Real-time PCR detection of Aeromonas salmonicida by amplification of specific vapA gene

In order to implement the early and quick quantitative determination of Aeromonas salmonicida, a SYBR Green I Real-time PCR method of A. salmonicida was established based on the pathogen sequence information. Based on the vapA of virulence array protein gene sequence of A. salmonicida, a pair of primers was designed and used in a real time quantitative polymerase chain reaction (qPCR) assay. The specificity, sensitivity, repeatability and application of the system were also evaluated. The results showed that A. salmonicida and its subspecies can be clearly discriminated from the other 10 bacteria species by SYBR Green I Real-time qPCR, which indicated that the primer pair has good inter-species specificity. The standard curve established by recombinant plasmid showed a fine linear relationship between initial templates and threshold cycle, which can be described as y=–4.8345x+42.535 (R²=0.998). The sensitivity analysis showed that the detection limit was 34 copies/μL, which suggested that the sensitivity of Real-time qPCR was about 1000 times higher than that of the conventional PCR assay. The established method was applied to detect the samples in rainbow trout after artificial infection. Results showed that 15 of those samples were positive, which had complete agreement (100%) with bacteriological analysis by isolation and culture. In conclusion, the developed Real-time PCR assay for A. salmonicida is fast, highly specific, and sensitive. This method had a broad application for clinical diagnosis and disease surveillance in aquaculture.