Virulence genes and dulplex PCR and the LAMP methods for the detection of pathogenic Edwardsiella tarda

Edwardsiella tarda is widely distributed in aquatic environments and can be pathogenic to a broad range of hosts.It is currently one of the most important fish pathogens that affect the aquaculture industries worldwide.Though the pathogenesis of E.tarda is poorly understood for the present,previous studies have shown that several factors may contribute to the virulent mechanisms of E.tarda.The adhesive properties such as fimbrial protein precursor A(fimA)and fimbrial protein precursor B(fimB),glutamate decarboxylase isozyme(gadB),citrate lyase ligase(citC)were observed to be the virulence genes that correlated with the mortality of infected fish.In this study,we investigated the prevalence distribution of the four virulence-associated genes and that of E.tarda isolated from flounder(Paralichthys olivaceus)and turbot(Scophthalmus maximus),and established the methods of dulplex PCR and LAMP that will detect E.tarda.Four pairs of primers were designed according to the published nucleotide sequence of virulence genes(fimA,fimB,gadB and citC)for screening the virulence genes of pathogenic E.tarda,and the methods of dulplex PCR and LAMP that will detect E.tarda were established using fimA and gadB genes as molecular marker.The results showed fimA,fimB,gadB and citC genes were detected simultaneously in 10 pathogenic strains of pathogenic E.tarda,and 240,217,171 and 119 bp gene fragments from chromosomal DNA of E.tarda could be amplified,and no positive reaction was detected in 4 other control strains;the PCR primers designed by fimA and gadB genes could detect E.tarda at a level of as low as 3.0×103 CFU/mL using dulplex PCR method;the LAMP primers designed by fimA gene could detect E.tarda at a level of as low as 30 CFU/mL within 60 min under isothermal condition at 65 ℃ using the LAMP detection system.The green amplified products were observed directly by naked-eye in the reaction tube by addition of SYBR Green Ⅰ,and negative reaction(no amplified bands and with orange color)was detected in 4 kinds of control pathogenic bacteria,including V.anguillarum,V.harveyi,V.damsela and Aeromonas salmonicida.These methods could be used as the rapid diagnosis of the disease caused by E.tarda in aquaculture.