Rapid assay using recombinase polymerase amplification and lateral flow dipstick for identifying Agrilus mali, a serious wood-boring beetle of the western Tianshan Mountains in China

doi:10.21203/rs.3.rs-2744727/v1

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Rapid assay using recombinase polymerase amplification and lateral flow dipstick for identifying Agrilus mali, a serious wood-boring beetle of the western Tianshan Mountains in China

https://doi.org/10.21203/rs.3.rs-2744727/v1

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Agrilus mali is a major wood-boring pest of Northeast Asia. Recently, it destroyed Malus sieversii and caused the decline of the wild fruit forests of the Tianshan Mountains in the Xinjiang Uygur Autonomous Region, China. The pest beetle is often not identified due to the absence of affective rapid discrimination methods beyond adult morphological examination. This study reports a simple, rapid, specific and sensitive method based on recombinase polymerase amplification (RPA) combined with lateral flow dipstick (LFD), which is able to quickly define the beetle even when it is young. RPA primers were designed based on the internal transcribed spacer 1 region (ITS1). The introduction of the probe in the reaction eliminated the false-positive signals. The probe (Probe1-b) was specificity and sensitivity with as little as 10^-3 ng of purified DNA needed to detect A. mali. Moreover, the RPA reaction was carried out using crude extracted DNA, which was only extracted by immersion in sterile distilled water. The LFD is used to visualize the detection results. The results show that the whole process of the RPA-LFD assay can be completed at 38 °C in approximately 30 min, including 10 min for A. mali gDNA extraction and 15 min for RPA-LFD detection, and the amplification can be carried out in an individual’s hand; thus, this method has very good application prospects in the field of quarantine and pest management.

insect rapid identification

recombinase polymerase amplification

lateral flow dipstick

molecular method

Agrilus mali (Coleoptera: Buprestidae) is a serious wood-boring beetle and is thought to have originated in Northeast Asia, including Eastern Siberia and the Far East of Russia, Mongolia and North Korea (Sun et al. 2022). In the past few decades, A. mali has been found in wild fruit forests in the Tianshan Mountains in western China and caused the widespread death of the wild apple Malus sieversii, which is regarded as a key germplasm resource and primitive ancestor of cultivated apple (Cui et al. 2015; Kong et al. 2018; Zhang et al. 2020). Some studies believe that the introduction of apple seedlings from Shandong Province to Xinjiang led to the invasion of A. mali (Ji et al. 2004; Bozorov et al. 2019; Zhang et al. 2021); however, a new study has also shown that people ignore the early stages of infestation, which may not have been understood until recently (Sun et al. 2022). This result indicated that there are still some deficiencies in the early monitoring of A. mali. In particular, outbreaks of A. mali in the Yili Valley directly threaten the native stands of M. sieversii in the Tianshan Mountains region, as well as the forests of neighboring Kazakhstan and other countries in Central Asia (Volkovitsh et al. 2020). Accordingly, a specific and sensitive on-site rapid detection technology for A. mali in the early stage of infestation has been developed to improve pest management strategies and quarantine measures to prevent its spread.

In general, the identification based on morphological characteristics of the adult stage is laborious and requires specialized taxonomic knowledge (Ashfaq et al. 2010; Tian et al. 2013). A. mali is small in size, and the adult stage has sex specific morphological characteristics and is similar to other con-generic Agrilus species (Volkovitsh et al. 2020). It is difficult to distinguish with the naked eye in the wild. Molecular identification of insects based on polymerase chain reaction (PCR), by contrast, is not hindered by sex or stage of development of the target species, including random amplified polymorphic DNA (RAPD), restriction fragment length polymorphism (RFPL), species-specific COI (SS-COI), multiplex PCR, and so on (Zhang et al. 2012; Zhang et al. 2013; Chen et al. 2015; Shi et al. 2022). In addition, DNA barcoding technology has been applied in the identification of A. mali, and it can be well distinguished from its closely related species (Bozorov et al. 2018). However, those methods are only suitable for the laboratory and cannot meet the requirements for on-site rapid detection (Ahmed et al. 2018; Dai et al. 2019; Wu et al. 2020).

The development of isothermal amplification technology (IAT) makes on-site assays possible, including the most commonly used loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA), which are promising methods that do not require complicated and expensive equipment (Li and Macdonald 2015; Li et al. 2019). LAMP requires high temperature (approximately 65℃) and relatively expensive portable analysis equipment for field assays (Ahmed et al. 2018). However, RPA has greater advantages in that reactions are performed at temperatures closer to the body, the lyophilized reagents are commercialized and the results are available within 20–30 min; thus, RPA has developed rapidly and been used largely in the diagnosis of microorganisms and parasites (Boyle et al. 2013; Crannell et al. 2014; Daher et al. 2016; Kapoor et al. 2017; Cha et al. 2019; Dai et al. 2019; Li et al. 2019; Yang et al. 2019; Cha et al. 2020; Wu et al. 2020; Mabrok et al. 2021; Lu et al. 2022; Tang et al. 2022). The RPA reaction system includes recombinase, DNA polymerase, and single-stranded binding protein (SSB) (Piepenburg et al. 2006), which opens the DNA double helical structure by recombinase and performs amplification reactions under constant temperature, in contrast to PCR (Piepenburg et al. 2006; Daher et al. 2016; Cha et al. 2019; Li et al. 2019). The end-point readout of RPA combined with the lateral flow dipstick (LFD) allows for the direct interpretation of results by the naked eye within a few minutes (Wu et al. 2020; Zhou et al. 2022). In brief, it is a rapid detection technology based on an antigen-antibody immune reaction with gold nanoparticles (AuNPs) (Fig. 1).

However, it has been less applied to insect identification; one study used RPA combined with fluorescence for Thrips palmi identification, and another used RPA combined with LFD for detection of Aedes aegypti resistance to drugs (Priti et al. 2021; Ahmed et al. 2022). The present study provides an example of the application of RPA combined with lateral flow dipstick (RPA-LFD) rapid on-site insect identification in the field, particularly for the early monitoring of invasive or regulated pests. We have developed an on-site rapid assay based on RPA-LFD for distinguishing A. mali, which does not require complex and expensive equipment. The specific RPA primer was designed with a part of internal transcribed spacer 1 (ITS1). To visualize the results, two methods for LFD observation were tested separately. Finally, the probe was added to the RPA reaction system and optimized, and false-positive signals from nonspecific primer–probe complexes were eliminated. This method is sensitive and species specific, and the practicality was also analyzed by detecting A. mali with crude DNA. This will provide an effective diagnostic tool for use in the field.

2.1 Specimen collection and DNA extraction

Specimens of A. mali were collected in China (Table S1). The damaged branches of host trees were cut in wild fruit forests or orchards, and branches were cultured in vitro at room temperature until adult beetles emerged. Species identification was performed using morphological and molecular methods. Both larvae and adults were kept in centrifugal tubes containing anhydrous ethanol and stored at -20°C. Genomic DNA (gDNA) was extracted from thorax and leg muscle tissues using the TIANamp Genomic DNA Kit (TIANGEN, China) according to the manufacturer’s protocol. The DNA concentration and purity were quantified using a NanoDrop2000 spectrophotometer (Thermo Fisher Scientific, USA). The extracted DNA was stored at -20°C for future use.

2.2 PCR amplification, cloning and sequencing

We designed specific primers, ITS-3F/ITS-3R (Table 1), for the amplification of complete ITS using the conserved region of the ribosome small subunit (18S) and ribosome large subunit (28S) of A. planipennis in Primer Premier 5.0 (Lalitha 2000). PCR amplification was performed in a total volume of 25 µl with 1.5 µl gDNA, 1 µl each of forward and reverse primers (10 µmol/L), 12.5 µl 2× Flash PCR MasterMix (CWBIO, China), and 9 µl ddH₂O. PCR amplification was performed with an initial denaturation at 98°C for 1 min, 34 cycles of 10 s at 94°C, annealing at 51°C for 15 s, extension at 72°C for 30 s, and a final elongation at 72°C for 1 min.

Table 1

Primer and probe sequences used to amplify fragments of rDNA-ITS and in the RPA assay
Fragment or assay	Name	Sequence (5’-3’)	Primer length (nt)	Amplicon (bp)
rDNA-ITS	ITS-3	F: 5’-ATGTTGCTGGGAAGATGACC-3’ R: 5’-ACTCTCAAGCAACCCGACTC-3’	20 20	/
Basic-RPA	AMITS-1	F: 5’-ATCTCGTCATACAACAAACGCGTCGCCCCG-3’ R: 5’-GCTGGGCAACGACGATGTATTGAGAGAGAG-3’	30 30	335
	AMITS-2	F: 5’-CGAACAACGCGCATTCACCACGGCGAACCT-3’ R: 5’-GCTGGGCAACGACGATGTATTGAGAGAGAG-3’	30 30	194
	AMITS-3	F: 5’-CGAACAACGCGCATTCACCACGGCGAACCT-3’ R: 5’-GCTGGGCAACGACGATGTATTGAGAGAGAGAG-3’	30 32	194
	AMITS-4	F: 5’-CGAACAACGCGCATTCACCACGGCGAACCT-3’ R: 5’-GGGCAACGACGATGTATTGAGAGAGAGAGAGAGAG-3’	30 35	191
	AMITS-5	F: 5’- CGAACAACGCGCATTCACCACGGCGAACCT-3’ R: 5’-GGGCAACGACGATGTATTGAGAGAGAGAGAGAG-3’	30 33	191
	AMITS-6	F: 5’- ATCTCGTCATACAACAAACGCGTCGCCCCG-3’ R: 5’- AGGCTGGGCAACGACGATGTATTGAGAGAG-3’	30 30	337
RPA-LFD	AMITS-2	mF: 5’-[FAM]-CGAACAACGCGCATTCACCACGGCGAACCT-3’ mR: 5’-[Biotin]-GCTGGGCAACGACGATGTATTGAGAGAGAG-3’	30 30	194
	Probe1	5’-[FAM]-CATCCCACAACTCATCGAGCATCGCATCACA[THF]GACGC ACATACATA-[C3 Spacer]-3’	45	138
	Probe2	5’-[FAM]-TCCCACAACTCATCGAGCATCGCATCACACG[THF]CGCAC ATACATACA-[C3 Spacer]-3’	45	136

The PCR products were purified using a TIANgel Midi Purification Kit (TIANGEN, China). Then, purified PCR products were cloned into a pLB vector with an eight-hour ligation step at 16°C and transformed into Escherichia coli TOP10. After 12 hours of incubation in LB medium, pass-through sequencing was performed by Tsingke Biotechnology Co., Ltd. (Beijing, China). To confirm the accuracy of all acquired sequences, Geneious R 9.0.2 software was used (Kearse et al. 2012).

2.3 RPA-LFD procedure with forward and reverse primers

The conserved region of A. mali ITS1 was aligned using the default parameters of MAFFT online (Katoh et al. 2019). The RPA primers were designed following TwistAmp® assay design manual guidelines (https://www.twistdx.co.uk) using Primer Premier 5.0 (Lalitha 2000) and synthesized by Tsingke Biotechnology Co., Ltd. (Beijing, China).

The basic RPA reactions were performed following the manufacturer’s instructions of the RPA-basic kit (AMP-Future Biotech Co. Ltd., Weifang, China). A 50 µl reaction volume consisted of a lyophilized enzyme pellet, 29.4 µl rehydration buffer, 2 µl forward primer, 2 µl reverse primer (10 µM), 2 µl DNA template, and 12.1 µl ddH2O; the reaction was initiated by adding 2.5 µl of 280 mM magnesium acetate. The reaction mix was incubated at 38°C in a heating block for 30 min. gDNA was replaced with ddH2O in the no template control (NTC). The amplified product was purified using a TIANquick Midi Purification kit (TIANGEN, China) and electrophoresed on a 2% agarose gel stained with loading buffer.

The forward and reverse primers were modified with FAM and biotin at the 5'-end, respectively. The reaction was the same as mentioned above, and the primers were replaced with AMITS-2mF/AMITS-2mR (Table 1). After the RPA reaction was completed, the product was diluted 20 times and 50 µl was added to the sample pad of the LFD, with visual interpretation after 3–5 min.

2.4 Designing and optimization of the probe

The probes were designed using Primer Premier 5.0 (Lalitha 2000). The size of the probe was set as 46–53 nt, the GC content was 20–80%, and the T_m was set as 50–80℃. Maximum hairpin and primer dimer parameters were set at ≤ 4 bonds near the 3'-end. Other settings were set as default. Two original probes were designed (Table 1). All reverse primers and probes were assessed using the OligoAnalyzer Tool (IDT) and screened depending on the binding score for continuous base pairings.

When the probe was added to the reaction system, the false-positive signals were not eliminated. RPA assays can tolerate several base mispairings in primers without noticeably influencing accuracy (Daher et al. 2016; Liu et al. 2019). Therefore, base substitutions were introduced into the probe and reverse primer according to previous research. We used IDT online software to evaluate all possibilities of primer-probe complexes before synthesizing new mismatched probes to increase the probability of success of the experiment (Przybylska et al. 2015; Zhou et al. 2022). Judging the probable complexes of probes and reverse primers based on the score obtained by IDT, more than 4 sites were easy to identify for LFD. We designed six groups of probes and reverse primers (Table 2). The false-positive signals were evaluated using NTC. The probes were synthesized by Tsingke Biotechnology Co., Ltd. (Beijing, China).

Table 2

Base substitutions in Probe1 and AMITS2-mR. Mismatched sites are marked in bold and underlined.
Code	Name	Sequence (5’-3’)
Group1	Probe1-a	5’-[FAM]-CATCCCACAACTCATCGAGCATCGCATCACA[THF]GACGCACATTCATA-[ C3 Spacer]-3’
	AMITS2-mRa	5’-[Biotin]-GCTGGGCAACGACGCTGTATTGAGAGAGAG-3’
Group2	Probe1-b	5’-[FAM]-CATCCCACAACTCATCGAGCATCGCATCACA[THF]GACGCACTTTCATA-[ C3 Spacer]-3’
	AMITS2-mRa	5’-[Biotin]-GCTGGGCAACGACGCTGTATTGAGAGAGAG-3’
Group3	Probe1-c	5’-[FAM]-CATCCCACAACTCATCGAGCATCGCATCACA[THF]GACGCAGAGATATA-[ C3 Spacer]-3’
	AMITS2-mR	5’-[Biotin]-GCTGGGCAACGACGATGTATTGAGAGAGAG-3’
Group4	Probe1-d	5’-[FAM]-CATCCCACAACTCATCGAGCATCGCATCACA[THF]GACGCACATTCCTA-[ C3 Spacer]-3’
	AMITS2-mRb	5’-[Biotin]-GCTGGGCAACGACGATATATTGAGAGAGAG-3’
Group5	Probe1-e	5’-[FAM]-CATCCCACAACTCATCGAGCATCGCATCACA[THF]GACGCACATTCTTA-[ C3 Spacer]-3’
	AMITS2-mRc	5’-[Biotin]-GCTGGGCAACGACGATCTATTGAGAGAGAG-3’
Group6	Probe1-f	5’-[FAM]-CATCCCACAACTCATCGAGCATCGCATCACA[THF]GACGCACATCCTTA-[ C3 Spacer]-3’
	AMITS2-mRb	5’-[Biotin]-GCTGGGCAACGACGATATATTGAGAGAGAG-3’

2.5 RPA-LFD procedure with primers and a probe

The RPA-LFD assays were carried out in a 50 µl reaction volume using the RPA nfo kit (AMP-Future Biotech Co. Ltd., Weifang, China) according to the manufacturer’s instructions. The reaction mixture included a lyophilized enzyme pellet, 29.4 µl rehydration buffer, 2 µl forward primer, 2 µl reverse primer (10 µM), 0.6 µl nfo probe (10 µM), 2 µl DNA template, 11.5 µl ddH₂O and 2.5 µl of 280 mM magnesium acetate. After 10 min of incubation at 38°C, 10 µl of the amplification product was mixed with 190 µl of ddH₂O, and 50 µl of the mixture was added to the sample pad of the LFD.

2.6 Specificity and reaction limit of the RPA-LFD assay

The specificity of the RPA-LFD assay was evaluated between A. mali and three closely related species. The RPA-LFD assay was tested for reaction limits, including different reaction temperatures (20, 25, 30, 35, 40, 45, and 50℃), different reaction times (5, 10, 15, 20, 25, 30, and 35 min) and sensitivity (the DNA levels are 10, 1, 10^− 1, 10^− 2, 10^− 3, 10^− 4, and 10^− 5 ng), so that the assay can be used in any environment in the field. The gDNA concentration of the A. mali sample was diluted to 10 in advance. The NTC reaction was performed at 38℃ for 10 min.

2.7 Rapid DNA extraction and field simulation detection

Insect tissue of A. mali was crushed in sterile distilled water (SDW). The tube was incubated in boiling water for 10 min. Two microliters of supernatant were collected for the RPA-LFD assay. To make the visualization results clearer, the amplification product was diluted 10 times and 50 µL were taken for LFD detection; visual interpretation of results took place after 3–5 min. To make better use of the detection on site, we also tested reaction temperatures closer to body temperature.

3.1 Primer design and screening

We amplified and cloned the complete ITS sequence of A. mali using the primers ITS-3F/ITS-3R. The conserved region of ITS1 was selected as the target sequence. Six primers were screened for amplification performance with basic RPA reactions. However, only AMITS-2F/AMITS-2R produced specific bands and no primer-dimer bands on agarose gel (Fig. 2a). AMITS-2mF/AMITS-2mR was used for basic RPA and visualization by LFD. The LFD result indicated that the color and density of the test line from NTC was comparable to that from gDNA (Fig. 2b). Even though the basic RPA did not show any primer–dimer bands on the agarose gel, false-positive signals were still present. Therefore, it was necessary to add a probe in the next reaction to eliminate the primer-dimers produced.

3.2 Adding a probe to the RPA reaction

Using a probe in the reaction could increase the specificity of amplification (Piepenburg et al. 2006; Wu et al. 2020). According to the feedback from Primer Premier 5.0 software and the corresponding parameters, two nfo probes were obtained (Table 1). Both probes were 46 nt in length, with a FAM modification at the 5'-end, a C3 spacer at the 3'-end, and a THF spacer placed 15 nt from the 3'-end that replaces a cytosine or guanine. The primers were AMITS-2F/AMITS-2mR, as mentioned above. In the RPA reaction, after the probe is completely matched to one of the product strands, the THF site was cleaved by the nfo enzyme, thereby releasing the 3'-end for extension. It is worth noting that the nfo enzyme only works if the bases flanking the THF site exactly match the other strand (Zhou et al. 2022).

The performance of the two probes was detected through RPA-LFD with gDNA of A. mali and NTC (Fig. 2c). However, the amplification signals of the two probes were obvious, and the corresponding NTC also had false-positive signals. The results showed that false-positive signals were still present even with the use of the probe, so the probes needed to be optimized. Probe 1 was selected for optimization because the color and density of the test line were both light.

3.3 Elimination of false-positive signals with base mismatches

The specificity of RPA largely depends on the function of the recombinase when searching for homologous templates (Liu et al. 2019). Unfortunately, even introducing a probe can generate false-positive signals by the formation of complexes with the probe and reverse primer, which is considered a major drawback (Wu et al. 2020). PCR increases specificity through high annealing temperatures, whereas RPA is more sensitive and more likely to produce false-positive signals at lower temperatures (Wang et al. 2020). Furthermore, LFD cannot be used to determine the size of the amplification products. False-positive signals must be avoided due to the extremely high amplification efficiency of RPA.

The matched bases were located on two sides of the THF site, facilitating nfo enzyme cutting activity, causing nucleotide mismatches and reducing the stability of the primer-template complex (Higgins et al. 2022). However, the RPA system can tolerate some base mismatches between primers and probes without affecting the amplification efficiency (Przybylska et al. 2015; Daher et al. 2016; Liu et al. 2019). Visualization and prediction of the Probe 1 mismatch sites and sites of introducing mismatch bases were analyzed using the IDT online website. Base substitutions were introduced into Probe 1 and AMITS2-mR, and six groups of probes and reverse primers were designed (Table 2; Fig. 3c). Probe 1 and AMITS2-mR have six continuous base pairings, which can form a complex of probe and primer leading to false-positive signals. In Group 1, one base on Probe 1 and AMITS2-mR was replaced. Two base substitutions were made on Probe 1 in Group 3. Three base substitutions were made at the same time in Group2, Group4, Group5 and Group6, with two on Probe 1 and one on AMITS2-mR. The results show that Group 1, Group 3, Group 5, and Group 6 had false-positive signals, and Group 4 had weaker but visible false-positive signals; only Group 2 probes eliminated the false-positive signals (Fig. 3a). Furthermore, the results of the RPA-LFD assay using gDNA of A. mali showed that the introduction of mismatched bases had no significant effect on the amplification efficiency of RPA (Fig. 3b).

3.4 Analytical specificity of the RPA-LFD assay

Three closely related species were used as controls to evaluate the specificity of the primers and probe (AMITS-2F/Group2), including A. viduus, A. fleischeri and A. planipennis (Table S1). It is worth noting that A. viduus was discovered in the wild during the investigation of A. mali, and A. fleischeri and A. planipennis are well-known worldwide pests in the genus Agrilus. The test line and control line of dipsticks display red with the gDNA of A. mali, and eight A. mali samples were successfully detected, including larvae. In contrast, negative results were obtained for the three closely related species and the NTC (Fig. 4).

3.5 Reaction limit of the RPA-LFD assay

In this study, the optimum temperature and time were 38°C and 10 min, respectively. This was enough to satisfy the needs of rapid detection. However, it is necessary to understand the reaction limit to meet the demand of field detection. The RPA-LFD assays were tested in temperatures ranging from 20 to 50°C at 5°C intervals for 30 min. Test lines were visible on dipsticks between 30 and 50°C, and the color and density did not increase noticeably from 35 to 45°C (Fig. S1a). Moreover, the reaction time was tested in the range from 5 to 35 min in 5 min intervals at 38°C. Positive results were obtained when the reaction time was between 5 and 35 min, and the color and density did not increase noticeably except at 5 min (Fig. S1b). Finally, a 10-fold series dilution of A. mali ranging from 10 to 10^− 5 ng was tested in the sensitivity evaluation. The results showed a signal band with 10 to 10^− 3 ng of A. mali gDNA template, and no test line was observed on those with 10^− 4 and 10^− 5 ng of gDNA and NTC (Fig. S1c).

3.6 Establishment of the RPA-LFD assay system for A. mali

On the basis of previous studies, crude DNA was extracted by SDW (Priti et al. 2021; Jangra and Ghosh 2022). The insect tissue was collected into a centrifuge tube. Then, 20 µL of SDW was added and manually grinded with a grinding pestle. The tube was placed in boiling water for 10 min, and 2 µL of supernatant was added to the premixed RPA reaction system. The temperature of the clasped hand was high enough to meet the need for detection, and the reaction tube was held in an individual’s hand and allowed to react for 10 minutes. The amplified product was diluted 10 times and 50 µL was added to the sample pad of the LFD. Then, the results were observed by LFD after 3–5 min. Finally, the RPA-LFD assay system of A. mali was established; this assay can be fun within 30 min, including crude DNA extraction for 10 min, sample processing for 5 min, and RPA reaction and visualization of LFD results for 15 min (Fig. 5).

To simulate on-site detection, RPA-LFD assays were performed with five geographic populations of A. mali and three closely related species, including larvae of A. mali. The RPA-LFD assays were performed according to the previously described method, and the results were observed by LFD after holding in one's hand for 10 min. All A. mali samples were positive and the results were negative for three closely related species and the NTC (Fig. 6).

A. mali is a major insect that aggressively attacks species of the genus Malus and has recently caused serious damage to the wild apple tree M. sieversii in the western Tianshan Mountains in Xinjiang, China (Sun et al. 2022). Rapid distinguishment of the pest is the first step in implementing appropriate control measures. For insects, molecular identification can largely offset the deficiency of morphological identification, especially when they are young. Indeed, molecular methods based on DNA barcoding and specific PCR primers have recently gained attention as tools in the identification of insects and have been used alongside morphological identification (Hebert et al. 2003; DeSalle et al. 2019). However, neither of these methods can be performed in the field due to the need for trained personnel and expensive equipment. As an emerging isothermal amplification technique, the RPA combined LFD end-point readout method is a promising solution for on-site rapid detection (James and Macdonald 2015). Currently, RPA is rarely used for insect distinguishment (Gao et al. 2016; Priti et al. 2021), and morphological methods have been dominant for a long time as insects are more or less visible, unlike microbes. Nevertheless, the disadvantages of the method are prominent, as discussed above, particularly when the pest spreads widely with increasing commercial impacts worldwide.

Furthermore, the choice of detection target gene is crucial for specific detection. Regions with high nucleotide differences are commonly used to develop species-specific markers, especially the cox1 gene, which is frequently used for universal DNA barcoding for species classification (Hebert et al. 2003; Shi et al. 2022). The characteristics of the insect mitochondrial genome are conserved, while the insect ITS gene is also recommended as a candidate marker for animal species identification; the length of ITS is also moderate (Presa et al. 2002; Wei 2009; Si et al. 2020; Huang et al. 2022). There was no previous information on the ITS gene of the genus Agrilus, but the sequences on both sides are conserved, making it easy to design primers (Avise 2000; Pereira et al. 2009). Therefore, amplification primers for the complete ITS sequence were designed based on the 18S and 28S sequences of A. planipennis in the present study. A part of ITS1 was selected as the target gene.

This study showed that the strategy of generating primer-dimers in the amplification reaction is not suitable for the RPA-LFD assay because RPA is extremely sensitive and primer–dimer bands also create the risk of false-positive signals (Miao et al. 2019). Even if cautiously screening, reactions with the primer do not completely prevent the false-positive signals generated (Meagher et al. 2018); thus, a probe was introduced to improve the specificity. However, the problem of false-positive singles cannot be solved only by using a probe. False-positive singles also come from the complex of the probe and reverse primer rather than from primer-dimers. RPA is highly sensitive and cannot recognize the fragment size, and any interference from base matches of the probe and reverse primer may lead to false-positive signals (Safenkova et al. 2020). The RPA reaction activates the nfo enzyme, which cuts the probe through base pairing on both sides of the THF site to produce amplification products, so the pairing between the probe and the reverse primer truly affects the result. The RPA system can tolerate some base mismatches between primers and probes without affecting the amplification efficiency (Daher et al. 2016). Based on the fact that the RPA reaction can tolerate some base mismatches on the forward and reverse primers of the template (Liu et al. 2019), the introduction of mismatched bases between the probe and reverse primer can eliminate false-positive signals produced by probe and primer complexes (Przybylska et al. 2015). We carried out base substitution on the probe and reverse primer and selected the best combination of probe and reverse primer. The 3'-end of the reverse primer is an extension site, so a mismatched base near the 3'-end should not be introduced and replacing multiple remote bases on the probe and primer should be avoided to maintain the template recognition ability (Wu et al. 2020). In this study, the mismatch site was mainly located near the 5'-end of the reverse primer, so base modification had little effect. When three mismatched bases were introduced between the probe and the reverse primer, the complex of the probe and primer was completely eliminated. The color and density of the detected strip had no significant impact on the detection efficiency (Fig. 3b). In addition, it was beneficial to eliminate false-positive signals by eliminating the base pairing near the THF site on the probe (Fig. 3c). Some scholars have discussed the results of eight types of RPA amplification products on LFD visualization, as shown in Fig. 7 (Wang et al. 2019; Wu et al. 2020; Zhou et al. 2022). After evaluating the combination of each probe and reverse primer with IDT, we found that there may be another case that may also lead to false-positive LFD signals (Fig. 7h). This structure appeared in Group 4, and the test line with weak color and density was produced on the LFD (Fig. 3a). Selecting the base mismatch between the designed probe and reverse primer by IDT online testing can greatly reduce the cost of experimental failure.

After successfully eliminating the probe-primer dependent artifacts, the reaction limit of RPA-LFD detection was also tested to determine whether the assay could cope with any impact from environmental factors in the field. This method can detect as little as 10 − 3 ng of the DNA of A. mali. At an isothermal temperature of 30–40°C, the detection is completed within 30 min. In addition, repeated RPA assays in the laboratory will produce aerosol pollution, which will not occur in the field.

Nucleic acid extraction is the first and foremost step in molecular biology studies (Jangra and Ghosh 2022), and the most commonly used methods have been standardized or commercialized in the form of kits for the extraction of insect DNA (Asghar et al. 2015). This requires not only professional instruments in the laboratory but also laboratory technicians with experimental skills to complete the entire process. However, RPA is highly resistant to crude samples, suggesting that it can be used for on-the-spot field testing with crude nucleic acid extraction (Miao et al. 2019). RPA has good application prospects in the field of invasive insect inspection and quarantine, even though research on RPA rarely focuses on insect detection (Priti et al. 2021). Insect rapid field detection technology may be limited by DNA extraction. There is no doubt that the rapid and efficient method of insect DNA extraction will greatly promote the application of RPA technology for insect field detection.

Acknowledgements: The authors thank Dr. Qinzheng Zhou (China Agricultural University) for providing critical suggestions during the study. We also thank Professor (Associate) Yanlong Zhang (Ecology and Nature Conservation Institute, Chinese Academy of Forestry) for helping sample collections. This research was funded by the National Key Research and Development Program of China (2021YFD1400300).

Author contributions: Conceptualization, Methodology, Validation, Formal analysis, Data curation, Software, Visualization, Writing-original draft preparation: CJL; Data curation, Software: HQS; Resources, Writing-review and editing: XYW, WXZ; Conceptualization, Methodology, Investigation, Resources, Project administration, Funding acquisition, Writing-review and editing: RZL, YXY.

Availability of data and material: The datasets generated during and/or analyzed during the current study are available in the GenBank database, OQ654087 - OQ654097. Supplementary Materials: Table S1 for list of insect samples used in this study and Figure S1 for results of the reaction limit of the RPA-LFD assays.

Conflict of interests: The authors have no relevant financial or non-financial interests to disclose.

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Rapid assay using recombinase polymerase amplification and lateral flow dipstick for identifying Agrilus mali, a serious wood-boring beetle of the western Tianshan Mountains in China

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Abstract

Figures

1 Introduction

2 Materials And Methods

2.1 Specimen collection and DNA extraction

2.2 PCR amplification, cloning and sequencing

2.3 RPA-LFD procedure with forward and reverse primers

2.4 Designing and optimization of the probe

2.5 RPA-LFD procedure with primers and a probe

2.6 Specificity and reaction limit of the RPA-LFD assay

2.7 Rapid DNA extraction and field simulation detection

3 Results

3.1 Primer design and screening

3.2 Adding a probe to the RPA reaction

3.3 Elimination of false-positive signals with base mismatches

3.4 Analytical specificity of the RPA-LFD assay

3.5 Reaction limit of the RPA-LFD assay

3.6 Establishment of the RPA-LFD assay system for A. mali

4 Discussion

Declarations

References

Additional Declarations

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