Development of a simple and accurate molecular tool forSpodoptera frugiperdaspecies identification using LAMP

Abstract

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 61℃ 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, 95℃ 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.

Development of a diamide resistance diagnostic method using LAMP based on a resistance-specific indel in ryanodine receptors forSpodoptera exigua(Lepidoptera: Noctuidae)

“…Sufficient DNA was released by incubation of larval and adult tissues, rendering this method, which requires only a heat block for temperature control, suitable in the field. LAMP, as a time-and labor-efficient protocol, has been utilized in various fields, including ecology, medicine, diagnostic of plant viruses inside insect bodies, and identification of pest species such as Spodoptera frugiperda (Lee et al 2017;Kim et al 2020), as well as in insecticide resistance allele diagnosis (Badolo et al 2012(Badolo et al , 2015Choi et al 2018…”

“…The general protocol of LAMP was conducted in a 25 μL reaction mixture using a WarmStart® LAMP Kit (New England Biolabs, Ipswich, MA, USA) following the manufacture's guideline (Kim et al 2020). To test the DNA releasing technique for insect tissue, approximately 10 mg of larval tissue or an adult leg or antenna was incubated at 95 ℃ for 5 min with 30 μL nuclease-free water, and 2 μL of the resulting supernatant was then used in the LAMP assay with each primer set.…”

“…6). Using the DNA releasing technique reported by Kim et al (2020), sufficient DNA was obtained in the supernatant from larvae and adult tissue after their incubation at 95 ° C for 5 min (Fig. 7).…”