Establishment and application of colony multiplex PCR for Aeromonas hydrophila

Abstract:

[Background] Aeromonas hydrophila is pathogenic to aquatic animals, livestock, poultry, and human. Hemolysin, aerolysin, and enterotoxin are major virulence factors particularly important in the early detection and prevention of A. hydrophila. There are few reports using colony PCR to extract templates for multiplex PCR. [Objective] Based on colony PCR, a multiplex PCR assay was established for rapid detection of five genes including hemolysin gene, enterotoxin gene, and 16s rRNA gene of A. hydrophila. [Methods] We used the selective Rimler-Shotts (RS) medium to enrich, isolate, and identify A. hydrophila in the sample. Subsequently, we established and optimized the multiplex PCR conditions for the 16S rRNA, ast, alt, aerA, and act of A. hydrophila and compared the results of multiplex PCR with the DNA templates extracted by different methods in colony PCR. Finally, we evaluated the specificity of the established method by using A. veronii, A. sobria and A. salmonicida. [Results] After 16S rRNA identification of single colonies on RS plates, the colony morphology of A. hydrophila and other cultivable bacteria was preliminarily characterized, and the enrichment degree of A. hydrophila was visually identified. The optimization results of the multiplex PCR system showed that the optimal primer concentration ratio was 16s rRNA:ast:alt:aerA:act=1:2:2:3:4. PCR for fresh bacterial liquids treated with both boiling-refrigerated centrifugation method and boiling-centrifugation method could produce clear bands, while the PCR of single colony required the former method. The established multiplex PCR method was specific. [Conclusion] Colony multiplex PCR can be used to easily and intuitively detect A. hydrophila and its virulence genes without the need of kit extraction of DNA templates, and it was specific.

Key words:multiplex PCR;Aeromonas hydrophila;colony PCR;virulence gene