Detection of pathogenic Aeromonas hydrophila by quadruple PCR

Aeromonas hydrophila can infect an array of animals,including fish,amphibians,reptiles,birds and mammals,and lead to hemorrhagic septicaemia.To develop a rapid and specific quadruple PCR method for the detection of pathogenic Aeromonas hydrophila,four pairs of primers were designed based on the conservative sequences of 16SrRNA gene,serine-protease(ahpA)gene,aerolysin(aerA)gene and hemolysin(hlyA)gene of Aeromonas hydrophila.After optimalizing the reaction conditions,specificity,sensitivity and detection rate of the quadruple PCR method were studied.Results indicated that this method has a high specificity in detecting pathogenic strains of Aeromonas hydrophila but not other irrelative bacteria,and can detect as less as 100fg DNA of Aeromonas hydrophila.Nine Aeromonas hydrophila strains and fifty-six clinical samples were tested by this quadruple PCR and conventional microbiology methods,and both of the methods could identify these nine Aeromonas hydrophila strains;the detection rate of the fifty-six clinical samples by quadruple PCR was 21.4%,which was higher than that by conventional microbiology methods(16.1%),and their coherence was 94.6%.It could be concluded that the quadruple PCR method which can simultaneously detect 16SrRNA gene and three virulent genes of pathogenic Aeromonas hydrophila was well established,and this method is a reliable and convenient method to detect Aeromonas hydrophila and identify specific type of pathogenic or nonpathogenic strains.