A peptide nucleic acid probe-based multiplex qPCR assay for rapid and accurate detection and quantification of fish-pathogenic Edwardsiella species.

Edwardsiellosis causes severe economic losses in the aquaculture industry worldwide. Accurate identification of the fish pathogenic Edwardsiella species is crucial for preventing disease spread and providing effective treatment strategies in the aquaculture industry. These species have high phenotypic and genetic similarities (> 99% in 16S rRNA sequences), which makes their differentiation challenging. In this study, we developed a multiplex real-time PCR diagnostic assay based on the single-copy DNA gyrase subunit B (gyr B) gene using a peptide nucleic acid (PNA) probe to discriminate among four fish pathogenic Edwardsiella species, E. tarda, E. piscicida, E. anguillarum, and E. ictaluri. Through phylogenetic analysis based on gyrB sequences (1462 bp), 57 Korean Edwardsiella isolates were affiliated to E. piscicida, E. anguillarum, and E. tarda , accounting for host and geographical origin specificity. The assay targeted a conserved region in gyr B, and designed four probes, each capable of distinguishing among the four Edwardsiella species, even in cases of 1–3 nucleotide mismatch, based on differences in melting temperature. The assay exhibited high specificity and sensitivity, with reaction efficiency in the range 85–100% and a reliable quantifiable limit of 102 copies per reaction for all fluorescence signals. In addition, it could detect and quantify the four species, even in mixed infections, allowing accurate identification within 2 h in a single reaction. This PNA probe-based multiplex qPCR assay could be a practical and reliable diagnostic tool in the field and provide correct taxonomic assignment for further research on the widened host range and niche, pathogenicity, and susceptibility of each Edwardsiella species, to help establish appropriate treatment and vaccine strategies. • A PNA probe-based multiplex qPCR assay developed in our study allows to rapid and accurate identification and quantification of four fish-pathogenic Edwardsiella species, E. tarda , E. anguillarum , E. piscicida , and E. ictaluri. • This assay exhibits high specificity and sensitivity (E = 85–100%, R2 > 0.99, and quantifiable limit = 102 copies/reaction) with highly repeatable and reproducible (CV < 2.0). • Perfectly hybridized PNA probe enables specific discrimination among the four Edwardsiella species even in 1–3 nucleotide variation and in a mixed infections by their melting temperature (T m). [ABSTRACT FROM AUTHOR]