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

Abstract

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 o C for 10 min before an aliquot was added to the LAMP reaction. After 40 min incubation at 65 o C, 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, 1 st instar larva, 4 th 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 1 st instar Ae. aegypti larva could also be detected in a pool of 1,000 non-target 1 st 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.

Development and Validation of a Loop-Mediated Isothermal Amplification Diagnostic Method to Detect the Quarantine Potato Pale Cyst Nematode, Globodera pallida

“…In recent years, we have seen an increasing need for early detection methods, mainly for emerging and invasive organisms and plant pathogens, either regulated or non-regulated, in all areas of diagnostics [57]. Among many new methods and technologies, LAMP is one of the most explored techniques to detect invasive and quarantine species both at the laboratory level and on site (farms, water resources, border inspection points) [19,58,59].…”

Loop‐mediated isothermal amplification (LAMP) assays for Aedes and Culex species and evaluation of a simple DNA preparation method for field application

“…In conclusion, the LAMP assays developed in the present work together with the earlier described ones (Bonizzoni et al, 2009;Huang et al, 2020;Kamber et al, 2020;Schenkel et al, 2019)…”

“…japonicus Theobald, 1901; and Ae. koreicus Edwards, 1917) (Huang et al., 2020; Schenkel et al., 2019) and for several Anopheles funestus group and An. gambiae complex species (Bonizzoni et al., 2009; Kamber et al., 2020).…”

Assessing the Risk of Exotic Mosquito Incursion through an International Seaport, Newcastle, NSW, Australia

“…There may be potential in incorporating alternative surveillance methods including citizen science-based programs to expand and/or enhance formal surveillance programs. Community led surveillance incorporating affordable mosquito trapping and smartphone technology has been shown to work effectively [ 74 ] and the incorporation of molecular techniques to specimen processing may be benefit in rapid detection of exotic mosquitoes [ 75 , 76 ] It is important that nuisance biting reported to local authorities is investigated. The suburbs within the study area are likely to be impacted by mosquitoes dispersing from nearby estuarine wetlands, as major pest species such as Ae.…”