From laboratory to point of entry: development and implementation of a loop-mediated isothermal amplification (LAMP)-based genetic identification system to prevent introduction of quarantine insect species.

Simon Blaser,Jennifer Tomlinson,Michael Andreou,Neil Boonham,Hanspeter Diem,Andreas Bühlmann,Jürg Utzinger,Andreas Von Felten,Jürg E Frey,Morgan Gueuning,Pie Müller

doi:10.1002/ps.4866

Abstract

BACKGROUNDRapid genetic on‐site identification methods at points of entry, such as seaports and airports, have the potential to become important tools to prevent the introduction and spread of economically harmful pest species that are unintentionally transported by the global trade of plant commodities. This paper reports the development and evaluation of a loop‐mediated isothermal amplification (LAMP)‐based identification system to prevent introduction of the three most frequently encountered regulated quarantine insect species groups at Swiss borders, Bemisia tabaci, Thrips palmi and several regulated fruit flies of the genera Bactrocera and Zeugodacus.RESULTSThe LAMP primers were designed to target a fragment of the mitochondrial cytochrome c oxidase subunit I gene and were generated based on publicly available DNA sequences. Laboratory evaluations analysing 282 insect specimens suspected to be quarantine organisms revealed an overall test efficiency of 99%. Additional on‐site evaluation at a point of entry using 37 specimens performed by plant health inspectors with minimal laboratory training resulted in an overall test efficiency of 95%. During both evaluation rounds, there were no false‐positives and the observed false‐negatives were attributable to human‐induced manipulation errors. To overcome the possibility of accidental introduction of pests as a result of rare false‐negative results, samples yielding negative results in the LAMP method were also subjected to DNA barcoding.CONCLUSIONOur LAMP assays reliably differentiated between the tested regulated and non‐regulated insect species within <1 h. Hence, LAMP assays represent suitable tools for rapid on‐site identification of harmful pests, which might facilitate an accelerated import control process for plant commodities. © 2018 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Highlights

The unintended spread of invasive insect species by global trade leads to considerable economic losses in agriculture.[1,2,3] Numerous insect species have been introduced into Europe, including harmful plant pests such as the western corn rootworm (Diabrotica virgifera) and the Colorado potato beetle (Leptinotarsa decemlineata).[1]
3.1 Primer design and laboratory evaluation of the loop-mediated isothermal amplification (LAMP) assays The primer sets of the LAMP assays were designed based on the mitochondrial c oxidase subunit 1 (COI) gene, where in silico analyses revealed taxa-specific regions for the target organisms
In the first evaluation of the LAMP assays, a total of 282 insect specimens suspected to be quarantine organisms were analysed by LAMP under laboratory conditions (Table 1A)

Summary

Introduction

The unintended spread of invasive insect species by global trade leads to considerable economic losses in agriculture.[1,2,3] Numerous insect species have been introduced into Europe, including harmful plant pests such as the western corn rootworm (Diabrotica virgifera) and the Colorado potato beetle (Leptinotarsa decemlineata).[1]. On-site LAMP-based identification of quarantine pests www.soci.org and lorries), which assist the dispersal along trade networks, including anthropogenic corridors such as canals and railways.[2,8,9] Besides trade, international tourism, as well as changes in climate and land use govern the movement of invasive species.[10]. Rapid genetic on-site identification methods at points of entry, such as seaports and airports, have the potential to become important tools to prevent the introduction and spread of economically harmful pest species that are unintentionally transported by the global trade of plant commodities. This paper reports the development and evaluation of a loop-mediated isothermal amplification (LAMP)-based identification system to prevent introduction of the three most frequently encountered regulated quarantine insect species groups at Swiss borders, Bemisia tabaci, Thrips palmi and several regulated fruit flies of the genera Bactrocera and Zeugodacus

Methods

Results

Discussion

Conclusion