A real-time PCR targeted to the upstream regions of HlyB for specific detection of Edwardsiella tarda

摘要

Edwardsiella tarda has become one of the most important emerging pathogens in aquaculture industry.Therefore,a rapid,reproducible,and sensitive method for detection and quantification of this pathogen is needed urgently.To achieve this purpose,we developed a TaqMan-based real-time PCR assay for detection and quantification of E.larda.The assay targets the hemolysin activator HlyB domain protein of E.tarda.Our optimized TaqMan assay is capable of detecting as little as 40 fg of genomic DNA per reaction.A standard curve was generated from the threshold cycle values(y)against log10(E.tarda genomic DNA concentration)as x.The intra-and inter-assay coefficient of variation(CV)values were less than 2.06％ and 1.05％ respectively,indicating that the assay had good reproducibility.This method is highly specific to E.tarda strains,as it shows no cross-reactivity to Edwardsiella ictaluri,a member of the same genus,or to nine other fish-pathogenic bacteria species belonging to three other genera.This sensitive and specific real-time PCR assay provides a valuable tool for diagnostic quantitation of E.tarda in clinical samples.