Detection Of Aeromonas Hydrophila By The SPA Co-agglutination Test And Polymerase Chain Reaction

Abstract: A bacterial diagnosis solution of Staphylococcus protein A(SPA) was prepared by using a SPA international standard bacterial strain Cowan I labbelled with super antisera to 11 strains of the Aeromonas hydrophila in freshwater fishes, and a SPA Co-agglutination test(SPA-CoA) was established for rapid detection of the main bacterial diseases in freshwater fishes. The results of detection could be reported within 3 mm by SPA-CoA after samples were cultivated on agarplates for 16梚 8h. The sensitivity of the test was 25-50 times higher than that of slide agglutination, and the tests has no cross-reaction with other aeromonas bacteria, The SPA-CoA was proved to be effective when used to the diagnosis and epidemiologic survey of bacterial diseases in freshwater fishes. In order to detect the Aeromonas hydrop hi/a more specific, synthetic oligonucleotide primers were used in a polymerase chain reaction (PCR) technique to detect the gene for aerolysin in strains of A eromonas hydrophila and to screen for identical genes in other related bacteria strains. PCR was performed by using 20 ul of a PCR mixture containing 1 ul of template, each primer 2u1(50mmol/L), dNTP 4u1(2.Smg/L) and Taq enzyme 3U. The amplification was performed with a GeneAmp 9600PCR system(Perkin-Elmer) by using the following temperature program: I cycle of denaturation for 6 mm at 96 0C, 32 cycles of melting at 95 0C for SOs, annealing at 55X2 for SOs, and elongation at 720C for SOs, and a final extension round at 720C for 10mm. Primers targeted a 421-bp fragment of the aer gene coding for the beta-hemolysin and detected template DNA only in the PCR using DNA or cells from 20 hemolytic strains of A. hydrophila. PCR amplification of DNA and br cells from 12 other bacteria strains was consistently negative. The detection limit for the aerolysin gene by PCR amplifiction was 100 cfu of bacteria cells. The PCR clearly identifid aerolysin-producing strains of A. hydrophila and may use as a species-specific virulence test. The result of detect clinical isolates of A. hydrophila suggest that the aer gene commonly present in aerononads and the method was rapid and effective.