PCR cloning and identification of the β-haemolysin gene of Aeromonas hydrophila from freshwater fishes in China

Abstract

In this study, β-haemolysin gene (AHTPS30HEM) of Aeromonas hydrophila was cloned from diseased fish in mainland China. AHTPS30HEM gene ( AB021152) resulted in a 1589 bp fragment which covers the open reading frame (ORF) in region 5–1486 coding for 493 amino acids. Multiple alignment of AHTPS30HEM with other β-haemolysin amino acid sequences showed 18 amino acid substitutions between AHTPS30HEM and A. hydrophila β-haemolysin. Although the ORF sizes between AHTPS30HEM and Aeromonas species are different, four cysteins and four potential N-glycosylation sites were conserved. To identify the β-haemolysin-producing virulent or pathogenic A. hydrophila, a specific PCR to amplify 208 bp target DNA of β-haemolysin gene was established. Twenty strains containing pathogenic A. hydrophila, Aeromonas sobria, Vibrio anguillarum, Cytophaga columnaris, Pseudomonas fluorescens, and Yersinia yuckeri were investigated by PCR. Based on the cloned β-haemolysin sequences, the specific PCR method for identification of the β-haemolysin gene of A. hydrophila was established, and surveyed on those samples. The results indicated that β-haemolysin-specific PCR might be useful in the detection of pathogenic A. hydrophila.