6株鱼源嗜水气单胞菌的分子鉴定与毒力基因分析检测

Molecular characterization of six strains of Aeromonas hydrophila isolated from fish as well as detection and analysis of virulence genes

为了分子鉴定6株鱼源嗜水气单胞菌,并从分子层面验证通过检测毒力基因以推测嗜水气单胞菌潜在致病性的可行性。实验采用PCR扩增16S rDNA和gyrB基因并结合系统发育树的构建和分析进行菌种的分子鉴定,检测气溶素(aerolysin,aer)、溶血素(haemolysin,hly)、丝氨酸蛋白酶(serine protease,ahp)、热稳定细胞肠毒素(heat-stable cytotonic enterotoxin,ast)和热敏感细胞肠毒素(heat-labile cytotonic enterotoxin,alt)5种毒力基因,且使用Mega 5.2对核苷酸和氨基酸序列进行分析。结果显示6株菌均为嗜水气单胞菌Aeromonas hydrophila,检测出5种毒力基因中的至少4种,其中均检测出溶血素和2种肠毒素,序列分析表明气溶素、溶血素和丝氨酸蛋白酶的氨基酸序列高度保守。本研究基于16S rDNA和gyrB基因可以准确地对嗜水气单胞菌进行分子鉴定,6株菌的毒力基因丰富预示着一定的致病性,aer、hly和ahp基因相对保守,编码的毒力因子高度同源,在临床分子诊断中建议使用aer、ahp和hly基因对嗜水气单胞菌的潜在致病性进行检测。

Six strains of Aeromonas hydrophila isolated from dying fish were molecularly characterized as well as five virulence genes were detected and analyzed in order to evaluate the feasibility of detecting the virulence genes to infer the potential pathogenicity of A. hydrophila at the molecular lever. 16S rDNA and gyrB gene were used to characterize A. hydrophila,five virulence genes including aerolysin（ aer） ,haemolysin（ hly） ,serine protease（ahp）,heat-stable cytotonic enterotoxin（ast） and heat-labile cytotonic enterotoxin（alt） were de-tected by PCR,and the sequences of gene and virulence were analyzed using Mega 5. 2. All of the strains were characterized as A. hydrophila,and four of five virulence genes at least were detected as positive,including the genes of haemolysin and two kinds of cytotonic enterotoxin. By the sequences analysis, we found the amino acid sequences of high similarity among aerolysin,haemolysin and serine protease. A. hydrophila could be well characterized using 16S rDNA and gyrB gene sequences,and the varied virulence genes indicated the potential pathogenicity. Through the sequences analysis,the genes of aer,hly and ahp were relatively conservative,and the encoding virulences were highly conserved. Detecting the virulence genes of aer,hly and ahp could infer the potential pathogenicity of A. hydrophila in the clinical molecular diagnosis.