Multiplex PCR Assay For The Detection Of Aeromonas Hydrophila And Edwardsiella Tarda

Aeromonas hydrophila and Edwardsiella tarda are two important pathogenic bacteria in many aquatics and involved in causing gastrointestinal illnesses.In humans, they cause acute gastroenteritis, septicaemia, wound infections, otitis media and peritonitis. Therefore, the detection of the presence of A. hydrophila and E. tarda have greater clinical significance.Since previous srudies have suggested that the combined effect of aerolysin (aerA), haemolysin (hlyA) and glutamate decarboxylase (gadB) contributes to virulence in A. hydrophila and E. tarda, respectively.In present paper, a new techvique based on multiplex PCR for detection virulence A. hydrophila and a multiplex PCR for detection of A.hydrophila and E.tarda were established.Then a detecting of submitted samples and water samples of aquaculture nurseries were carried out base on the mutiplex PCR and microbiological methods.1 Cloning and Analyzing sequences the correlated virulence genes from A.hydrophila and E.tardaThree pairs of specific primers were designed and multiplex PCR assay was developed to amplify the aerA, hlyA and gadB genes. We reclaimed and purified the produces from amplifying vir PCR,which cloned into pMD18-T carrier.Then they were measured sequences by TaKaRa.The sequences were compared the homologous of nucleotide acid sequences with that of GenBank.The result is that there are high homologous between the sequences of the three genes(aerA, hlyA and gadB) and that of the GenBank .The nucleotide sequence of aerA is 99.8 % identical to that of AF539467 and that of hlyA is 97.9 % identical to that of AF146599,while that of gadB is 87.1% identical to that of AY078505.The results testify the validity and dependability of these primers.2 Establishment of multiplex PCR for detection of pathogenic A. hydrophilaThree 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. 8 A.hydrophila strains isolated and the other sixteen strains were tested by multiplex PCR. The result showed that nonpathogenetic A. hydrophila strains were not amplified the virulence genes of hlyA or aerA, but that pathogenetic ones were amplified the genes of hlyA at least. 40 aquatic livestock specimen were tested by the multiplex PCR and conventional microbiology methods and their coherence was 97.5 %. It can be concluded that the multiplex PCR was specific and sensitive and could be used in quick diagnose and epidemiology investigation of A.hydrophila.3 Establishment the multiplex PCR of virulence genes in A. hydrophila and E. tardaA multiplex PCR assay was designed to amplify the A.hydrophila aerolysin (aerA), hemolysin (hlyA) genes and E. tarda glutamate decarboxylase (gadB) gene. The reaction conditions of the multiplex PCR were optimized. Finally, we established the multiplex PCR of virulence genes in A.hydrophila and E.tarda. 7 A.hydrophila strains isolated and 3 E.tarda strains isolated were tested by multiplex PCR. The result showed that nonpathogenetic A, hydrophiIa and E.tarda strains were not amplified the virulence genes, but pathogenetic A. hydrophila strains were amplified the genes of hlyA at least and pathogenetic E.tarda strains were amplified the genes of gadB. 40 aquatic livestock specimen and 12 water samples were tested by the multiplex PCR and conventional microbiology methods.Their coherence was 100 % (E.tarda) and 98.1% (A. hydrophila). It can be concluded that the multiplex PCR was specific and sensitive and could be used in quick diagnose and epidemiology investigation of A.hydrophila and E.tarda.4 Isolation and Identification pathogenic A.hydrophila from waterOne pathogeny bacteria were isolated from water in one eel mill of Fujian province.The bacteria are gram-negative bacilli, dynamic, oxigenation enzyme positive and produced indole, that can react with glucose, galactose, saccharose, amritarose, etc and V-P positive. Further identification showed that they are A.hydrophila. The pathogenicity test shows that the isolated bacteria were pathogenesis. They are sensitive to Kanamycin, Chloronycetin and Furazolidone etc Kui promise ketone medicine.5 Expression of gyrA Gene from A. hydrophilaQuinolone drug interdict their DNA reproduction, which infects the approach of DNA separation and resetion according to the gyrA gene.while the gyrA gene is mutated, the quinolones drug resistance occurs. Primers were designed based on the gyr A gene ,with the specific primers, one target fragment anout 539 bp of gyrA gene was amplified from A. hydrophila genomic DNA via PCR.The gyrA gene was transformed to E.coli, then was espressed with vector pET30 a (+) by IPTG. The recombinant gyrA exhibited a size of 28.0 kD with SDS-PAGE.