A novel multiplex PCR assay for rapid detection of virulent Aeromonas in cultured eels.

Abstract

Under intensive and stressful aquaculture conditions, cultured eels are highly susceptible to virulent Aeromonassp. infections. To rapidly and simultaneously confirm Aeromonas isolate and its virulence, a two-tube multiplex PCR (mPCR) assay incorporating gyrB gene for genus-specific recognition and seven major virulence genes for virulence assessment was developed. Eight pairs of primers were designed and divided into two groups-gyrB, ahpA, epr and aerA in tube 1 and alt, act, ast and hlyA in tube 2. The optimized mPCR conditions were the same except for their final concentrations. The specificity of the mPCR was validated by the extracted DNA of 10 Aeromonas and 8 non-Aeromonas species, or mixed DNA templates. Detection limits were determined to be 200 copies per μl in tube 1 and 20 copies per μl in tube 2. The mPCR reproducibility was tested by both artificial challenge and clinical samples. The results showed this two-tube mPCR assay was rapid, specific, sensitive and reliable. To our knowledge, this is the first report to distinguish virulent Aeromonas isolates from nonvirulent ones by seven popular and major virulence genes at the genus-specific level. And it will be useful for large-scale screening of virulent Aeromonas sp. in cultured eels.