Development and validation of a triplex qPCR assay for simultaneous detection of Xylella fastidiosa in plant matrices and insect vectors

Xylella fastidiosa (Xf) is a plant quarantine bacterium that threatens Mediterranean agricultureand natural ecosystems. The possible presence of Xf variants may undermine the ability of monoplex PCRs assays to correctly detect the presence of this bacterium in the matrices analyzed. This study presents a triplex qPCR assay for simultaneous detection of Xf in plant and insect matrices. A triplex qPCR targeting three genomic loci (cysK, gyrB, and 16S rRNA genes using FAM, HEX, and CY5 labels, respectively) was developed and validated according to EPPO PM7/98(5) and ISO 16140-2 guidelines. The assay was tested on 77 Xf strains (belonging to five subspecies), 197 non-target bacteria, and naturally Xf subsp. multiplex ST87 infected samples: 38 plant samples (Spartium junceum, Rhamnus alaternus, Prunus dulcis, Lavandula angustifolia) and 23 insect vectors (Philaenus spumarius, Neophilaenus campestris). The assay demonstrated 100% analytical specificity with no cross-reactivity. Analytical sensitivity (LOD) was 1.28 × 10⁻⁵ ng/µL for all targets; the 16S rRNA gene showed superior sensitivity with Cq values 2–3 cycles lower respect to cysK and gyrB. Amplification efficiencies were 93–102%, with excellent linearity (R² > 0.996) and precision (SD ≤ 0.3 Cq). On naturally infected samples, the assay detected Xf in all 38 plant samples and in 21 out of 23 insect vectors (91.3%), achieving 100% diagnostic concordance with reference assays. The triplex format provides critical advantages: multilocus targeting reduces false-negative risk, suitability for both plant and insect matrices enable integrated surveillance, and a single reaction replaces three monoplex assays, reducing time and costs. This robust, versatile protocol is particularly suitable for large-scale surveillance and routine diagnostics by phytosanitary services.