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

Abstract

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.

A simple PCR-based quick detection of the economically important oriental fruit fly, Bactrocera dorsalis (Hendel) from India

“…Numerous contemporary methodologies have emerged for fruit fly identification, encompassing diverse assays such as real-time PCR/qualitative PCR ( Koohkanzade et al., 2018 ), multiplex PCR ( Chen et al., 2016 ), loop-mediated isothermal amplification (LAMP) ( Blacket et al., 2020 ), restriction fragment length polymorphism (RFLP) ( Barr et al., 2006 ), simple-sequence repeat (SSR) markers ( Ding et al., 2018 ), and conventional PCR ( Jiang et al., 2013 ), each offering unique applications and advantages. Notably, Rizzo et al. (2024) recently devised a qPCR assay targeting species-specific primers derived from mtCOI gene sequences to discriminate B. dorsalis , catering specifically to the prevailing pest dynamics in Europe.…”