Establishment of a multiplex PCR assay to detect five major freshwater bacteria

Abstract

A multiplex polymerase chain reaction (mPCR) method for simultaneous detection of Aeromonas hydrophila, Streptococcus agalactiae, Klebsiella pneumoniae, Edwardsiella tarda, and E. ictalur was developed to rapidly and accurately identify the five most common bacteria that infect aquatic animals. The expected amplicons for ahe2 gene of A. hydrophila, cpsE gene of S. agalactiae, khe gene of K. pneumoniae, mukF gene of E.tarda, and the serC gene of E. ictaluri were 853 bp, 685 bp, 428 bp, 356 bp, and 124 bp, respectively. In the single PCR assays, the minimum detectable DNA contents were 13.2 pg for A. hydrophila, 27.4 pg for S. agalactiae, 1.95 pg for K. pneumoniae, 1.63 pg for E. tarda, 1.02 pg for E. ictalur. The detection limits of the multiplex PCR were 0.66 ng, 1.91 ng, 0.68 ng, 0.41 ng, 0.71 ng for A. hydrophila, S. agalactiae, K. pneumoniae, E. tarda and E. ictalur, respectively. The established multiplex PCR is significant for the rapid detection of common pathogenic bacteria of aquatic animals and provides the basis for the diagnosis of fish diseases.