Detection and quantification of Aeromonas schubertii in Channa maculata by TaqMan MGB probe fluorescence real-time quantitative PCR.

Abstract

Aeromonas schubertii is a major epidemiological agent that threatens cultured snakeheads (Channidae) and has caused great economic losses in fish-farming industries in China in recent years. In present study, a specific TaqMan minor groove binder (MGB) probe fluorescence real-time quantitative PCR (qPCR) assay was developed to rapidly detect and quantify A.schubertii. A pair of qPCR primers and a TaqMan MGB probe were selected from the rpoD gene, which were shown to be specific for A.schubertii. A high correlation coefficient (R2 =0.9998) in a standard curve with a 103% efficiency was obtained. Moreover, the qPCR method's detection limit was as low as 18copies/μl, which was 100 times more sensitive than that of conventional PCR. The detection results for the A.schubertii in pond water and fish tissue were consistent with those of the viable counts. Bacterial load changes detected by qPCR in different tissues of snakeheads infected with A.schubertii showed that the gills and intestines may be the entry for A.schubertii, and the spleen and kidney are major sites for A.schubertii replication. The established method in present study should be a useful tool for the early surveillance and quantitation of A.schubertii.