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

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.

A LAMP-based colorimetric assay to expedite field surveillance of the invasive mosquito species Aedes aegypti and Aedes albopictus

“…A heating block or water bath can be battery-powered for locations where electrical mains power is not available. Test results are obtained by visual inspection, without the need for gel-based or cell phone visualization [16,22]. Critically, LAMP assays are cheap, being priced at < $US3.00 per test, which is considerably lower than the � $US20 per test required to extract and perform a real-time TaqMan PCR.…”

“…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].…”

“…albopictus were designed for this work and the primers used to detect Ae. aegypti were published previously [16] (Table 1).…”

“…The Ae. aegypti assay was adapted from [16], whilst we developed the Ae. albopictus assay as part of the current study.…”

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

“…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.…”