暗纹东方鲀非01霍乱弧菌的鉴定及毒力基因检测

在对养殖暗纹东方病害调查中，从呈典型症状病鱼腹腔和肠道粘液中分离到2株（J-1和H-3）菌株，经生化特性和血清型鉴定为非01群霍乱弧菌(Non-01 Vibrio cholerae)。注射、创伤和浸泡3种方式进行人工感染试验表现出较强的致病性，出现症状与自然发病鱼相同。为进一步确认分离菌株的分类地位，使用P1、P2引物，以非01群霍乱弧菌N92001菌株和01群霍乱弧菌N16961菌株为对照，对分离菌株进行PCR扩增，得到451bp的DNA片段。根据现有霍乱弧菌肠毒素ctxA基因序列设计引物P3,P4，以N16961菌株为阳性对照，PCR扩增结果均得到大小为400 bp的DNA片段。在对H-3菌株的扩增片段进行纯化、克隆和测序，得到407bp序列，该菌株与N16961菌株中肠毒素ctxA基因序列的完全一致，证实该片段源于ctxA基因。进一步说明从暗纹东方体内分离到的J-1和H-3菌株具有霍乱肠毒素ctxA，有一定的致病力。

Separation and determination on virulence genes of Vibrio cholerae in Fugu obscurus

During the survey on diseases of Fugu obscurus from 2002 to 2004,two strains(J-1 and H-3) of pathogenic bacteria with typical symptoms were isolated from abdominal cavity and intestinal mucus of suffering Fugu obscurus.The two isolated strains were identified as non-01strains of Vibrio cholerae through biochemical test and serotype identification.It could be inferred that they are pathogens of bacterial enteritis of Fugu obscurus since the results of artificial infection are the same as the natural infection.In order to testify the classification of the isolated strains,the isolated strains were amplified by polymerase chain reaction(PCR) with two primers designed according to the sequence of Vibrio.The PCR products were DNA sequence of 451 bp.The result showed that systematic status between J-1,H-3 and N92001,N16961 was close.Enterotoxin with active subunit A named ctxA is one of the primary infection agents of Vibrio cholerae.While the pathogenicity is related with ctxA,the test of gene of enterotoxin is the most important step of diagnosis.Using N16961 strains as positive control,the same PCR DNA sequence of 400 bp was obtained in the PCR analysis.By sequencing and alignment,this 407 bp sequence of H-3 strains is identical with ctxA,which indicated that J-1 strains and H-3 strains were the pathogenic bacteria of F.obscurus.Bacterial diseases are common and frequent in process of aquaculture especially the intensive aquiculture.This paper shows introduces how to combine common method and PCR to isolate,identify and study bacteria. The pollution of non-01strains of V cholerae in aquatic product reflects in some way the degree of environmental pollution.Thus the study of pathogenicity is meaningful to evaluate if bacteria would cause disease and to limit its prevalence,so as to study and control the zoonosis.