粘附素(aha1)、气溶素(aerA)和细胞兴奋性肠毒素(alt)是气单胞菌的主要毒力因子.根据aha1、aerA和alt基因序列设计三对引物建立了可同时检测三种毒力基因的多重PCR方法(MPCR).该方法扩增出气单胞菌的aha1大小为1087 bp,aerA 为721 bp,alt为480 bp,其敏感度为102 CFU?mL-1.而对金黄色葡萄球菌、恶臭假单胞菌、拟态弧菌以及非致病性气单胞菌均未扩增出任何条带.用限制性内切酶EcoRⅤ、BamHⅠ和FbaⅠ分别酶切PCR扩增产物,均获得与预期一致的酶切图谱.用建立的MPCR对15株水生动物源气单胞菌安徽分离株进行毒力基因检测,结果显示在13株致病性气单胞菌中10株细菌的毒力基因型为alt aha1 aerA ,2株为alt aha1-aerA-,1株为alt aha1 aerA-;alt、aha1和aerA基因在气单胞菌中的携带率分别为100%、84.62%和76.92%.表明气单胞菌安徽分离株的主要毒力基因型是alt aha1 aerA 的高毒力表型,alt毒力基因普遍存在于不同表型种气单胞菌中.

Adhesion gene （ahal）, aerolysin gene （aerA） and cytotonic enterotoxin gene （alt） were considered as major virulence genes of Aeromonas. According to ahal, aerA and alt gene sequences, a multiplex polymerase chain reaction （MPCR） was developed to detect the above virulence genes simultaneously. The ahal, aerA and adt genes were 1087 bp, 721 bp and 480 bp PCR products respectively. Digested with EcoRV, BamnHI and Fbal, the sizes of digestive fragments of each PCR product were consistent with expectation. Using the same pairs, no PCR product was detected from S. aureus, P. putide, V. minicus and unpathogenic Aeromonads. The sensitivity of the MPCR was 100 CFU?mL^-1 of the bacteria. At the same time, multiplex PCR analysis was performed on 15 strains Aeromonas spp. isolated from different aquatic animals in Anhui and the results showed there were 10 strains with virulence genotype alt^＋ ahal^＋ aerA^＋ , 2 strains with virulence genotype alt^＋ ahal^- aerA^- and 1 strain with virulence genotype alt^＋ ahal^＋ aerA^- within 13 strains pathogenic Aeromonas spp. The positive rates of alt, ahal and aerA genes were 100%, 84. 62% and 76.92% respectively. These results indicate that the major virulence genotype of Anhui isolation strains is the high virulence phenotype of alt^＋ ahal^＋ aerA^＋ and the virulence gene alt generally exists in different Aeromonas phenospecies.

安徽省自然科学基金，安徽省高校拔尖人才基金