Loop-mediated isothermal amplification (LAMP) assays for Anopheles funestus group and Anopheles gambiae complex species.

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).

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

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.