The Rapid Identification Technology Research Of Aeromonas In Aquaculture Pond

Members of the genus Aeromonas are gram-negative bacteria,belonging to Gammaproteobacteria、 Aeromonadales、Aeromonadaceae、Aeromonas. Aeromonas bacteria is a kind of important pathogenic bacteria in fresh water aquaculture, which can cause a variety of aquatic animal disease. Aquatic animals infected by Aeromonas show different symptoms such as bleeding, ulcers and soft shells. The study explored the rapid identification methods of Aeromonas and analysis the ecological distribution of Aeromonas in fresh water aquaculture.The main results are as follows:1. The rapid identification methods of Aeromonas are established.The Aeromonas bacterias were selected by ADA(Ampicillin- dextrin Agar) selective medium from henan province aquaculture water and sediment.The bacteria were preliminarily screened by oxidase and the fermentation of glucose oxidation test. Then the suspected Aeromonas were further screened by aeromonas specific primers(gyrB specific gene fragment) and there are 267 Aeromonas strains fianlly.The separation strains of Aeromonas can be divided into different groups according to stripe by 16SrRNA-RFLP method, then the representative strains of different groups were to submitted to sequence analysis of 16 SrRNA gene and the proportion of different aeromonas specieses of the sample were analyzed.There are 7 kinds of aeromonas species in the sample including A.veronii、A.hydrophila、A.sobria、A.caviae、A.salmonicida、A.media and A.trota.The majority of aeromonas separated from sample were A.veronii,even more than 90 %.2. Multiple polymerase chain reaction(PCR) were established to detect Aerononas strains 、A.hydrophila and vibrio strains.The primers of aeromonas specific gene(gyrB)、A.hydrophila specific gene(16SrRNA) and vibrio specific gene(Vlb) were used in the study.The results show that Aeromonas and Vibrios can be distinguished by Aeromonas gyrB specific primers and vibrios specific Vlb primers.In addition,A.hydrophila can be selected specifically by A.hydrophila specific primers.The detection limitation of A.hydrophila DNA 、 Aeromonas and vibrio were 5.6 ╳ 10-3 ng/μL 、 9.6×10-3 ng/μL and 0.56 ng/μL,respectively.3. The A.veronii separated were typed and their virulence genes were detected.77 strains of A.veronii were typed by ERIC-PCR.For the result,2~4 brands could be amplified between 1000~4000 bp,typing strains into 3 groups and 6 classes according to the clustering figure.In addition,6 virulence genes were detected by PCR,the results show that their are few virulence genes in aeromonas separated from environment.There were no Aer、Aha and Hem genes were detected.33.3 % of omp gene、89 % ahp and 2.7 % of Alt were detected among A.veronii strains.