Zoological Studies

Vol. 37 No. 3, 1998

PCR for Direct Detection of Edwardsiella tarda from Infected Fish and Environmental Water by Application of the Hemolysin Gene

Jau-Der Chen* and Shau-Yan Lai

Department of Aquaculture, National Taiwan Ocean University, Keelung, Taiwan 202

Jau-Der Chen and Shau-Yan Lai (1998) A DNA fragment associated with hemolysin production in the Edwardsiella tarda strain ET16 was labeled with nonradioactive DIG and used to probe chromosomal DNA from 40 E. tarda strains. The resulting hybridization patterns were classified into 2 groups, matching the group of strains which secreted or didn't secrete hemolysin, except for anomalous patterns produced by strain ET83 and reference strain ATCC 15947. Those strains yielding a 5.3-kb hybridization band, which corresponds to the original HindIII cloned fragment from the chromosome of ET16, were assigned to group I, while the presence of other hybridized HindIII bands from strains producing endo-hemolysin were characteristic of group II. When 2 oligomers selected from the beginning region of ORF II and the end region of ORF III were used as primers for PCR assay, a 1109-bp PCR product was generated by all 40 examined E. tarda strains. Thus, the hemolysin gene sequence spanning the region of ORF II and ORF III was concluded to be conservative. Live bacterial cells from the visceral organs and blood of infected fish, as well as bacteria in environmental water were detected by a direct PCR assay which yielded the 1109-bp fragment.

Key words: Edwardsiellosis, PCR diagnosis.

*Correspondence: Tel: 886-2-24622192 ext. 5239. Fax: 886-2-24628918. E-mail: jdchen@ntou66.ntou.edu.tw