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

Abstract

Molecular-based species identification tools are helpful to identify tiny insect and lepidopteran pests that show morphological similarities in the larval stage and are essential for quarantine as well as agricultural research. Here, we focused on four major Spodoptera pests: S. exigua, S. frugiperda, S. litura, and S. littoralis. S. exigua and S. litura mitochondrial genome sequences were newly identified and species-specific sequence regions were identified in the cytochrome c oxidase subunit II and III regions. Species primers were designed and applied in loop-mediated isothermal amplification (LAMP) and PCR to identify Korean field-collected or overseas samples. The optimal incubation conditions for LAMP were 61 °C for 60 min with four LAMP primers. Additional loop primers increased the amplification efficiency for S. exigua, and the nonspecific amplification for other species. The LAMP assay could detect a wide range of DNA concentrations, with the range 1 ng–1 pg in dependence of four LAMP primers. The DNA-releasing technique, without DNA extraction, in the LAMP assay involved larval or adult tissue sample incubation at 95 °C for 5 min. The entire process takes approximately 70 min. This new molecular diagnostic method is simple and accurate, with application in the field and laboratory and for monitoring and ecological studies.

Development of Molecular-Based Species Identification and Optimization of Reaction Conditions for Molecular Diagnosis of Three Major Asian Planthoppers (Hemiptera: Delphacidae)

“…To overcome the limitations of morphological identification methods, several studies have explored the potential of DNA-based molecular markers to distinguish between insect species with indistinct morphological characteristics, such as cryptic species or immature life stages [ 46 , 47 , 48 ]. In particular, mitochondrial genes have been used as a source of target-specific primers in recent studies due to their high copy number and inherent resistance to degradation [ 30 , 43 ]. In this study, we developed field-level diagnostic LAMP, multiplex PCR, and conventional PCR markers based on the allelic polymorphism of aligned mitochondrial DNA sequences.…”

“…In point-of-care settings, a molecular diagnosis is an important tool for developing efficient insect pest management strategies [ 30 ]. Therefore, appropriate primer design and validation using sufficient samples are required.…”

“…The LAMP assay was carried out according to the manufacturer’s instructions using a WarmStart ® LAMP Kit (New England Biolabs, Ipswich, MA, USA). The LAMP conditions were selected based on our previous studies [ 30 , 40 ]. The LAMP assay was run for 60 min at 61 °C, 63 °C, and 65 °C with four primers (F3, B3, FIP, and BIP) to optimize the reaction temperature using an Applied Biosystems ProFlex PCR system (Thermo Fisher Scientific).…”

“…In contrast, a multiplex PCR assay is useful for detecting more than two species [ 27 ]. Furthermore, as demonstrated in our previous study [ 30 ], the LAMP assay is useful for field diagnosis without genomic DNA (gDNA) isolation. LAMP is a highly specific technique, and its high sensitivity, speed, cost-effectiveness, reproducibility, and detection ability are the most important characteristics for such diagnostic assays.…”

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 ].…”