Isolation And Identification Of Edwardsiella In South Catfish And Establishment Of The Duplex PCR For Detection Of Edwardsiella

Edwardsiella tarda (E. tarda) and Edwardsiella ictaluri (E. ictaluri) from sick South catfish were isolated and identified in order to protecting the two diseses. A duplex PCR assay for detecting E.tarda and E.ictaluri was established aim to fast diagnosis.Aseptic techniques were employed to isolate the dominant bacteria from liver and kindey of the diseased South catfish which collected from Leshan southern catfish farms, two dominant bacterial strains named CH0406 and H0701 were isolated from the diseased fish;The isolation bacterial was confirmed by a series of artificial infection, physics and chemistry characteristics test,the result show that they were proved to be the pathogen of the disease by artificial infection test, CH0406 colonies were circular, smooth-surface, whiteand 0.5-1mm sized after incubation at 28℃ for 24h on LB. H0701 colonies were colorless, circular, smooth-surface, regular and pin-head sized after incubation at 28℃ for 24h on BHI. The CH0406 biochemical characteristics consistent with E.tarda, and the H0701 biochemical characteristics consistent with E.ictaluri.Used primers (F:5’-AGAGTTTGATCCTGGCTCAG-3’, R:5’-TACGGCTACCTTGTTACGAC-3’)designed according to conesrved sequences in bacterial 16S rDNA,amplified the fragments with 1500bp. The 16S rDNA gene sequencing analysis by BALST in GenBank indicated that both two isolates showed high levels of similarity to E.tarda and E.ictaluri (more than 99%), In the phylogenetic tree the CH0406 isolates and other E.tarda strains constituted a branch,the H0701 isolates and other E. ictaluri strains constituted a branch. Two sets of specific primers (gadB:F:5’-ATTTGGATTCCCGCTTTGGTTCA-3’, R:5’-GCAC GACGCCGATGGTGTTCTC-3’;eip:F:5’-TCATCACATCTCTAGCGATT-3’,R:5’-C TTTACACAGATAGGCGATAC-3’)were designed according to the published gadB gene sequences of E.tarda and eip gene sequences of E. ictaluri in the GenBank, both two isolates were positive in a specific PCR detection of E.tarda based on gadB gene and E. ictaluri based on eip gene.Based on the above results, CH0406 was identified as E.tarda, H0701 was identified as E.ictaluri.A duplex PCR methods were established for detecting the mixed samples which contained E.tarda and E.ictaluri.Optimize the reaction conditions, the simultaneous and sensitivity. After optimize the duplex PCR reaction system:10×PCR Buffer 2.5μL,10 mmol/L dNTPs 1.0μL, Taq DNA polymerase0.5μL, Mg2+ 1-2.5mmol/L, E.tarda primers and E.ictaluri primers 0.7μmol/L, DNA 2.0 ng/μL,add ddH2O to 25uL; Reaction conditions:94℃ 5min,94℃ 1min,58℃ 1min,72℃ 1min,30 cycles,72℃ 5min; The duplex PCR methods amplified the special fragments with 582bp (E.tarda) and 276bp (E.ictaluri) which consistent with the PCR results. Specific experiments show the control strains did not amplify any fragments. The lowest bacterial content that can be detected was 1×102cfu/mL of E.tarda and 1×102cfu/mL of E. ictaluri.Above all, the duplex PCR method established in the study was a specific, high-efficient, sensitive, faster method for detecting E.tarda and E.ictaluri.It can be used to detect the clinical tissue.