Development of an on‐site diagnostic LAMP assay for rapid differentiation of the invasive pest Phthorimaea absoluta (Meyrick) using insect tissues

Abstract

BACKGROUNDThe tomato leafminer, Phthorimaea absoluta (Meyrick) (Lepidoptera: Gelechiidae), is a destructive invasive pest that originated in South America and has spread within China since 2017. A rapid method for on‐site identification of P. absoluta is urgently needed for interception of this pest across China.RESULTSWe developed a loop‐mediated isothermal amplification (LAMP) technique to differentiate P. absoluta from L. sativae, C. horticola, and P. operculella using extracted genomic DNA, which was then refined to create an on‐site LAMP diagnostic method that can be performed under field conditions without the need for laboratory equipment.CONCLUSIONIn the present research, we developed an on‐site diagnostic method for rapid differentiation of P. absoluta from other insects with similar morphology or damage characteristics in China.This article is protected by copyright. All rights reserved.

Development of LAMP and droplet digital PCR methods for differentiation between Cydia pomonella (L.) and Grapholita molesta (Busck)

“…The mtCOI sequences of C. pomonella and G. molesta, and other borer insects were downloaded from the NCBI database: C. pomonella (MK759945.1), G. molesta (GU096466.1), Dichocrocis punctiferalis (KX860179.1), and Carposina sasakii (HM377790.1). The method to design the LAMP primers for C. pomonella was followed as described by Yang et al 21 Brie y, the sequences were aligned using CLUSTAL W, and speci c points were marked with ESPript3.0 software. Speci c primers for C. pomonella COI were designed using PrimerExplorer V5.…”

“…Reaction system and conditions of LAMP and ddPCR. The reaction system and conditions of LAMP were conducted as described by Yang et al 21 Brie y, the tube was placed in a constant-temperature water bath at 63℃ for 45 min for the reaction and at 80℃ for 10 min to inactivate the enzyme. After ampli cation, the reaction tube was brie y centrifuged, or the SYBR Green I dye was brie y shaken into the bottom of the tube and mixed with the reaction solution.…”

“…Speci city and sensitivity of the LAMP assay. The speci city and sensitivity of the LAMP assay were conducted as described by Yang et al 21 Brie y, the speci city of the LAMP primers was tested using DNA extracted from adult C. pomonella collected in several geographic regions and G. molesta. Distilled water without nuclease was used as the non-template control (CK).…”

“…However, these molecular methods need a complicated DNA extraction process and a laboratory setting, which is not suitable for on-site diagnostics 20 . Additionally, these molecular methods may have low e ciency in detecting the environmental DNA (eDNA) from the frass when its number size is small in the early stage of spreading 21 . Thus, it is necessary to develop a rapid on-site diagnostic method that is more e cient for the detection of the eDNA of C. pomonella.…”

“…Recent research has shown that loop-mediated isothermal ampli cation (LAMP) has the potential for eld use, offering an advantage over conventional PCR, which requires laboratory equipment 20 . Insect tissue can also be used in the LAMP method, which does not require DNA extraction 21,22 . Until now, the LAMP assays have successfully been used to diagnose other plant pests [23][24][25][26][27][28] .…”