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 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 Species Diagnostic Molecular Tool for an Invasive Pest, Mythimna loreyi, Using LAMP

“…As previously reported, the sensitivity of LAMP may vary depending on the temperature and reaction time [ 33 ]. Therefore, the reaction was performed at 65, 63, and 61 °C to find the optimal reaction conditions ( Figure 3 ).…”

“…Therefore, it could be sufficiently used for species diagnosis. The basic species diagnosis primer production strategy is the same as the previously reported species diagnosis development method of S. frugiperda [ 33 ]. We targeted the open reading frame (ORF) region because the ORF region is more intraspecies conserved than the non-coding region.…”

“…As with other invasive pests found in the field, they are often similar in morphology to allied species, making the investigation of initial density and pest management difficult. Species diagnosis methods for invasive pests, such as S. frugiperda , have been developed and utilized for this reason [ 33 ]. Currently, the molecular biology method for diagnosing M. loreyi species is only using the mtCO1 universal primer (LCO1490, HCO2198) and process PCR and sequencing to compare the degree of homology.…”

“…Therefore, we developed a molecular diagnosis method that could diagnose species within a short time without a separate DNA extraction process, and only a heat block is needed that can control temperature ( Figure 5 ). The basis diagnosis strategy is very similar to the S. frugiperda species diagnosis method [ 33 ]. This method can complete all experimental procedures and verify the results within 1 h and 30 min right after obtaining a sample.…”

Shining a LAMP on the applications of isothermal amplification to monitoring illegal wildlife trade

“…A large focus area is the detection of fruit fly species (Blaser et al 2018a;Blaser et al 2018b;Huang et al 2009;Sabahi et al 2018) (Blaser et al 2018a). Several studies have focused on the detection of fall armyworm (Agarwal et al 2022;Congdon et al 2021;Kim et al 2021). The most recent is based on the CO1 gene, with high specificity and sensitivity down to 2.4 pg of DNA (Agarwal et al 2022).…”