Specificity and Sensitivity of a Rapid LAMP Assay for Early Detection of Emerald Ash Borer (Agrilus planipennis) in Europe

Abstract

Buprestids are an emerging threat to broadleaf forests across the world. Species such as emerald ash borer (EAB, Agrilus planipennis) seriously threaten ash (Fraxinus spp.) in North America and Europe. As it continues spreading west from European Russia, native European ash populations will suffer dramatic losses. Due to their cryptic lifestyle of the egg and larval stages on developing bark and vascular tissue, buprestids and other wood borers can be difficult to detect. Early detection tools are vital to implement fast eradication measures, and prevent the establishment of invasive species populations. Detection methods using polymerase chain reaction (PCR) assays to target specific taxa can be extremely timely to obtain results especially since samples need to be transported to the laboratory first. However, loop-mediated isothermal amplification (LAMP) eDNA assays are highly specific and sensitive providing results within 30 min after sample extraction. In this study, we investigated the specificity and sensitivity of an EAB LAMP assay as an early detection tool in Europe. The assay was specific to EAB when tested against 12 European Agrilus spp., five buprestids, two Scolytinae, and five cerambycids (n = 24). The LAMP assay sensitivity amplified DNA from a concentration as low as 0.02 pg/µL. These results demonstrate that the LAMP assay is a highly specific, sensitive tool that can be used to detect and monitor EAB in European forests and urban settings.

1. Introduction

Non-native insects and microorganisms that are introduced to new environments can cost billions of US dollars annually in lost production and value, and threaten native biodiversity and the provision of ecosystem services upon which humans rely [1,2,3]. Global trade is the main source of these accidental introductions which have increased exponentially during the last century [4,5,6] and unfortunately, there appears to be no sign of saturation in the number of alien species introductions that will be introduced around the world in the future [7]. Failure to intercept and quickly eradicate a new invasion allows the organism to establish a viable breeding population when environmental barriers are overcome and subsequently spread making eradication efforts impossible [8]. The ecological and economic consequences of biological invasions can be reduced or avoided by investing in biosecurity tools that prevent the establishment of potentially harmful pests by detecting them early which will allow for rapid response to curb their establishment and potential impact [8,9,10].

Buprestids are an emerging group of pests affecting a broad range of tree species worldwide. Historically, buprestids in their native ranges are secondary pests that attack and kill native host trees stressed by other biotic or abitoics factors [11,12]. Over the last several decades, these beetles have been increasingly observed and detected due to major damage inflicted on evolutionarily naïve hosts. Bronze birch borer (Agrilus anxius Gory), for example, started killing healthy Eurasian birch (Betula spp.) trees when these plants were introduced to North America [13]. Similarly, gold-spotted oak borer (A. coxalis Waterhouse) was discovered in the 2000s attacking and killing healthy oak (Quercus spp.) trees in southern California [14]. The main example of a buprestid pest is emerald ash borer (EAB), A. planipennis (Fairmaire). This insect was discovered in 2002 after an accidental introduction to Michigan, USA and Ontario, Canada, where it was found killing evolutionarily naïve ash (Fraxinus spp.) by girdling the plant via larval feeding on the phloem [15]. Since then, EAB has become one of the most costly biological invasions of forests in North America [2,16]. In East Asia, EAB is a secondary pest and only attacks and kills stressed Manchurian ash (F. mandshurica Rupr.) and Chinese ash (F. chinensis Roxb.). But in North America, EAB attacks and readily kills more than 99% of North American ash trees [17]. A separate introduction of EAB to European Russia occurred around 2003 [18], which has since spread in a westward direction, recently arriving in Ukraine [19]. Based on evidence from North America, EAB is expected to continue spreading unhindered as a result of natural dispersion and the unintentional movement of wood products [20], putting the fate of European ash (F. excelsior L.) across the rest of Europe at risk if no measures are taken to contain its spread. The potential impact of EAB on European ash, a valuable and ecologically important species, is critically important as it is known as a keystone species in temperate broadleaf forest ecosystems. European ash is already under threat due to the fungal pathogen Hymenoscyphus fraxineus (Baral, Queloz & Hosoya) which was introduced to Europe decades earlier and has substantially reduced the ash population across Europe, impacting critical biodiversity that depends on ash for its habitat [21]. In addition, there is a strong likelihood that the expanding EAB population will heavily impact other European Fraxinus species including F. angustifolia (Vahl) and F. ornus (L.) as well as other Oleaceae species such as olive (Olea europaea L.) in the southern parts of Europe and possibly other related ornamental landscape trees [22,23].

Biosurveillance tools that are rapid, accurate, and portable allowing for on-site detection are a critical way forward to prevent the introduction and establishment of harmful pests and pathogens [8,24]. Detection of Buprestids is difficult due to their cryptic lifestyle, with larvae feeding on the vascular tissue of their host trees under the bark. Species such as EAB often can be present for 3–8 years in a novel environment before they are discovered [20,25]. A major limitation of using traditional surveys is to detect and identify taxa that are rare, in low abundance, or both [8,24,26]. The morphological identification of some buprestid adults and larvae, such as Agrilus species, can be extremely challenging for non-experts, which is made worse by the fact that the group is highly speciose [27]. Traditionally, plant diagnostics are also labor-intensive and time-consuming since it requires a high level of sampling and multiple processing steps that must be handled by experienced staff in laboratories. This makes it both expensive and difficult to detect potentially invasive forest insects at an early enough stage that would allow mitigation measures to be implemented quickly and effectively [28]. However, surveys using environmental DNA (eDNA) can be a reliable tool for improving the detection probability of target species [29,30]. Multiple studies in recent years have demonstrated the power of eDNA over traditional survey methods for monitoring terrestrial pest populations [31,32,33]. While significant advances have been made with portable molecular techniques to detect organisms directly in the field and offer the possibility to scale up their usage in practice, progress has been largely curtailed by the need to optimize and validate on-site methods, ensure specificity and accuracy of detection, and increasing high throughput capacity for screening large numbers of pests. Portable DNA-based equipment that operates with great simplicity, sensitivity, specificity, and high speed, that can detect pests in the early stages of attack when symptoms are not yet entirely visible, or detect traces of their presence in other forms, would significantly enhance both diagnostic and surveillance applications to help control outbreak populations. Environmental DNA, which can detect trace evidence of DNA shed by pests on different plant substrates [32,34], shows high promise to advance current biosecurity efforts and the capacity to detect cryptic species such as Buprestids, including EAB. This technology has the potential to be adapted to rapid and throughput in-field detection using a portable system called loop-mediated isothermal amplification (LAMP) [35]. A LAMP assay was recently developed for EAB in North America [36], but in order for it to be used for early detection in European conditions, the protocol must be able to discriminate EAB from European insect fauna, and its sensitivity needs to be determined to understand the limit of detection (LOD), i.e., the lowest DNA concentration that can be determined by the molecular assay. The objective of this study was to determine the ability of the EAB LAMP assay to be specific in relation to European wood borers, other Agrilus spp., Cerambycids, and Scolytinae; and to determine a lower threshold of DNA detection for field application.

2. Methods

2.1. Collection and Extraction of Specimens

In this study, all insect specimen used for specificity testing were adults and trapped in broadleaf forests in Europe and North America (Table 1). Insects were collected in multifunnel traps in a mix of 50/50 monopropylene glycol and water; a common method used for the collection of buprestids and EAB [37,38] and in connection with the Euphresco project (2020-A-337-‘Developing and assessing surveillance methodologies for Agrilus beetles’) in Europe.

Genomic DNA was extracted from EAB and A. anxius specimens from North America at Rutgers University (New Brunswick, NJ, USA) in December 2021 using the DNeasy Blood and Tissue Kit (Qiagen) and stored in a −20 °C freezer. Extracted DNA was shipped on dry ice in January 2022 to the Institute for Sustainable Plant Protection—National Research Council (IPSP-CNR, Florence, Italy) and then transferred to a −20 °C freezer until subsequent processing. All other specimens from France and Italy were shipped to IPSP-CNR and stored in 70%–95% ethanol. For all specimens (Table 1), each sample was triple rinsed with DI water to wash any potential exterior DNA contamination. Using flame-sterilized forceps, we removed legs with attached muscle tissues, and total genomic DNA was extracted. The number of specimen legs that were used ranged from three for Cerambycid adults to a maximum of six for smaller Agrilus individuals. A full body extraction was performed with the two smaller ambrosia beetles (Xyleborinus saxesenii and Anisandrus dispar) to obtain enough genetic material for subsequent PCR amplification. For EAB and A. anxius (three-five individuals/species), at least three legs were manually removed and crushed with a sterilized metal forceps inside the 1.5 mL microcentrifuge tube. For all other specimens (one per insect species), legs or full body tissue was pulverized by adding two metal sterile beads to 1.5 mL tubes and running the tubes in a TissueLyser (Qiagen) for 1–2 min at 300 oscillations/min. DNA extraction was performed from homogenized samples by using the standard protocol for insect extractions from two different commercial kits: Invitrogen JetQuick Blood and Cell Culture and DNeasy Blood and Tissue Kit (Qiagen) (Table 1). DNA quantity and quality were checked using a Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA).

2.2. PCR Amplification and Sequencing of Samples

DNA extracted from insects were amplified with PCR in a final volume of 20 μL containing, 12.5 μL Reaction buffer, GoTaq (Promega), 0.4 μL Primer Forward (10 μM final concentration, Eurofins Genomics, Ebersberg, Germany, see Table 1), 0.4 μL Primer Reverse (10 μM final concentration, Eurofins Genomics, see Table 1), 2 μL of extracted DNA, and 4.7 μL of nuclease-free distilled water. EAB DNA was amplified using the primer set EAB_COIF and EAB_COIR designed to capture potential polymorphism of EAB within a conserved region for this species [36] (Table 2). The EAB PCR reactions were run with an initial denaturation step (94 °C for 1 min) followed by five cycles of denaturation (94 °C for 40 s), annealing (45 °C for 40 s), and extension (72 °C for 60 s), and then 35 cycles of denaturation (94 °C for 45 s), annealing (60 °C for 40 s), and extension (72 °C for 60 s) and a final extension (72 °C for 10 min). All other species used the primers LCO1490-HCO2198 [39], rrnl mtDNA (16A)-rrnl mtDNA (16B), and 28S_D1D2.3.a_up-28S_D1D2.3.a_low (Table 2) with an initial denaturation (94 °C 2 min), denaturation (94 °C 30 s), annealing (45 °C 1 min), extension (72 °C 2 min), 34 cycles, and a final extension step (72 °C 5 min). Amplification products were separated by electrophoresis on gels containing 1% (w/v) of agarose LE (Genespin). Each sample (2.5 μL amplified DNA and 2.5 μL SYBR™ Safe DNA Gel Stain-Invitrogen) was run at 90 V for 30 min. The approximate length (bp) of the amplification products was determined using the 1 kbp DNA ladder Ready to Load (Genespin). For a several species that did not initially amplify, we ran the DNA at a lower concentration (1/10 dilution) to show successful amplification (Figure S1).

2.3. Specificity and Sensitivity Testing of EAB Assay

The LAMP assay (see Table 2) was tested using the extracted DNA from closely related species (Table 1). The reactions were carried out in a final volume of 25 μL containing 15 μL of isothermal master-mix (OptiGene, Horsham, UK); 0.5 μL of F3 and B3 (Table 2; final conc. 0.2 μM each) (Eurofins Genomics, Vienna, Austria); 1.0 μL of Loop F and Loop B (Table 2; final conc. 0.4 μM each) (Eurofins Genomics, Vienna, Austria); 2.0 μL FIP and BIP (Table 2; final conc. 0.8 μM each) (Eurofins Genomics, Vienna, Austria); and 3.0 μL of extracted DNA. LAMP amplification reactions were performed at 65 °C for 30 min using the Genie^® II (OptiGene Limited, Horsham, UK) according to methods in [42]. Each species was run in duplicate. In each run, one positive (EAB) control and one negative (No Template Control, NTC) were also included to verify the success of the reaction and ensure there was no DNA contamination.

A standard curve was generated from five-fold serial dilutions (ranging from 2 ng/μL to 0.02 pg/μL) to determine the lower limit of detection. Each dilution was run in duplicate under the same conditions previously described.

3. Results and Discussion

To adequately detect target species, molecular assays need to be specific to the species of concern, highly sensitive to low or degraded DNA quantities, and free from environmental inhibitors [43]. Specificity is key to successfully designing eDNA assays. Since Agrilus is the largest described animal genus [27], it is particularly important to demonstrate that specific primers can be designed to readily and accurately detect the target taxa. In the study by Kyei Poku et al. [36], the similarity of the targeted gene region for EAB was compared to other congeners and other taxa from an online database to make the assay species-specific; however, specificity of a molecular assay, such as LAMP, needs to be validated with extracted DNA of other taxa. Kyei Poku et al. [36] conducted specificity testing with extracted DNA of mostly North American co-occurring fauna including four Agrilus and two Scolytinaes, and none amplified with the EAB LAMP primers. In order for EAB LAMP to be specific in European conditions and to better understand the risks of obtaining false positives as a result of having a different suite of co-occurring species within European forests, we tested the extracted DNA of additional Agrilus spp. native to Europe to determine specificity.

The EAB LAMP assay was tested on DNA extracted from 25 species (24 non-target; DNA concentration range of 5 to 160 ng/µL): 13 Agrilus (12 native to Europe) and 12 non-Agrilus (Table 1). We confirmed by PCR amplification that the DNA of EAB and the non-EAB specimens were suitable for specificity and sensitivity testing using the LAMP assay (Table 1). All EAB DNA samples were successfully amplified using the PCR primers EAB_COIF and EAB_COIR [36]. Three sets of primers were used to amplify the DNA of other specimens because DNA can be difficult to amplify for specific Agrilus spp. (personal communication Amanda Roe, Great Lakes Forestry Centre, Sault Ste. Marie, ON, Canada). The majority of the insect species were amplified with LCO1490-HCO2199 (Table 1), a common, universal primer set, and ten samples were successfully amplified with other common insect primers.

Of the non-target species tested, none were amplified by LAMP primers (Table 1). Our findings provide additional data supporting that the LAMP assay for EAB is highly specific and can be used to distinguish EAB DNA from other Buprestids originating from both North America and Europe. Overall, 30 species have been examined for specificity testing including those from Kyei-Poku et al. [36]. While these results are promising for two geographic regions of the world, other locations warrant further investigation because closer relatives of EAB, such as those in eastern Asia, can be more genetically similar [27].

Similar to Kyei-Poku et al. [36], we demonstrated that the DNA of A. sulcicollis (Lacordaire) and A. anxius did not amplify by the LAMP assay. In addition, we also found that non-buprestids that co-occur in European broadleaf forests did not amplify using the designed EAB LAMP primers, and DNA extracts of two additional Scolytinaes and five Cerambycid were negative for amplification. Overall, our data and that by Kyei-Poku et al. [36] demonstrate that the LAMP primers appear highly specific to the targeted fauna (EAB), even among very speciose genera Agrilus, and can be used as a diagnostic tool for early detection and monitoring on both continents.

Critical to the use of eDNA assays is the ability to detect low concentrations of target DNA. If the threshold for adequate detection is too high, then the ability to detect a species at the early stages of invasion may be jeopardized. Insect DNA can be detected at quite low levels; for example, spotted lanternfly (Lycorma delicatula White) DNA reached a limit of detection of 0.14 pg/μL with qPCR [33]. For LAMP studies, levels have reached as low as 0.61–16 pg/μL using the DNA of long-horned beetles [44,45]. Our study revealed similar levels of sensitivity with a serial dilution experiment detecting as low as 0.02 pg/μL with the EAB LAMP assay when starting from a higher level of 2.0 ng/μL (Figure 1). This concentration is lower but comparable to those results found when running qPCR assays [33]. Our findings are similar to other studies where LAMP outperforms traditional PCR methods [46,47]. The results clearly demonstrate that LAMP assays can not only be specific, portable, and provide rapid results, but also detect lower DNA amounts than some qPCR assays. The benefits and trade-offs of using eDNA LAMP assays in early detection and surveillance efforts for EAB are evident. Conventional PCR is more expensive due to the thermocycler equipment and reagent needs, the necessity of highly trained staff, and the time needed to prepare samples. This results in an overall higher cost per sample than running LAMP protocols [48]. In contrast, LAMP equipment (i.e., Genie^® II) is generally cheaper to produce, smaller in size, portable due to isothermal amplification, and amplification are much faster (generally less than 30 min). Thus, this LAMP assay is providing a user-friendly tool for stakeholders to use. One potential advantage of PCR over LAMP is the influence of plant-produced chemicals that can inhibit DNA amplification. qPCR can outperform LAMP in terms of sensitivity when inhibitors are present [49] and LAMP is known to be influenced by some plant chemicals [50]. Thus, future research should consider the influence of host plant chemistry, or other inhibitors present in the environmental sample, on the sensitivity and false negatives.

Insects are a hyper-diverse group of organisms; beetles (Coleoptera) being the largest order of all described insects and the genus Agrilus, is the largest among animals [27]. In recent decades, Agrilus spp. have become major forest pests where they have been introduced outside of their native range, whereas in their native range, they are usually considered only secondary pests, killing or attacking weakened or stressed host plants. With so many species, ~3000 in the genus Agrilus, it is often difficult to accurately identify taxa to a species level and often taxonomic experts are needed, but even then species may go undetected for decades as was the case with EAB in North America [51]. Reliable methods such as LAMP, as demonstrated in our study, are therefore needed to rapidly detect the species of concern, and reduce the time from incursion to detection and subsequent mitigation efforts. As EAB continues to spread throughout Europe, the developed and validated LAMP assay can play an important role to monitor for satellite populations or delimit the expanding range in forests and urban settings. With the current location of EAB in eastern Europe, now on the fringe of invading the rest of Europe, LAMP could provide a critical role for sampling to assist in containment efforts, since the Genie^® II or III instruments allow users to conduct on-site testing which can lead to more rapid decisions for mitigation.

Targeted sampling methods for eDNA collection of woodboring insects such as EAB are still needed for larval stages but some methods for collecting eDNA of adults already exist for sampling foliage and bark from trees in forests (e.g., using a backpack sprayer or a paint rolling pole) [31,34]. Overall, our results demonstrated, validated, and expanded the usability of the EAB LAMP assay for European conditions, and is highly recommended that the LAMP assay be adopted by plant protection organizations throughout Europe to aid in the early detection of A. planipennis in surveillance efforts that ultimately aim to save European ash from its demise.

4. Conclusions

In conclusion, our study determined that the emerald ash borer LAMP assay appears specific to EAB with none of the non-target species DNA being amplified. The LAMP assay has the capacity to detect A. planipennis in European and North American conditions. Furthermore, the assay is quite sensitive, being able to detect low concentrations of DNA. Future validation of this or other LAMP assays in the field is being developed and needs to be implemented to demonstrate the capacity to obtain this technology for detection in both forested and urban settings.

Author Contributions

Conceptualization, D.L.P., A.S. (Alberto Santini), N.L. and M.C.; methodology, D.L.P., K.K., A.S. (Aurélien Sallé), F.P., D.M., A.S. (Alberto Santini), N.L. and M.C.; investigation, D.L.P. and F.P.; resources, D.L.P., K.K., A.S. (Aurélien Sallé), F.P., D.M., A.S. (Alberto Santini), N.L. and M.C.; writing—original draft preparation, D.L.P. and M.C.; writing—review and editing D.L.P., K.K., A.S. (Aurélien Sallé), F.P., D.M., A.S. (Alberto Santini), N.L. and M.C.; visualization, D.L.P. and F.P.; funding acquisition, D.L.P., A.S. (Alberto Santini), N.L. and M.C. All authors have reviewed, read, and accept the published version of the manuscript. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement

The data from this study is available upon reasonable request to the corresponding author.

Acknowledgments

We would like to thank Anthony Vastano at Rutgers University for coordination and shipment of DNA extractions from USA to Europe, Amanda Roe at the Great Lakes Forestry Centre, Canada for advice on DNA amplification and primers and BBB samples, Toby Petrice from USDA Forest Service for BBB specimen, and Davide Rassati from University of Padova and the Euphresco project 2020-A-337-‘Developing and assessing surveillance methodologies for Agrilus beetles’ for other non-target specimen.

Conflicts of Interest

The authors declare no conflict of interest.

Footnotes

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Figure and Tables

Figure 1. Fluorescence of EAB DNA in a 1:5 serial dilution to determine the lower detection threshold of the EAB LAMP assay.

Table 1

Insect species used for specificity testing for EAB LAMP primers for use in European conditions including insect collection location, collection date, and the DNA extraction kit and PCR primer set to amplify DNA. * represents Figure S1 showing PCR amplification in gels.

Insect Species	Label Name	Collection Date(mm-yr)	Insect Locality	Longitude Latitude	Extraction Kit	PCR Primers(A–F *)	EAB LAMP Detection
Buprestidae
1. Agrilus angustulus	AA	21 May	Vierzon, France	2.0327°47.2611°	Invitrogen	LCO1490-HCO2198 (B)	-
2. Agrilus anxius	BBB	14,15 June	CT, USA; ON, Canada		Qiagen	LCO1490-HCO2199 (A, D, E)	-
3. Agrilus ater	AAT	2020	Vierzon, France	2.0327°47.2611°	Qiagen	28 s up–28 s low (F)	-
4. Agrilus convexicollis	AC	21 June	Orléans campus, France	1.9383°47.8456°	Qiagen	16a–16b (F)	-
5. Agrilus curtulus	Acu	20 August	France		Qiagen	28 s up–28 s low (F)	-
6. Agrilus graminis	AG	20 August	Orléans campus, France	1.9383°47.8456°	Qiagen	LCO1490-HCO2199 (D)	-
7. Agrilus hastulifer	AH	21 June	Orléans campus, France	1.9383°47.8456°	Qiagen	28 s up–28 s low (F)	-
8. Agrilus laticornis	AL	21 June	Orléans campus, France	1.9383°47.8456°	Qiagen	28 s up–28 s low (F)	-
9. Agrilus obscuricollis	AO	21 June	Orléans campus, France	1.9383°47.8456°	Qiagen	28 s up–28 s low (F)	-
10. Agrilus olivicolor	AD	21 June	Orléans campus, France	1.9383°47.8456°	Qiagen	LCO1490-HCO2198 (B)	-
11. Agrilus planipennis	EAB		NY, RI, VT, USA		Qiagen	LCO1490-HCO2198 (A) EAB_COIF EAB_COIR (C)	+
12. Agrilus roscidus	AR	2020	France		Qiagen	LCO1490-HCO2199 (E)	-
13. Agrilus sulcicollis	AS	21 May	Vierzon, France	2.0327°47.2611°	Invitrogen	LCO1490-HCO2198 (A)	-
14. Agrilus viridis	AV	21 June	Orléans campus	1.9383°47.8456°	Qiagen	28 s up–28 s low (F)	-
15. Anthaxia nitidula	AN	21 May	Marcenat	3.3619°46.2461°	Qiagen	LCO1490-HCO2199 (E)	-
16. Chrysobothris affinis	CA	20 June	Vierzon, France	2.1544°47.2899°	Qiagen	LCO1490-HCO2198 (E)28 s up–28 s low (F)	-
17. Coraebus undatus	CU	21 June	Orléans campus, France	1.9383°47.8456°	Qiagen	LCO1490-HCO2198 (B)28 s up–28 s low (F)	-
18. Lamprodila mirifica	LM	Summer 2021	Friuli Venezia Giulia Italy	13.120926°45.791784°	Qiagen	28 s up-28 s low (F)	-
Curculionidae: Scolytinae
19. Anisandrus dispar	A dis	Summer 2021	Friuli Venezia Giulia, Italy	13.120926°45.791784°	Qiagen	LCO1490-HCO2198 (C)	-
20. Xyleborinus saxesenii	XS	Summer 2021	Friuli Venezia Giulia Italy	13.120926°45.791784°	Qiagen	LCO1490-HCO2199 (C)	-
Cerambycidae
21. Aegomorphus clavipes	Aeg	Summer 2021	Friuli Venezia Giulia, Italy	13.120926°45.791784°	Qiagen	28 s up–28 s low (F)	-
22. Exocentrus punctipennis	EP	Summer 2021	Friuli Venezia Giulia, Italy	13.120926°45.791784°	Qiagen	LCO1490-HCO2199 (C)	-
23. Leiopus nebulosus	LN	Summer 2021	Friuli Venezia Giulia Italy	13.120926°45.791784°	Qiagen	LCO1490-HCO2199 (C)	-
24. Saperda punctata	SP	Summer 2021	Friuli Venezia Giulia Italy	13.120926°45.791784°	Qiagen	LCO1490-HCO2199 (C)	-
25. Trichoferus pallidus	TN	21 June	Orléans campus, France	1.9383°47.8456°	Qiagen	LCO1490-HCO2199 (E)28 s up–28 s low (F)	-

Table 2

General and specific PCR and LAMP primers used to amplify and confirm insect DNA.

PCR Primer	Sequence (5′–3′)	Gene Type	Reference
EAB_COIF	AGG AAT AGT AGG AAC AGC CCT TAG A	COI	[36]
EAB_COIR	TAT TTC ATC TAA GGT AGG CAT CTG G	COI
LCO1490	GGT CAA CAA ATC ATA AAG ATA TTG G	COI	[39]
HCO2198	TAA ACT TCA GGG TGA CCA AAA AAT CA	COI
28S_D1D2.3.A_UP	GGA ATC CGC TAA GGA GTG TGT AA	28S	[40]
28S_D1D2.3.A_LOW	AGG GCC TCG CTG GAG TAT TT	28S
RRNL MTDNA (16A)	CGC CTG TTT AAC AAA AAC AT	mtDNA	[41]
RRNL MTDNA (16B)	CCG GTC TGA ACT CAG ATC ATG T	mtDNA
LAMP PRIMERS
EAB1_F3	CTC CCT CCC TCT TTA ACA TTA C	COI	[36]
EAB1_B3	GAT CAG ACT AGT AGA GGT GT	COI
EAB1_FIP	ATA TTA GCC GCT AAT GGT GGG AAT AGT CGA AAG AGG AGC AG	COI
EAB1_BIP	GGC TCT GTT GAC TTA GCA AAG GTT ATT CCT ATT GCT CGC	COI
EAB1_LF	ATA TAC TGT CCA ACC AGT CC	COI
EAB1_LB	CTG GAA TCT CCT CAA TTC TAG G	COI

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/f14020436/s1, Figure S1: Gels of amplified PCR DNA of insects used for specific testing of EAB LAMP primers. All primers used to confirm DNA were LCO1490-HCO2198 (A–E) in gel except the final gel (F) shows DNA amplified with 28S_D1D2.3.a_up-28S_D1D2.3.a_low (Top) and rrnl mtDNA (16A)-rrnl mtDNA (16B) (Bottom). Species with plus mark (+) were considered amplified. See Table 1 for the species codes.

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