Development of a simple and accurate molecular tool for Spodoptera frugiperda species identification using LAMP

Abstract

BACKGROUND: The fall armyworm, Spodoptera frugiperda is a native species of the Americas. First detected in western and central Africa in early 2016, it has become one of the most serious invasive lepidopteran pests in many African and Asian countries. S. frugiperda has spread very quickly; however, there are no molecular-based, simple and accurate diagnostic tools for identification of this species in the field. Methods to identify invasive S. frugiperda are urgently needed because farmers and agricultural managers have no prior experience with this pest. RESULTS: Based on mitochondrial genome sequence alignment, a S. frugiperda-specific sequence region was identified in the transfer RNA-coding region between NADH dehydrogenase, ND3, and ND5. Using this unique region, species-diagnostic primers were designed and applied in a loop-mediated isothermal amplification (LAMP) assay and a conventional polymerase chain reaction to identify field-collected samples of S. frugiperda. The optimal incubation conditions for the LAMP assay were 61°C for 90 min with four LAMP primers; an additional loop primer increased the amplification efficiency. A response was obtained for a wide range of DNA concentrations in the LAMP assay and the minimum detectable DNA concentration was 10 pg.CONCLUSIONS: We developed a new LAMP-based molecular diagnostic method that it is easy to use and accurate. The LAMP assay was used with a DNA-releasing technique for larval and adult samples, without a DNA extraction step, by incubating the tissue sample at 95°C for 5 min. This method can be applied in intensive field monitoring of S. frugiperda and its ecological studies.

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

“…Therefore, a method was developed without a separate DNA extraction process that could be combined with the LAMP method. A part of the antenna or leg tissue from BAW, FAW, and TCW adults or larvae was cut and reacted at 95 • C for 5 min to release DNA, similar to the method reported previously for FAW [10]. By measuring the amount of released DNA using a nanodrop system, the DNA concentration was found to be at least 1 ng or more, even though the concentration differed depending on the amount of tissue in the initial reaction.…”

“…Field population samples were used to confirm the selected primer sets and LAMP application. The two African populations of S. frugiperda originating from Nigeria and Benin, as well as three Korean field populations, were used [10]. The S. litura population was collected from a potato field (Gangneung, Korea; 37 • 46 23" N, 128 • 56 08" E) and used for mitochondrial genome sequencing.…”

“…The general protocol of LAMP was carried out according to the manufacturer's guidelines in a 25 µL reaction. The LAMP conditions were used according to a previous study on S. frugiperda [10]. LAMP amplifications were carried out in an Applied Biosystems ProFlex PCR system (ThermoFisher Scientific, Waltham, MA, USA).…”

“…In contrast, loop-mediated isothermal amplification (LAMP) has been verified as a method that can be used in the field. Recently, a species diagnosis method was developed for S. frugiperda using LAMP [10]. The developed species diagnosis method had high utility in the field, and there was additional demand for the development of a species diagnosis method for related species that occur in the cornfields simultaneously (personal communication) [11].…”

A diagnostic LAMP assay for rapid identification of an invasive plant pest, fall armyworm Spodoptera frugiperda (Lepidoptera: Noctuidae)

“…Recently a LAMP assay was published for identification of FAW, targeting a tRNA region of the mitochondrial genome 37 . This assay involves colorimetric detection to identify positive samples through extended incubation of LAMP reactions for 1 ½ hours.…”