3.2 Conditions for diagnostic PCR and LAMP assay
Standard PCR with diagnostic primers showed that S. frugiperdaand S. exigua could be readily distinguished (Fig. 4). The LAMP assay with four primers was tested with various incubation temperatures ranging from 59 to 65℃. Best results were obtained with 50ng of total DNA, incubated at 61℃ for 90 minutes in a 25 μl reaction volume (Fig. 5). A positive reaction can be seen under visible light without any treatment (Fig. 5A) or confirmed under ultraviolet light with Cyber Green (Fig. 5B), or gel electrophoresis (Fig. 5C). Under 100 bp sized bands are not the LAMP product. That’s aggregated primers. So that generated even in negative control.
The two loop primers were tested for possible enhancement of the LAMP reaction, as suggested by Nagamine et al. (2002) (Nagamine et al., 2002). Addition of LB enhanced the reaction, producing the same amount of product in ten fewer minutes (Fig. 6C). Addition of LF inhibited the LAMP reaction with LB (Fig. 6D) or without LB (Fig. 6B).
The detection limits of the four LAMP primers and LB were tested on purified DNA. The color change under visible light could be detected with as little as 1 ng of DNA (containing genomic DNA and mitochondrial DNA resulting from a standard DNA isolation protocol, Fig. 7A). With UV light in reaction tubes or gel electrophoresis, as little as 10 pg of DNA could be detected (Fig. 7B and C).
To test the DNA releasing technique on insect tissue, approximately 10 mg of larval tissue, or an adult leg or antenna, was incubated at 95℃ for 5 minutes with 30 μl nuclease free water (Fig. 8B). Then 2 μl of the supernatant was used in the LAMP assay with 5 primers. Results could clearly be seen with visible light (Fig. 8A), UV light with Cyber Green (Fig. 8C) or gel electrophoresis (Fig. 8D).
DISSCUSSION
Methods to identify invasive S. frugiperda are urgently needed, as farmers and agricultural managers have no prior experience with this pest. The LAMP assay with the modifications described here can fulfil that need. It is not necessary when other species, such as corn borerOstrinia species, can be distinguished morphologically in the field.
As previously reported, the amount of DNA is generally not the limiting factor for the LAMP assay (Choi, Hur, Heckel, Kim, & Koh, 2018). Varying the incubation time can generally compensate for different amounts of DNA. The incubation temperature was more important for specificity. At temperatures less than 59℃, M. separata produced false positives (data not shown). There are five mismatches between the primer FAW_F3 and the priming sequence of M. separata as well as that of S. exigua . However, there was no false positive reaction in the optimal incubation condition, 61℃ during 90 minutes. On the other hand, incubation temperatures higher than 62℃ required longer incubation times to produce the same amount of product (data not shown).
In another attempt to decrease the incubation time, we applied two other loop primers, loop forward (LF) between the F2 and F1 regions and loop backward (LB) between the B1 and B2 regions, which have been suggested to amplify additionally from the loops in the dumbbell forms (Choi et al., 2018). Finding suitable loop primers was difficult because of the high AT content, and the strategy of adding overhanging ‘G’s to the 5’ end to increase the melting temperature was hard to use in the inner primers.
Alternatively, conventional PCR also can be used for S. frugiperda species detection as well as S. exigua (Fig. 3, Fig. 5). The simplicity, accuracy and adaptability for high throughput of the LAMP assay are distinct advantages (Notomi, Mori, Tomita, & Kanda, 2015; Zhang, Lowe, & Gooding, 2014) (Mori & Notomi, 2009). Moreover, recently LAMP applied in many ecology studies in and outside of the lab (Lee, 2017).
The origin of S. frugiperda populations in Korea has not been identified as of 2019. Based on meteorological predictions, they might migrate from some part of China and East Asia to Korea at least twice (end of May to end of June and July) (Li et al., 2020; Wu et al., 2019). Mitochondrial genome sequencing of three Korean populations implies that there are at least two genetically different populations of possibly different origin which invaded Korea. The next plan of this study is to identify the S. frugiperda species using this new LAMP assay and to analyze the identified SNPs, indels and the insecticide resistant related mutations of various field collected S. frugiperdapopulations, which may be useful in tracking gene flow and for insecticide resistance management.