A Loop-mediated Isothermal Amplification (LAMP) Assay for Rapid Identification of <em>Bemisia tabaci</em>

Abstract

The whitefly Bemisia tabaci (Gennadius) is an invasive pest of considerable importance, affecting the production of vegetable and ornamental crops in many countries around the world. Severe yield losses are caused by direct feeding, and even more importantly, also by the transmission of more than 100 harmful plant pathogenic viruses. As for other invasive pests, increased international trade facilitates the dispersal of B. tabaci to areas beyond its native range. Inspections of plant import products at points of entry such as seaports and airports are, therefore, seen as an important prevention measure. However, this last line of defense against pest invasions is only effective if rapid identification methods for suspicious insect specimens are readily available. Because the morphological differentiation between the regulated B. tabaci and close relatives without quarantine status is difficult for non-taxonomists, a rapid molecular identification assay based on the loop-mediated isothermal amplification (LAMP) technology has been developed. This publication reports the detailed protocol of the novel assay describing rapid DNA extraction, set-up of the LAMP reaction, as well as interpretation of its read-out, which allows identifying B. tabaci specimens within one hour. Compared to existing protocols for the detection of specific B. tabaci biotypes, the developed method targets the whole B. tabaci species complex in one assay. Moreover the assay is designed to be applied on-site by plant health inspectors with minimal laboratory training directly at points of entry. Thorough validation performed under laboratory and on-site conditions demonstrates that the reported LAMP assay is a rapid and reliable identification tool, improving the management of B. tabaci.

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].…”

“…Because of its speed, robustness and simplicity, the use of LAMP is gaining popularity for diagnostics in plant health. LAMP-based assays have been developed for the detection of plant pathogenic viruses and phytoplasmas [16][17][18], insects [19], fungi [20][21][22][23][24][25][26] and bacteria [23,27].…”

Significance and interpretation of molecular diagnostics for insecticide resistance management of agricultural pests

“…The majority of molecular diagnostics used for monitoring insecticide resistance in agricultural pests (described in Table 2 [16,[59][60][61][62][63][64]) are based on nucleic acid detection (DNA and/or RNA). Simple/low-tech versions of PCR-(AS-PCR, PCR-RFLP) and isothermal LAMP are used to detect the presence of known mutations or differentially expressed genes (targeted analysis).…”

Loop-Mediated Isothermal Amplification assays for on-site detection of the main sweetpotato infecting viruses

“…RNA/DNA concentrations were standardized to 100 ng/μL before use in amplification assays. For LAMP, we also used the alkaline polyethylene glycol (APEG) quick extraction method ( Chomczynski and Rymaszewski, 2006 ; Blaser et al, 2018 ) with slight modifications. Briefly, the APEG buffer was prepared by combining 60 g PEG 200 (Sigma-Aldrich) with 0.93 mL of 2 M KOH and 39 mL water and pH adjusted to 13.5.…”