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

Abstract

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. 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. 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.