Diagnostic Tool for the Identification of Bactrocera dorsalis (Hendel) (Diptera: Tephritidae) Using Real-Time PCR.

Accurate identification of Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), commonly known as the Oriental fruit fly, is a significant challenge due to the morphological convergence and taxonomic uncertainties of species belonging to the same genus. This highly polyphagous species poses a significant threat to fruit crops. With its potential establishment in Europe becoming a growing concern, there is an urgent need for rapid and efficient diagnostic methods. The study presented here introduces a diagnostic protocol based on real-time PCR using a TaqMan probe for the early and reproducible identification of B. dorsalis. Specimens representing the genetic diversity of the Italian population were collected and analyzed. Specific primers and probe were designed based on the conserved regions and an in silico analysis confirmed their specificity. The assay conditions were optimized, and analytical sensitivity, specificity, repeatability, and reproducibility were evaluated. The protocol showed high sensitivity and specificity, accurately detecting low DNA concentrations of B. dorsalis. This standardized method provides a reliable tool for routine diagnostics, enhancing the accuracy and efficiency of identifying the Oriental fruit fly at all stages of its development, thereby facilitating effective pest management measures. The development of this diagnostic protocol is crucial for monitoring and supporting efforts to prevent the passive spread of B. dorsalis in Europe, particularly in light of the recent active infestations detected in Italy.

Site-Selective Functionalized PD-1 Mutant for a Modular Immunological Activity against Cancer Cells.

Targeting immune checkpoints is a well-established strategy in cancer therapy, and antibodies blocking PD-1/PD-L1 interactions to restore the immunological activity against cancer cells have been clinically validated. High-affinity mutants of the PD-1 ectodomain have recently been proposed as an alternative to antibodies to target PD-L1 on cancer cells, shedding new light on this research area. In this dynamic scenario, the PD-1 mutant, here reported, largely expands the chemical space of nonantibody and nonsmall-molecule inhibitor therapeutics that can be used to target cancer cells overexpressing PD-L1 receptors. The polyethylene glycol moieties and the immune response-stimulating carbohydrates, used as site-selective tags, represent the proof of concept for future applications.

Theoretical and experimental studies on the interaction of biphenyl ligands with human and murine PD-L1: Up-to-date clues for drug design.

Today it is widely recognized that the PD-1/PD-L1 axis plays a fundamental role in escaping the immune system in cancers, so that anti-PD-1/PD-L1 antibodies have been evaluated for their antitumor properties in more than 1000 clinical trials. As a result, some of them have entered the market revolutionizing the treatment landscape of specific cancer types. Nonetheless, a new era based on the development of small molecules as anti PD-L1 drugs has begun. There are, however, some limitations to advancing these compounds into clinical stages including the possible difficulty in counteracting the PD-1/PD-L1 interaction in vivo, the discrepancy between the in vitro IC50 (HTFR assay) and cellular EC50 (immune checkpoint blockade co-culture assay), and the differences in ligands' affinity between human and murine PD-L1, which can affect their preclinical evaluation. Here, an extensive theoretical study, assisted by MicroScale Thermophoresis binding assays and NMR experiments, was performed to provide an atomistic picture of the binding event of three representative biphenyl-based compounds in both human and murine PD-L1. Structural determinants of the species' specificity were unraveled, providing unprecedented details useful for the design of next generation anti-PD-L1 molecules.

Combining Solid-State NMR with Structural and Biophysical Techniques to Design Challenging Protein-Drug Conjugates.

Several protein-drug conjugates are currently being used in cancer therapy. These conjugates rely on cytotoxic organic compounds that are covalently attached to the carrier proteins or that interact with them via non-covalent interactions. Human transthyretin (TTR), a physiological protein, has already been identified as a possible carrier protein for the delivery of cytotoxic drugs. Here we show the structure-guided development of a new stable cytotoxic molecule based on a known strong binder of TTR and a well-established anticancer drug. This example is used to demonstrate the importance of the integration of multiple biophysical and structural techniques, encompassing microscale thermophoresis, X-ray crystallography and NMR. In particular, we show that solid-state NMR has the ability to reveal effects caused by ligand binding which are more easily relatable to structural and dynamical alterations that impact the stability of macromolecular complexes.

Cerebrospinal fluid lipoproteins inhibit α-synuclein aggregation by interacting with oligomeric species in seed amplification assays.

BACKGROUND: Aggregation of α-synuclein (α-syn) is a prominent feature of Parkinson's disease (PD) and other synucleinopathies. Currently, α-syn seed amplification assays (SAAs) using cerebrospinal fluid (CSF) represent the most promising diagnostic tools for synucleinopathies. However, CSF itself contains several compounds that can modulate the aggregation of α-syn in a patient-dependent manner, potentially undermining unoptimized α-syn SAAs and preventing seed quantification. METHODS: In this study, we characterized the inhibitory effect of CSF milieu on detection of α-syn aggregates by means of CSF fractionation, mass spectrometry, immunoassays, transmission electron microscopy, solution nuclear magnetic resonance spectroscopy, a highly accurate and standardized diagnostic SAA, and different in vitro aggregation conditions to evaluate spontaneous aggregation of α-syn. RESULTS: We found the high-molecular weight fraction of CSF (> 100,000 Da) to be highly inhibitory on α-syn aggregation and identified lipoproteins to be the main drivers of this effect. Direct interaction between lipoproteins and monomeric α-syn was not detected by solution nuclear magnetic resonance spectroscopy, on the other hand we observed lipoprotein-α-syn complexes by transmission electron microscopy. These observations are compatible with hypothesizing an interaction between lipoproteins and oligomeric/proto-fibrillary α-syn intermediates. We observed significantly slower amplification of α-syn seeds in PD CSF when lipoproteins were added to the reaction mix of diagnostic SAA. Additionally, we observed a decreased inhibition capacity of CSF on α-syn aggregation after immunodepleting ApoA1 and ApoE. Finally, we observed that CSF ApoA1 and ApoE levels significantly correlated with SAA kinetic parameters in n = 31 SAA-negative control CSF samples spiked with preformed α-syn aggregates. CONCLUSIONS: Our results describe a novel interaction between lipoproteins and α-syn aggregates that inhibits the formation of α-syn fibrils and could have relevant implications. Indeed, the donor-specific inhibition of CSF on α-syn aggregation explains the lack of quantitative results from analysis of SAA-derived kinetic parameters to date. Furthermore, our data show that lipoproteins are the main inhibitory components of CSF, suggesting that lipoprotein concentration measurements could be incorporated into data analysis models to eliminate the confounding effects of CSF milieu on α-syn quantification efforts.

First Report of Xanthomonas hydrangeae Causing Leaf Spot on Oakleaf Hydrangea (Hydrangea quercifolia) in Tuscany (Italy).

Hydrangeas (Hydrangea L.) are popular ornamental plants used in urban landscapes and gardens worldwide for the beauty of their large flowers. In June 2022, dark brown/purple and irregular water-soaked spots coalescing into large areas of necrosis were observed on the leaves of potted Hydrangea quercifolia Bartr. plants growing in two ornamentals nurseries in Pistoia, Tuscany, Italy. Isolations, using two symptomatic plants/nursery, were performed by excising small portions of leaf tissue from the margin of the lesions, and macerating them in 100 µl of sterile distilled water (SDW). After 15 min of incubation, a loopful of the resulting suspension was streaked on yeast extract-dextrose-CaCO3 agar (YDCA) amended with 60 mg L-1 cycloheximide. Mucoid, convex and yellow colonies appeared on YDCA after incubation at 28°C for 48h. After colony purification on yeast extract-nutrient-agar (YNA), two isolates from each nursery were subject to amplification and sequence analysis of the 16S rRNA using universal primers FD1/RD1, for genus identification (Vauterin et al. 2000; Weisburg et al. 1991). All 16S rRNA sequences (OP441051) were identical and BLASTn searches indicated that the isolates belong to the genus Xanthomonas [99.9% nucleotide identity with X. hydrangeae strain LMG 31885 (LR990741.1) and 99.8% with strain LMG 31884T (NR_181958.1)]. For classification at species level, fragments of the housekeeping genes gyrB, rpoD, dnaK, and fyuA, were amplified according to Young et al. (2008). Both strands were sequenced and the consensus sequences aligned using MUSCLE as implemented in MEGA X (Kumar et al. 2018). Homologous sequences were once again identical between the isolates. A neighbor joining phylogenetic analysis of the concatenated fragments, was carried out, using the Tuscan isolate HyQ-Tu1, the type/pathotype strains of the seven pathovars of X.hortorum, proposed by Morinière et al. (2020), the four X.hydrangeae strains characterized by Dia et al. (2021) and the type strain of X.populi as the outgroup. The analysis indicated that HyQ-Tu1 isolate clusters within the X. hydrangeae branch of the recently described X. hortorum - X. hydrangeae species complex (Morinière et al. 2020; Dia et al. 2021; 2022). In agreement with this result, isolates tested positive to the LAMP assay specific for members of the complex's clade C (X. hydrangeae) (Dia et al. 2022). Based on molecular evidence, the isolates were identified as X. hydrangeae (Dia et al. 2021; Oren and Garrity, 2022). Three healthy H. quercifolia potted plants were inoculated by rubbing a 10 µl droplet of a bacterial suspension of X. hydrangeae HyQ-Tu1 adjusted to an OD600 of 0.3 (approx. 108 CFU/ml) in SDW on the adaxial surface of two leaves per plant. Two control leaves/plant were inoculated with SDW. Each inoculated leaf was enclosed for 24h in a polyethylene bag and all plants were maintained in a greenhouse at 28°C. Nine days post inoculation (DPI), leaf spots similar to those observed on naturally infected plants started to become evident on the bacteria-inoculated leaves while control leaves remained asymptomatic throughout the trial (21 DPI). Koch's postulates were fulfilled by re-isolating the bacterium from the symptomatic tissues, obtaining a positive amplification with the clade C-specific LAMP assay (Dia et al. 2022), and confirming that the gyrB sequence was 100% identical to that of X. hydrangeae HyQ-Tu1. Housekeeping gene sequences were submitted to GenBank (OP456006-9). Members of the X. hortorum - X. hydrangeae species complex have been reported to affect H. quercifolia in the USA (Uddin et al. 1996) and H. quercifolia and H. arborescens L. in Belgium (Cottyn et al. 2021). To the best of our knowledge, this is the first documentation of X. hydrangeae causing disease on H. quercifolia in Italy. Further work is required to verify the presence of the bacterium in other European countries and to assess the economic impact that it causes within and outside nurseries.

Large Protein Assemblies for High-Relaxivity Contrast Agents: The Case of Gadolinium-Labeled Asparaginase.

Biologics are emerging as the most important class of drugs and are used to treat a large variety of pathologies. Most of biologics are proteins administered in large amounts, either by intramuscular injection or by intravenous infusion. Asparaginase is a large tetrameric protein assembly, currently used against acute lymphoblastic leukemia. Here, a gadolinium(III)-DOTA derivative has been conjugated to asparaginase, and its relaxation properties have been investigated to assess its efficiency as a possible theranostic agent. The field-dependent 1H longitudinal relaxation measurements of water solutions of gadolinium(III)-labeled asparaginase indicate a very large increase in the relaxivity of this paramagnetic protein complex with respect to small gadolinium chelates, opening up the possibility of its use as an MRI contrast agent.

Quantitative Real-Time PCR Based on SYBR Green Technology for the Identification of Philaenus italosignus Drosopoulos & Remane (Hemiptera Aphrophoridae).

The use of molecular tools to identify insect pests is a critical issue, especially when rapid and reliable tests are required. We proposed a protocol based on qPCR with SYBR Green technology to identify Philaenus italosignus (Hemiptera, Aphrophoridae). The species is one of the three spittlebugs able to transmit Xylella fastidiosa subsp. pauca ST53 in Italy, together with Philaenus spumarius and Neophilaenus campestris. Although less common than the other two species, its identification is key to verifying which role it can play when locally abundant. The proposed assay shows analytical specificity being inclusive with different populations of the target species and exclusive with non-target taxa, either taxonomically related or not. Moreover, it shows analytical sensibility, repeatability, and reproducibility, resulting in an excellent candidate for an official diagnostic method. The molecular test can discriminate P. italosignus from all non-target species, including the congeneric P. spumarius.

DNA Extraction Methods to Obtain High DNA Quality from Different Plant Tissues.

DNA extraction from plant samples is very important for a good performance of diagnostic molecular assays in phytopathology. The variety of matrices (such as leaves, roots, and twigs) requires a differentiated approach to DNA extraction. Here we describe three categories of matrices: (a) symptomatic bark/wood tissue; (b) residues of frass resulting from insect woody trophic activities, portions of the galleries produced in the wood, and tissues surrounding exit holes; and (c) leaves of different plant species. To improve the performances of diagnostic assays, we here describe DNA extraction procedures that have been optimized for each matrix type.

Loop-Mediated Isothermal Amplification (LAMP) and SYBR Green qPCR for Fast and Reliable Detection of Geosmithia morbida (Kolarik) in Infected Walnut.

Walnut species (Juglans spp.) are multipurpose trees, widely employed in plantation forestry for high-quality timber and nut production, as well as in urban greening as ornamental plants. These species are currently threatened by the thousand cankers disease (TCD) complex, an insect-fungus association which involves the ascomycete Geosmithia morbida (GM) and its vector, the bark beetle Pityophthorus juglandis. While TCD has been studied extensively where it originated in North America, little research has been carried out in Europe, where it was more recently introduced. A key step in research to cope with this new phytosanitary emergency is the development of effective molecular detection tools. In this work, we report two accurate molecular methods for the diagnosis of GM, based on LAMP (real-time and visual) and SYBR Green qPCR, which are complimentary to and integrated with similar recently developed assays. Our protocols detected GM DNA from pure mycelium and from infected woody tissue with high accuracy, sensitivity, and specificity, without cross-reactivity to a large panel of taxonomically related species. The precision and robustness of our tests guarantee high diagnostic standards and could be used to support field diagnostic end-users in TCD monitoring and surveillance campaigns.

Epitope Mapping and Binding Assessment by Solid-State NMR Provide a Way for the Development of Biologics under the Quality by Design Paradigm.

Multispecific biologics are an emerging class of drugs, in which antibodies and/or proteins designed to bind pharmacological targets are covalently linked or expressed as fusion proteins to increase both therapeutic efficacy and safety. Epitope mapping on the target proteins provides key information to improve the affinity and also to monitor the manufacturing process and drug stability. Solid-state NMR has been here used to identify the pattern of the residues of the programmed cell death ligand 1 (PD-L1) ectodomain that are involved in the interaction with a new multispecific biological drug. This is possible because the large size and the intrinsic flexibility of the complexes are not limiting factors for solid-state NMR.

Not only manganese, but fruit component effects dictate the efficiency of fruit juice as an oral magnetic resonance imaging contrast agent.

Several fruit juices are used as oral contrast agents to improve the quality of images in magnetic resonance cholangiopancreatography. They are often preferred to conventional synthetic contrast agents because of their very low cost, natural origin, intrinsic safety, and comparable image qualities. Pineapple and blueberry juices are the most employed in clinical practice due to their higher content of manganese(II) ions. The interest of pharmaceutical companies in these products is testified by the appearance in the market of fruit juice derivatives with improved contrast efficacy. Here, we investigate the origin of the contrast of blueberry juice, analyze the parameters that can effect it, and elucidate the differences with pineapple juice and manganese(II) solutions. It appears that, although manganese(II) is the paramagnetic ion responsible for the contrast, it is the interaction of manganese(II) with other juice components that modulates the efficiency of the juice as a magnetic resonance contrast agent. On these grounds, we conclude that blueberry juice concentrated to the same manganese concentration of pineapple juice would prove a more efficient contrast agent than pineapple juice.

Seed amplification assays for diagnosing synucleinopathies: the issue of influencing factors.

Background: The prion-like misfolding and aggregation of α-synuclein (α-syn) is involved in the pathophysiology of Parkinson's disease and other synucleinopathies. Seed amplification assays (SAAs) are biophysical tools that take advantage on the peculiar properties of prion proteins by amplifying small amounts of aggregates in biological fluids at the expense of recombinant monomeric protein added in solution. SAAs have emerged as the most promising tools for the diagnosis of synucleinopathies in vivo. However, the diagnostic outcome of SAAs depends on the aggregation kinetics of α-syn, which in turn is influenced by several experimental variables. Methods: In our work, we analysed the impact on SAAs of some of the most critical experimental factors by considering models that describe the aggregation kinetics of α-syn. Results: We started our analysis by making simulations to understand which kinetic models could explain the aggregation kinetics of α-syn during incubation/shaking cycles. Subsequently, under shaking/incubation cycles similar to the ones commonly used in SAAs, we tested the influence of some analytical variables such as monomer concentration, presence/absence of glass beads, pH, addition of human cerebrospinal fluid, and use of detergents on α-syn aggregation. Conclusions: Our investigation highlighted how optimization and standardization of experimental procedures for α-syn SAAs is of utmost relevance for the ultimate goal of applying these assays in clinical routine. Although these aspects have been evaluated with specific SAA protocols, most of the experimental variables considered influenced very general aggregation mechanisms of α-syn, thus making most of the results obtained from our analyses extendable to other protocols.

Evaluation of the Higher Order Structure of Biotherapeutics Embedded in Hydrogels for Bioprinting and Drug Release.

Biocompatible hydrogels for tissue regeneration/replacement and drug release with specific architectures can be obtained by three-dimensional bioprinting techniques. The preservation of the higher order structure of the proteins embedded in the hydrogels as drugs or modulators is critical for their biological activity. Solution nuclear magnetic resonance (NMR) experiments are currently used to investigate the higher order structure of biotherapeutics in comparability, similarity, and stability studies. However, the size of pores in the gel, protein-matrix interactions, and the size of the embedded proteins often prevent the use of this methodology. The recent advancements of solid-state NMR allow for the comparison of the higher order structure of the matrix-embedded and free isotopically enriched proteins, allowing for the evaluation of the functionality of the material in several steps of hydrogel development. Moreover, the structural information at atomic detail on the matrix-protein interactions paves the way for a structure-based design of these biomaterials.

The Rapid Identification of Anoplophora chinensis (Coleoptera: Cerambycidae) From Adult, Larval, and Frass Samples Using TaqMan Probe Assay.

A molecular diagnostic method using TaqMan probe qPCR is presented for the identification of Anoplophora chinensis (Förster) (Coleoptera: Cerambycidae) from whole body insects (adults and larvae) and frass samples stored under different conditions. The results showed a perfect amplification of DNA from all samples; the repeatability and reproducibility of the protocol were very good, with standard deviations of inter-run and intra-run variability less than or equal to 0.5. The assay allowed to discern all A. chinensis samples from those of the other non-target wood-borer species, with 100% correspondence to the homologous sequences. No amplification or cross reactions were observed with A. glabripennis (Motschulsky) (Coleoptera: Cerambycidae), which is the most related species among those tested. The protocol was validated by an internal blind panel test which showed a good correspondence between the results obtained by different operators in the same lab. The analytical sensitivity for the lab frass with the Probe qPCR, namely the lowest amount of A. chinensis DNA that can be detected (LoD), was 0.64 pg/µl with a Cq of 34.87. The use of indirect evidence for the identification of a pest is an important feature of the method, which could be crucial to detect the presence of wood-boring insects. This diagnostic tool can help prevent the introduction of A. chinensis into new environments or delimit existing outbreak areas thanks to indirect frass diagnosis.

Rapid diagnostics for Gnomoniopsis smithogilvyi (syn. Gnomoniopsis castaneae) in chestnut nuts: new challenges by using LAMP and real-time PCR methods.

Nuts of the sweet chestnut (Castanea sativa) are a widely appreciated traditional food in Europe. In recent years producers and consumers reported a drop of nut quality due to the presence of rot diseases caused by Gnomoniopsis smithogilvyi. Early detection of this pathogen is fundamental to the economic viability of the chestnut industry. In the present study, we developed three molecular methods based on real-time portable LAMP, visual LAMP and qPCR assays for G. smithogilvyi. The molecular assays were specific for G. smithogilvyi and did not amplify the other 11 Gnomoniopsis species and 11 other fungal species commonly associated with chestnuts. The detection limit of both the qPCR and real-time portable LAMP (P-LAMP) assays was 0.128 pg/µL, while the visual LAMP (V-LAMP) assay enabled the detection up to 0.64 pg/µL. By using these newly developed molecular tools, the pathogen was detected in symptomatic and asymptomatic nuts, but not in leaves. The reliability of these molecular methods, including the P-LAMP assay, was particularly useful in detecting G. smithogilvyi of harvested nuts in field, even in the absence of rot symptoms.

Rapid Detection of Pityophthorus juglandis (Blackman) (Coleoptera, Curculionidae) with the Loop-Mediated Isothermal Amplification (LAMP) Method.

The walnut twig beetle Pityophthorus juglandis is a phloem-boring bark beetle responsible, in association with the ascomycete Geosmithia morbida, for the Thousand Cankers Disease (TCD) of walnut trees. The recent finding of TCD in Europe prompted the development of effective diagnostic protocols for the early detection of members of this insect/fungus complex. Here we report the development of a highly efficient, low-cost, and rapid method for detecting the beetle, or even just its biological traces, from environmental samples: the loop-mediated isothermal amplification (LAMP) assay. The method, designed on the 28S ribosomal RNA gene, showed high specificity and sensitivity, with no cross reactivity to other bark beetles and wood-boring insects. The test was successful even with very small amounts of the target insect's nucleic acid, with limit values of 0.64 pg/µL and 3.2 pg/µL for WTB adults and frass, respectively. A comparison of the method (both in real time and visual) with conventional PCR did not display significant differences in terms of LoD. This LAMP protocol will enable quick, low-cost, and early detection of P. juglandis in areas with new infestations and for phytosanitary inspections at vulnerable sites (e.g., seaports, airports, loading stations, storage facilities, and wood processing companies).

Rapid and Sensitive Detection of Tomato Brown Rugose Fruit Virus in Tomato and Pepper Seeds by Reverse Transcription Loop-Mediated Isothermal Amplification Assays (Real Time and Visual) and Comparison With RT-PCR End-Point and RT-qPCR Methods.

Tomato brown rugose fruit virus (ToBRFV) represents an emerging viral threat to the productivity of tomato and pepper protected cultivation worldwide. This virus has got the status of quarantine organism in the European Union (EU) countries. In particular, tomato and pepper seeds will need to be free of ToBRFV before entering the EU and before coming on the market. Thus, lab tests are needed. Here, we develop and validate a one-step reverse transcription LAMP platform for the detection of ToBRFV in tomato and pepper leaves, by real-time assay [reverse transcription loop-mediated isothermal amplification (RT-LAMP)] and visual screening (visual RT-LAMP). Moreover, these methods can also be applied successfully for ToBRFV detection in tomato and pepper seeds. The diagnostic specificity and sensitivity of both RT-LAMP and visual RT-LAMP are both 100%, with a detection limit of nearly 2.25 fg/µl, showing the same sensitivity as RT-qPCR Sybr Green, but 100 times more sensitive than end-point RT-PCR diagnostic methods. In artificially contaminated seeds, the proposed LAMP assays detected ToBRFV in 100% of contaminated seed lots, for up to 0.025-0.033% contamination rates in tomato and pepper, respectively. Our results demonstrate that the proposed LAMP assays are simple, inexpensive, and sensitive enough for the detection of ToBRFV, especially in seed health testing. Hence, these methods have great potential application in the routine detection of ToBRFV, both in seeds and plants, reducing the risk of epidemics.

TaqMan probe assays on different biological samples for the identification of three ambrosia beetle species, Xylosandrus compactus (Eichoff), X. crassiusculus (Motschulsky) and X. germanus (Blandford) (Coleoptera Curculionidae Scolytinae).

Molecular assays based on qPCR TaqMan Probes were developed to identify three species of the genus Xylosandrus, X. compactus, X. crassiusculus and X. germanus (Coleoptera Curculionidae Scolytinae). These ambrosia beetles are xylophagous species alien to Europe, causing damages to many ornamental and fruiting trees as well as shrubs. DNA extraction was carried out from adults, larvae and biological samples derived from insect damages on infested plants. For X. compactus, segments of galleries in thin infested twigs were cut and processed; in the case of X. crassiusculus, raw frass extruded from exit holes was used, while DNA of X. germanus was extracted from small wood chips removed around insect exit holes. The assays were inclusive for the target species and exclusive for all the non-target species tested. The LoD was 3.2 pg/µL for the frass of X. crassiusculus and 0.016 ng/µL for the woody matrices of the other two species. Both repeatability and reproducibility were estimated on adults and woody samples, showing very low values ranging between 0.00 and 4.11. Thus, the proposed diagnostic assays resulted to be very efficient also on the woody matrices used for DNA extraction, demonstrating the applicability of the protocol in the absence of dead specimens or living stages.

Development of a loop-mediated isothermal amplification (LAMP) assay for the identification of the invasive wood borer Aromia bungii (Coleoptera: Cerambycidae) from frass.

The red-necked longhorn beetle Aromia bungii (Faldermann, 1835) (Coleoptera: Cerambycidae) is native to east Asia, where it is a major pest of cultivated and ornamental species of the genus Prunus. Morphological or molecular discrimination of adults or larval specimens is required to identify this invasive wood borer. However, recovering larval stages of the pest from trunks and branches causes extensive damage to plants and is timewasting. An alternative approach consists in applying non-invasive molecular diagnostic tools to biological traces (i.e., fecal pellets, frass). In this way, infestations in host plants can be detected without destructive methods. This paper presents a protocol based on both real-time and visual loop-mediated isothermal amplification (LAMP), using DNA of A. bungii extracted from fecal particles in larval frass. Laboratory validations demonstrated the robustness of the protocols adopted and their reliability was confirmed performing an inter-lab blind panel. The LAMP assay and the qPCR SYBR Green method using the F3/B3 LAMP external primers were equally sensitive, and both were more sensitive than the conventional PCR (sensitivity > 103 to the same starting matrix). The visual LAMP protocol, due to the relatively easy performance of the method, could be a useful tool to apply in rapid monitoring of A. bungii and in the management of its outbreaks.