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.

Aleurocanthus spiniferus (Quaintance) (Hemiptera: Aleyrodidae) has rapidly spread, mainly in the central and eastern Mediterranean coastal area, infesting various new host plants alongside known ones. This invasive species poses a significant threat to agricultural ecosystems, necessitating urgent action to monitor and control outbreaks in previously pest-free areas. While entomological and morphological recognitions are crucial for initial detection, challenges often arise in quickly identifying different developmental stages or genus-level distinctions, particularly in surveys conducted by personnel with limited entomological skills. Due to these challenges, a qPCR probe protocol was developed to enhance the diagnostic capacity of laboratories responsible for the territorial control of pests. This biomolecular tool integrates morphological surveys, enabling prompt and reliable proof of A. spiniferus presence in free areas, delimited territories, or during phytosanitary import inspections. The protocol’s high analytical specificity, inclusivity, and exclusivity ensure accurate identification of A. spiniferus, while its low limit of detection and high repeatability and reproducibility reinforce its utility as a standardized diagnostic method. By facilitating prompt and targeted control efforts, this innovative approach strengthens the resilience of agricultural systems against the widespread threat of A. spiniferus infestations.

This study presents a qPCR-based molecular diagnostic method utilizing a TaqMan probe to ac-curately identify Agrilus auroguttatus Schäffer (Coleoptera: Buprestidae) from adult insects, ex-creta samples, and frass residues. The method demonstrated excellent DNA amplification across all sample types, highlighting strong analytical specificity. The protocol exhibited high repeata-bility and reproducibility, with inter-run and intra-run variability standard deviations consist-ently at or below 0.5. This assay distinguished A. auroguttatus samples from non-target species with 100% sequence correspondence. Analytical sensitivity (LoD) for adult and frass samples was determined at 8 pg/µL and at a 1:5 dilution from an initial concentration of 5 ng/µL, yield-ing mean Cq values of 35.14 ± 0.71 and 37.07 ± 0.59, respectively. The indirect diagnostic capabil-ity of this method is particularly valuable for detecting the presence of wood-boring insects, po-tentially aiding in the early detection of A. auroguttatus in new environments or in managing cur-rent outbreak areas. This diagnostic tool could thus play a critical role in biosecurity efforts to limit the spread of this invasive species.

Ips typographus is one of the most destructive bark beetles affecting conifer forests in Europe, where climatic disturbances and the movement of infested wood can rapidly shift populations from endemic levels to severe outbreaks. Early detection through border inspections and forest monitoring is essential to prevent new introductions and limit the spread of established populations. Here, we developed and validated a probe-based TaqMan qPCR assay, targeting the mitochondrial COI barcode region, for the rapid and species-specific detection of I. typographus from both insect material and environmental DNA recovered from frass and exit-hole wood chips. Validation followed EPPO PM7/98(5) guidelines, assessing analytical specificity, sensitivity, repeatability, reproducibility, and inter-laboratory transferability. High analytical specificity was demonstrated against a broad panel of non-target species, and reliable amplification was obtained across different tested matrices. The method showed strong analytical sensitivity, with limits of detection of 0.32 pg/µL for adult-derived DNA and 1.6 pg/µL for artificial frass. Repeatability, reproducibility, and inter-laboratory blind testing further confirmed the diagnostic reliability of the method. This validated qPCR assay provides a rapid and sensitive molecular tool for the early detection of I. typographus, supporting border inspection and phytosanitary diagnostic laboratories in forest biosecurity activities.