So far studies have focused on bacteria isolation aimed at establishing causes of fish diseases and medication methods.Now,more and more attention has been given to the composition of the microflora,its variations in time,and effect on whiteleg shrimp(Litopenaeus vannamei).This is why the problem of bacterial flora in water ought to be studied.The major role of ammonifying bacteria is to break down nitrogenous organic matter to ammoniac nitrogen.The research of ammonifers in the pond water of Litopenaeus vannamei is increasing with the demand for environment friendly aquaculture.Four bacterial strains(No.zjs01,zjs02,zjs03 and zjs04),isolated from the pond water of Litopenaeus vannamei in Jinshan district of Shanghai,were cultured in ammonifying bacteria rich medium and identified by two methods.One is the sequence analysis of 16S rDNA,the other is bacteria identification system.First,sequence analysis of the 16S rDNA was done.Genomic DNA of four strains was isolated respectively,then their full length of the 16S rDNA were amplified by PCR respectively,using universal primers to the 16S rDNA.After purification by gel extraction,the PCR products were cloned and subsequently sequenced by Shanghai Invitrogen Biotechnology Company(SIBC).The phylogenetic trees were constructed,based on the result of online alignment.At the same time,the four sequences of 16S rDNA were submitted to NCBI(http://www.ncbi.nlm.nih.gov) in order to obtain accession number of strains of zjs01,zjs02,zjs03 and zjs04.Then the API 2000 Bacteria Identification System(Biomerieux Company) was applied in assessment the identification result of zjs01,zjs02,zjs03 and zjs04 by molecular method.Finally the 4 strains were identified as,zjs01: Brevundimonas diminuta,zjs02 and zjs03: Alcaligenes faecalis,zjs04: Enterobacter aerogenes.And the accession number of the strains(zjs01,zjs02,zjs03 and zjs04) is DQ857897,DQ857898, DQ857895,DQ857896 respectively.The method of detecting bacterial 16S rDNA using PCR technique is specific,sensitive,rapid and accurate in detecting bacterium in culture pond.Also an interesting thing should be indicated,it is absolutely necessary that the 16S rDNA PCR products should be cloned before DNA sequencing.This paper will establish theory groundwork for application of ammonifers microbiological preparation to bioremediation of the polluted culture water.
