Establishment of a Multiplex Fluorescence Quantitative T aq M an PCR Approach for the Detection of Aeromonas veronii , Aeromonas sobria and Edwardsiella ictaluri

ABSTRACT Pelteobagrus fulvidraco , which is popular with consumers because of its fine meat and appreciated taste, occupies an important position in freshwater fish in China. In recent years, P. fulvidraco has been seriously infected by Aeromonas veronii , Aeromonas sobria and Edwardsiella ictaluri , which has caused great economic losses. However, the current detection methods for these three pathogens are difficult to meet the needs of disease prevention and control. To address this challenge, this study established a TaqMan multiplex real‐time fluorescence quantitative PCR method capable of simultaneously detecting three pathogens based on the conserved genes gyrB of A. veronii , A. sobria and E. ictaluri . This method has strong specificity and no cross reactivity between different pathogens. In addition, this method has high sensitivity, with detection limits of 10 1 , 10 0 and 10 0 copies/μL for A. veronii , A. sobria and E. ictaluri , respectively. Using this method to detect 100 clinical specimens collected in 2023–2024, the detection rates of A. veronii , A. sobria and E. ictaluri were higher than those of conventional PCR. In conclusion, the TaqMan multiplex qPCR method developed in this study provides a new and powerful tool for early diagnosis, prevention, control and purification of A. veronii , A. sobria and E. ictaluri . This study is of great significance in improving the efficiency of P. fulvidraco culture and ensuring the healthy development of the aquaculture.