Development of a Species Diagnostic Molecular Tool for an Invasive Pest, Mythimna loreyi, Using LAMP

Abstract

The Mythimna loreyi (Duponchel) is one of the well-known invasive noctuid pests in Africa, Australia, and many Asian countries. However, it is difficult to identify the invasive and morphologically similar species, Mythimna separate, which occur at the cornfield in the larvae stage. Currently, the molecular biology method for diagnosing M. loreyi species is only using the mtCO1 universal primer (LCO1490, HCO2198), which requires a lot of time and effort, such as DNA extraction, PCR, electrophoresis, and sequencing. In this study, the LAMP assay was developed for rapid, simple, effective species identification. By analyzing the mitochondrial genome, the species-specific sequence was found at the coding region of the NADH dehydrogenase subunit 5 gene. Based on this unique sequence, four LAMP primers and two loop primers were designed. The F3 and B3 primers were able to diagnose species-specific, in general, and multiplex PCR and specifically reacted within the inner primers in LAMP assay. The optimal incubation condition of the LAMP assay was 61 °C for 60 min with four LAMP primers, though additional loop primers, BF and LF, did not significantly shorten the amplification time. The broad range of DNA concentration was workable in LAMP assay, in which the minimum detectable DNA concentration was 100 pg. DNA releasing method was applied, which took five minutes of incubation at 95 °C without the DNA extraction process. Only some pieces of tissue of larvae and adult samples were needed to extract DNA. The incidence of invasive pests is gradually diversifying. Therefore, this simple and accurate LAMP assay is possibly applied in the intensive field monitoring for invasive pests and integrated management of Mythimna loreyi.

Development of a LAMP-Based Molecular Species Diagnosis Method for Four Major Agricultural Pests in the Genus Spodoptera (Lepidoptera: Noctuidae)

“…Traps were set in Pyeongchang (37 • 40 53" N, 128 • 43'49" E), Hongchen (37 • 43 35" N, 128 • 24 33" E), and Gangneung (37 • 36 56" N, 128 • 45'59" E) near the corn fields in 2020 and 2021. Most of these species caused frequent or occasional damage in Korean corn fields [12]. S. littoralis does not exist in Korea.…”

Detection and Control of Pantoea agglomerans Causing Plum Bacterial Shot-Hole Disease by Loop-Mediated Isothermal Amplification Technique

“…The gyrB gene sequence was selected for designing LAMP primers by using the Primers Explorer V5 software program (available online at http://primerexplorer.jp/e/ ). The LAMP primer set (Nam et al, 2020 ) ( gyrB -2) has six primers ( Supplementary Figure S3 ), including outer primers F3 and B3, inner primers FIP and BIP (FIP containing F1 and F2, BIP containing B1 and B2), loop primers LF and LB ( Supplementary Table S1 ).…”

A LAMP Assay for the Detection of Thecodiplosis japonensis, an Alien Gall Midge Species Pest of Pine Trees

“…This method has been widely used in the detection of many kinds of pests, such as Myzus persicae , Spodoptera frugiperda , etc. [ 23 , 24 , 25 , 26 , 27 , 28 ].…”