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

The fall armyworm, Spodoptera frugiperda is a native species in the Americas. However, nowadays it is one of the most serious invasive lepidopteran pests in African and Asian countries. S. frugiperda has been spread very quickly after the first outbreak was reported in many countries. Based on mt genome sequence alignment, S. frugiperda specific sequence region was identified in tRNAs coding region between NADH dehydrogenase, ND3 and ND5. By using this unique region, species diagnostic primers were designed and applied in LAMP (lamp loop mediated isothermal amplification) assay as well as conventional PCR to identify the field-collected samples of S. frugiperda. Optimal incubation condition of LAMP assay was 61oC for 90 minutes with 4 LAMP primers, and additional loop primer increased the amplification efficiency. Also, wide range of DNA concentration responded in LAMP assay and minimum detectable DNA concentration was 10 pg. This LAMP assay was also applied in DNA releasing technique from larval and adult sample, without DNA extraction, 95oC incubation for five minutes of the tissue sample. This new molecular diagnostic method is easy to use and accurate. It possibly applied in intensive field monitoring of S. frugiperda and its ecological studies.