致病性嗜水气单胞菌多重PCR检测方法的建立

Development of multiplex polymerase chain reaction for detection of pathogenic aeromonas hydrophila and its preliminary application

致病性嗜水气单胞菌(Aeromonas hydrophila)是近年中国各地大规模流行的淡水养殖鱼类暴发性疾病的主要病原,本研究针对GenBank中登录的致病性嗜水气单胞菌的气溶素基因(hlyA)、溶血素基因(aerA)以及为气单胞菌属所特有的内参照基因16S rRNA保守区设计了3对特异性引物,通过进行多重PCR反应体系优化,多重PCR产物的测序鉴定与特异性和敏感性实验,试图建立一种检测致病性嗜水气单胞菌的多重PCR检测方法。对8株嗜水气单胞菌、16株相关菌株进行多重PCR检测,结果显示,非致病性分离株均未扩增出毒力基因hlyA和aerA,而致病性分离株则至少含有hlyA基因;对40份送检的水产动物样品进行多重PCR检测,结果与常规微生物学检测符合率为97.5%。多重PCR检测方法具有较高的敏感性与特异性,最低可检测模板量为10 ng的样品。该方法的建立对水产动物嗜水气单胞菌病的快速诊断和分子流行病学的调查有重要意义。

Aeromonas hydrophila is gram-negative bacteria of the family Aermonadaceae that are often found in association with hemorrhagic septicemia in cold-blooded animals including fish, reptiles and amphibians. At present,a number of virulence factors derived from A. hydrophila have been proposed in an effort to explain the pathogenesis of infections due to these organisms. Exotoxin, protease and outer-membrane protein have been described in A. hydrophila and aerolysin（aerA ）, hemolysin（hlyA ）,hemolytic toxin and cytolytic enterotoxin are all exotoxins. Some studies suggested that all virulent A. hydrophila isolates carried both hlyA and aerA genes,so aerA and hlyA genes were considered as primary virulence genes of A. hydrophila. The 16s rRNA gene belonging to Aeromonas characteristically acted as an internal control gene. Three pairs of specific primers were designed and multiplex PCR assay was developed to amplify the aerA, hlyA genes and 16s rRNA gene. The reaction conditions of the multiplex PCR were optimized and PCR products were sequenced. Specificity and sensitivity of multiplex PCR were studied. Eight A. hydrophila strains were isolated and the other sixteen strains were tested by multiplex PCR. The results showed that the virulence genes of hlyA and aerA could not be amplified from nonpathogeneti A. hydrophila strains, but the pathogenetic ones contained the genes of hlyA at least. Forty aquatic animal specimen were tested by multiplex PCR and conventional microbiokgy methods and their coherence was 97.5 %. It can be concluded that the multiplex PCR is specific and sensitive and can be used in quick diagnose and epidemiokgy investigation of A. hydrophila. [Journal of Fishery Sciences of China,2007,14（5） ：749 - 755]