Availability and implementation: AliView is released as open-source software under the GNU General Public License, version 3.0 (GPLv3), and is available at GitHub (www.github.com/AliView). The program is cross-platform and extensively tested on Linux, Mac OS X and Windows systems. Downloads and help are available at http://ormbunkar.se/aliview Contact: anders.larsson@ebc.uu.se Supplementary information: Supplementary data are available at Bioinformatics online.
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Invasive aedine mosquito species have become a major issue in many parts of the world as most of them are recognised vectors or potentially involved in transmission of pathogens. Surveillance of these mosquitoes (e.g. Ae. aegypti, Yellow fever mosquito, Aedes albopictus, Asian tiger mosquito) is mainly done by collecting eggs using ovitraps and by identification of the larvae hatched in the laboratory. In order to replace this challenging and laborious procedure, we have evaluated matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) for easy and rapid species identification. Individual protein profiles were generated using five eggs each of nine aedine species (Ae. aegypti, Ae. albopictus, Ae. atropalpus, Ae. cretinus, Ae. geniculatus, Ae. japonicus, Ae. koreicus, Ae. phoeniciae, Ae. triseriatus) from various geographical origins, and species-specific biomarker mass sets could be generated. A blinded validation using our reference data base for automated egg identification was performed. In addition, pools of 10 aedine eggs (132 two-species and 18 three-species pools) in different ratios were evaluated. Specific biomarker mass sets comprising 18 marker masses could be generated for eggs of nine container-inhabiting aedine species, including all the major invasive and indigenous species of Europe and North America. Two additional masses shared by all investigated aedine species are used as internal calibrators. Identification of single eggs was highly accurate (100% specificity, 98.75% sensitivity), and this method is also of value for the identification of species in pools of ten eggs. When mixing two or three species, all were identified in all pools in at least 2 or 1 of the 4 loaded replicates, respectively, if the "lesser abundant" species in the pool accounted for three or more eggs. MALDI-TOF MS, which is widely applied for routine identification of microorganisms in clinical microbiology laboratories, is also suited for robust, low-cost and high throughput identification of mosquito vectors in surveillance programmes. This tool can further be developed to include a wide spectrum of arthropods but also other Metazoa for which surveillance is required, and might become the method of choice for their centralised identification via online platforms.
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Background The recent notifications of autochthonous cases of dengue and chikungunya in Europe prove that the region is vulnerable to these diseases in areas where known mosquito vectors (Aedes albopictus and Aedes aegypti) are present. Strengthening surveillance of these species as well as other invasive container-breeding aedine mosquito species such as Aedes atropalpus, Aedes japonicus, Aedes koreicus and Aedes triseriatus is therefore required. In order to support and harmonize surveillance activities in Europe, the European Centre for Disease Prevention and Control (ECDC) launched the production of ‘Guidelines for the surveillance of invasive mosquitoes in Europe’. This article describes these guidelines in the context of the key issues surrounding invasive mosquitoes surveillance in Europe. Methods Based on an open call for tender, ECDC granted a pan-European expert team to write the guidelines draft. It content is founded on published and grey literature, contractor’s expert knowledge, as well as appropriate field missions. Entomologists, public health experts and end users from 17 EU/EEA and neighbouring countries contributed to a reviewing and validation process. The final version of the guidelines was edited by ECDC (Additional file 1). Results The guidelines describe all procedures to be applied for the surveillance of invasive mosquito species. The first part addresses strategic issues and options to be taken by the stakeholders for the decision-making process, according to the aim and scope of surveillance, its organisation and management. As the strategy to be developed needs to be adapted to the local situation, three likely scenarios are proposed. The second part addresses all operational issues and suggests options for the activities to be implemented, i.e. key procedures for field surveillance of invasive mosquito species, methods of identification of these mosquitoes, key and optional procedures for field collection of population parameters, pathogen screening, and environmental parameters. In addition, methods for data management and analysis are recommended, as well as strategies for data dissemination and mapping. Finally, the third part provides information and support for cost estimates of the planned programmes and for the evaluation of the applied surveillance process. Conclusion The ‘Guidelines for the surveillance of invasive mosquitoes in Europe’ aim at supporting the implementation of tailored surveillance of invasive mosquito species of public health importance. They are intended to provide support to professionals involved in mosquito surveillance or control, decision/policy makers, stakeholders in public health and non-experts in mosquito surveillance. Surveillance also aims to support control of mosquito-borne diseases, including integrated vector control, and the guidelines are therefore part of a tool set for managing mosquito-borne disease risk in Europe.
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The rapid divergence of repetitive sequences makes them desirable markers for phylogenetic studies of closely related groups, provided that a high level of sequence homogeneity has been maintained within species. Intraspecific polymorphisms are found in an increasing number of studies now, and this highlights the need to determine why these occur. In this study we examined intraindividual variation present in the first ribosomal internal transcribed spacer (ITS1) from a group of cryptic mosquito species. Individuals of the Anopheles punctulatus group contained multiple ITS1 length variants that ranged from 1.2 to 8.0kb. Nucleotide and copy number variation for several homologous internal repeats is common, yet the intraspecific sequence divergence of cloned PCR isolates is comparable to that of other mosquito species (~0.2–1.5%). Most of the length variation is comprised of a 5′-ITS1 repeat that was identified as a duplication of a conserved ITS2 region. Secondary structure conservation for this repeat is pronounced and several repeat types that are highly homogenized have formed. Significant interspecific divergence indicates a high rate of evolutionary change for this spacer. A maximum likelihood tree constructed here was congruent with previous phylogenetic hypotheses and suggests that concerted evolution is also accompanied by interpopulation divergence. The lack of interindividual differences and the presence of homogenized internal repeats suggest that a high rate of turnover has reduced the overall level of variation. However, the intraindividual variation also appears to be maintained by the absence of a single turnover rate and the complex dynamics of ongoing recombination within the spacer.
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Bemisia tabaci, the sweetpotato whitefly, is a globally invasive pest that causes serious agricultural damage by transmitting plant viruses. This pest forms a cryptic species complex that displays morphologically indistinguishable biotypes. Among them, the B and Q biotypes are the most important pests worldwide. Because they have different levels of insecticide resistance, these biotypes must be identified in order to achieve proper pest control. Therefore, a convenient, rapid and specific detection method for identifying the two biotypes is necessary. Loop-mediated isothermal amplification (LAMP) was employed for rapid identification of B. tabaci B and Q biotypes. By combining a quick DNA extraction method, identification of the two biotypes was achieved within 1 h of detection time. The LAMP assay was applied to study the dynamics of B. tabaci biotypes both in the field and in greenhouses. It was found that, while temperature may be important for population dynamics of the whitefly in the field, population dynamics in greenhouse conditions may be influenced by the types of insecticide. The newly designed LAMP assay is a simple, rapid and accurate method for identifying the B and Q biotypes. It can be conducted by non-specialists and can contribute to pest management.
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The main malaria vectors of sub-Saharan Africa, Anopheles gambiae sensu stricto and Anopheles arabiensis are morphologically indistinguishable, but often occur in sympatry and differ in feeding preference and vector competence. It is important to assess vector species identity for understanding the vectorial system and establishing appropriate vector control measures. The currently available species diagnosis methods for An. gambiae sensu latu require equipment to which public health practitioners in many African countries may not have access. This report describes a loop-mediated isothermal amplification technique (LAMP) for An. gambiae species diagnosis. The LAMP method was tested in single mosquito legs and whole body. The sensitivity and specificity of the LAMP method, in reference to the conventional rDNA-polymerse chain reaction (PCR) method, ranged from 0.93 to 1.00. The LAMP-based species identification method can be performed in a water bath and completed within 65 minutes, representing an alternative method for rapid and field applicable vector species diagnosis.
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The Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), ranks as one of the world's most destructive agricultural pests. This pest is also widespread and highly invasive; thus, it is a high priority for pest detection and quarantine programs. Although Mediterranean fruit fly adult and third-instar larvae can usually be identified and distinguished from other species by morphological keys, it is often difficult or impossible to identify or distinguish this species from other tephritids by using material from other stages of development. In such situations, use of a molecular technique known as loop-mediated isothermal amplification (LAMP) would be valuable as a rapid and robust alternative species diagnostic tool. This method uses isothermal conditions and requires only relatively inexpensive equipment. In this study we have developed a simple and rapid procedure that combines a Chelex-based DNA extraction procedure with LAMP to rapidly detect the presence of Mediterranean fruit fly DNA and discriminate it from other species, by using material from different stages of development. Amounts of DNA as little as that recovered from a single egg were shown to be adequate for the analysis, and LAMP itself required only 45 min to complete.
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In 2005, a widespread infestation of Aedes albopictus was discovered in the Torres Strait, the region between northern Australia and New Guinea. To contain this species, an eradication program was implemented in 2006. However, the progress of this program is impeded by the difficulty of morphologically separating Ae. albopictus larvae from the endemic species Aedes scutellaris. In this study, three real-time TaqMan polymerase chain reaction assays that target the ribosomal internal transcribed spacer 1 region were developed to rapidly identify Aedes aegypti, Ae. albopictus, and Ae. scutellaris from northern Australia. Individual eggs, larvae, pupae, and adults, as well as the species composition of mixed pools were accurately identified. The assay method was validated using 703 field-collected specimens from the Torres Strait.
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The ability to select short DNA oligonucleotide sequences capable of binding solely to their intended target is of great importance in developing nucleic acid based detection technologies. Applications such as multiplex PCR rely on primers binding to unique regions in a genome. Competing side reactions with other primer pairs or template DNA decrease PCR efficiency: Freely available primer design software such as Primer3 screens for potential hairpin and primer-dimer interactions while selecting a single primer pair. The development of multiplex PCR assays (in the range of 5 to 20 loci) requires the screening of all primer pairs for potential cross-reactivity. However, a logistical problem results due to the number of total number of comparisons required. Comparing the primer set for a 10-plex assay (20 total primer sequences) results in 210 primer-primer combinations that must be screened. The ability to screen sets of candidate oligomers rapidly for potential cross-reactivity reduces overall assay devlelopment time. Here we report the application of a familiar sliding algorithm for comparing two strands of DNA in an overlapping fashion. The algorithm has been employed in a software package wherein the user can compare multiple sequences in a single computational run. After the screening is completed, a score is assigned to potential duplex interactions exceeding a user-defined threshold. Additional criteria of predicted melting temperature (Tm) and free energy of melting (deltaG) are included for further ranking. Sodium counterion and total stand concentrations can be adjusted for the Tm and deltaG calculations. The predicted interactions are saved in a text file for further evaluation.
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Dengue outbreaks occur regularly in parts of northern Queensland, Australia, and there is concern that these outbreaks may spread with the introduction and range expansion of the two main vectors Aedes aegypti (L.) and Aedes albopictus (Skuse). Problems encountered in separating larvae of endemic and exotic container mosquito species resulted in the development of a polymerase chain reaction diagnostic procedure that uses a restriction enzyme to cut the internal transcribed spacer region 1 of the ribosomal DNA to separate Ae. aegypti and Ae. albopictus from a number of common local container mosquito species which can be used at any stage of the life cycle, including eggs up to 8 wk of age. Identification was possible using desiccated or alcohol-preserved specimens with a response time of < 24 h after receipt of the specimens.
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Foodborne bacterial infections and diseases have been considered to be a major threat for public health in the worldwide. Increased incidence of human diseases caused by foodborne pathogens have been correlated with growing world population and mobility. Loop-mediated isothermal amplification (LAMP) has been regarded as an innovative gene amplification technology and emerged as an alternative to PCR-based methodologies in both clinical laboratory and food safety testing. Nowadays, LAMP has been applied to detection and identification on pathogens from microbial diseases, as it showed significant advantage in high sensitivity, specificity and rapidity. The high sensitivity of LAMP enables detection of the pathogens in sample materials even without time consuming sample preparation. An overview of LAMP mainly containing the development history, reaction principle and its application to four kind of foodborne pathogens detection are presented in this paper. As concluded, with the advantages of rapidity, simplicity, sensitivity, specificity and robustness, LAMP is capable of applications for clinical diagnosis as well as surveillance of infection diseases. Moreover, the main purpose of this paper is to provide theoretical basis for the clinical application of LAMP technology.
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The significant strides made in reducing global malaria burden over the past decades are being threaten by the emergence of multi-drug resistant malaria. Mechanisms of resistance to several classes of antimalarial drugs have been linked to key mutations in the Plasmodium falciparum genes. Pyrimethamine targets the dihydrofolate reductase of the bifunctional dihydrofolate reductase thymidylate synthase (DHFR-TS), and specific point mutations in the dhfr-ts gene have been assigned to resistant phenotypes. Several molecular methods are available to detect the mutant genotypes including DNA sequencing and PCR-based methods. In this study, we report the development of PfSNP-LAMP to detect nucleotide polymorphism in the dhfr gene associated with N51I mutation and antifolate resistance. The PfSNP-LAMP method was validated with genomic DNA samples and parasite lysates prepared from sensitive and pyrimethamine resistant strains of P. falciparum.
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Rapid and accurate identification of potentially invasive taxa that may cause high economic losses or environmental damage is of critical importance. The onion thrips, Thrips tabaci Lindeman, ranks as one of the world’s most destructive agricultural pests and commonly found in imported agricultural products and field samples, but is prone to undetected transport because of its minute size as well as cryptic behavior. Although traditional taxonomic methods are pretty useful in straightforward assignment of specimens to the genus Thrips, identification in the species level is much more difficult and requires expertise, knowledge, and experience. Furthermore, it is often difficult or impossible to identify or distinguish this species from other thrips by using material from other stages of development. Based on the foregoings, use of a molecular technique known as loop-mediated isothermal amplification (LAMP) as a rapid and robust alternative species diagnostic tool would be valuable. In this study, a relatively quick and simple method was used to detect the presence of onion thrips DNA rapidly and discriminate it from other species, by using material from different stages of development. Not only LAMP itself required less than 1 h to complete but also amounts of DNA as little as that recovered from a single specimen were adequate for the detection. Another advantage of this identification system is that nonspecialists will be able to make faster and cheaper identifications.
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Loop-mediated isothermal amplification (LAMP), a newly developed gene amplification method, combines rapidity, simplicity, and high specificity. Several tests have been developed based on this method, and simplicity is maintained throughout all steps, from extraction of nucleic acids to detection of amplification. In the LAMP reaction, samples are amplified at a fixed temperature through a repetition of two types of elongation reactions occurring at the loop regions: self-elongation of templates from the stem loop structure formed at the 3′-terminal and the binding and elongation of new primers to the loop region. The LAMP reaction has a wide range of possible applications, including point-of-care testing, genetic testing in resource-poor settings (such as in developing countries), and rapid testing of food products and environmental samples.
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There are currently five invasive Aedes mosquito species known to be established in Europe, namely Aedes albopictus, Aedes aegypti, Aedes japonicus, Aedes atropalpus and Aedes koreicus. Aedes albopictus and Aedes aegypti are the incriminated vectors in the recent outbreaks of chikungunya and dengue fever in Europe. However, both laboratory experiments and field observations indicate that these invasive mosquitoes have a potential to also transmit other pathogens of public health importance. Increasing travel and pathogen introduction, expansion of vector distribution, and both environmental and climatic changes are likely to raise the risk of pathogen transmission by these invasive Aedes mosquitoes. Their vector status and their involvement in pathogen transmission are dynamic processes that shape the future of mosquito-borne disease epidemiology in Europe. Beside vector surveillance, enhanced disease surveillance will enable the early detection of cases and the prompt implementation of control measures.
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Loop-mediated isothermal amplification (LAMP) is a DNA-based analytical method which can be used as an isothermal alternative to polymerase chain reaction (PCR). In comparison to PCR, the advantage of LAMP is the possibility to perform the isothermal reaction without any sophisticated technical equipment - only a water bath is needed and naked eye detection is sufficient. Up to now, an application of LAMP methods for the detection of even closely related plant species in food or feed matrices is not described, whereas a large number of PCR methods for that topic are cited in the literature. The aim of the study was the evaluation of LAMP based methods for plant species identification with respect to method parameters like R2, LOD and LOQ. An existing (real-time) PCR method (for the detection of spices) was used for comparison. It could be shown that the developed LAMP methods have the potential as an alternative strategy to PCR in DNA based analysis.
Article
Several molecular methods have been developed for detection of Erwinia amylovora, the causal agent of fire blight in pear and apple, but none are truly applicable for on-site use in the field. We developed a fast, reliable and field applicable detection method using a novel target on the E. amylovora chromosome that we identified by applying a comparative genomics pipeline. The target coding sequences (CDS) are both uniquely specific for and all-inclusive of E. amylovora genotypes. This avoids potential false negatives that can occur with most commonly used methods based on amplification of plasmid gene targets, which can vary among strains. Loop-mediated isothermal AMPlification (LAMP) with Optigene GenieII chemistry and instrumentation proved to be an exceptionally rapid (under 15 minutes) and robust method for detecting E. amylovora in orchards, as well as simple to use in the plant diagnostic laboratory. Comparative validation results using plant samples from inoculated greenhouse trials and from natural field infections (of regional and temporal diverse origin) showed that our LAMP had equivalent or greater performance regarding sensitivity, specificity, speed and simplicity than real-time PCR (TaqMan), other LAMP assays, immunoassays and plating, demonstrating its utility for routine testing.
Article
Loop-mediated isothermal amplification (LAMP) is a method for amplification and detection of target organisms which, unlike polymerase chain reaction, does not require thermal cycling. LAMP assays can be developed in the laboratory for subsequent deployment in the field, where the simplicity of isothermal amplification makes LAMP a suitable method for rapid detection of phytoplasmas with levels of sensitivity and specificity approaching those of more complex and time-consuming laboratory methods.
Article
Biting midges of the genus Culicoides (Diptera, Ceratopogonidae) are vectors of several viruses of veterinary relevance, and they can cause insect bite hypersensitivity. As the morphological identification of these tiny insects is difficult, we aimed at developing real-time PCRs. Partial mitochondrial cytochrome oxidase I gene (mtCOI) sequences were determined from 380 Culicoides midges from three regions of Switzerland (Alps, Midland, Ticino) and from non-biting midges. Sequence variability (haplotypes) was observed in all 21 morphologically identified species. For each of C. grisescens and C. obsoletus, a cryptic species was identified. Further, a sister taxon to C. pulicaris was determined (Culicoides sp.). Alignments of available mtCOI sequences from Ceratopogonidae (GenBank, this study) were used to design real-time PCRs to distinguish C. chiopterus, C. deltus, C. dewulfi, C. grisescens, C. imicola, C. lupicaris, C. obsoletus (both species), C. pulicaris, C. scoticus and Culicoides sp. Specificities of the assays were tested with cloned targets from 1 to 4 haplotypes of 18 Culicoides spp. and 4 other Ceratopogonidae revealing no cross-reactivity when testing 5x106 gene copies. The sensitivities of two assays were tested by spiking single insects into pools of 99 or 999 randomly selected non-target Ceratopogonidae. Primers and probes of this study were devised to be suitable for multiplexed assays but these evaluations await to be performed.
Article
Vector-borne diseases, such as malaria and lymphatic filariasis, are co-endemic in large parts of the world. To develop a multiplex amplification method for the simultaneous detection of multiple insect-borne infectious diseases, we used LAMP with fluorescently labeled primers to identify the SPECT2 gene of Plasmodium berghei and the cytochrome oxidase subunit I gene of Dirofilaria immitis in mosquitoes. This technique could detect as few as 100 P. berghei-infected red blood cell-equivalents or one D. immitis microfilaria. Moreover, individual species of parasites in mosquitoes could be identified when a mixture of fluorescently labeled primer sets was used. These findings suggest that the multiplex LAMP assay is sensitive and specific enough to identify parasite-bearing mosquitoes in areas where several diseases occur simultaneously. This procedure could increase the efficiency and effectiveness of arthropod-borne disease elimination programs.
Article
An algorithm is presented for the multiple alignment of sequences, either proteins or nucleic acids, that is both accurate and easy to use on microcomputers. The approach is based on the conventional dynamic-programming method of pairwise alignment. Initially, a hierarchical clustering of the sequences is performed using the matrix of the pairwise alignment scores. The closest sequences are aligned creating groups of aligned sequences. Then close groups are aligned until all sequences are aligned in one group. The pairwise alignments included in the multiple alignment form a new matrix that is used to produce a hierarchical clustering. If it is different from the first one, iteration of the process can be performed. The method is illustrated by an example : a global alignment of 39 sequences of cytochrome c.
Article
Culex pipiens complex mosquitoes (Cx. p. pipiens and Cx. p. quinquefasciatus) are among the principal vectors of St. Louis encephalitis (SLE) virus in the eastern United States; Cx. restuans and Cx. salinarius play secondary roles in the transmission and maintenance of the virus cycle. Accurate identification of these three species in field collections is required for epidemiologic studies of SLE virus transmission. We have developed a polymerase chain reaction (PCR) assay for this purpose. Species-specific PCR primers were designed based on interspecies nucleic acid sequence variation in the first and second internal transcribed spacers (ITS1 and ITS2) of the nuclear ribosomal DNA gene array; however, insufficient variation was detected to differentiate between subspecies of the Cx. pipiens complex. The primers were used together in a single amplification reaction to correctly identify specimens to species using genomic DNA extracted from whole individual mosquitoes, DNA from triturated mosquito pools, or crude DNA from mosquito heads or legs.
Article
Until the eradication of malaria from Europe, members of the Anopheles maculipennis complex had been the major vectors for plasmodial parasites. With the possible reintroduction of Plasmodium species due to climate change and increased travel to and from countries where malaria is endemic, accurate identification of mosquito species will be essential for preventive studies. For this purpose, a diagnostic PCR system to differentiate between six of the seven A. maculipennis sibling species occurring in Europe was developed. The second internal transcribed spacer (ITS2) of the ribosomal DNA was amplified and sequenced for all six species. Based on differences in the nucleotide sequences, species-specific primers were constructed for PCR amplification of mosquito DNA that in combination with a universal primer generate amplification products of different length, each unique for one species.
Arthropods as Vectors of Emerging Diseases
  • N. Becker
  • B. Pluskota
  • A. Kaiser
  • F. Schaffner