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.