Abstract:
For rapid diagnosis of early infection with
Pseudomonas anguilliseptica (PA), we established SYBR Green I Real-time quantitative PCR (qPCR) and recombinase-mediated isothermal amplification combined with lateral flow dipstick (RAA-LFD) based on
recA gene for rapid and specific detection. A pair of qPCR specific primers, a pair of RAA specific primers and a RAA probe were designed and screened based on the
recA gene of PA. The standard quality plasmid pUC18-
recA was constructed by homologous recombination to establish the two detection methods. The established methods were applied to detect PA-infected largemouth bass (
Micropterus salmoides) tissue samples and PA load was determined. The minimum DNA detection concentration of the qPCR method was 2.816×10
2 copies·μL
−1. There was a good linear relationship between the template amount and Ct value in the standard curve (
r2=0.999 2), and the method had strong specificity and stability. The minimum DNA detection concentration of the RAA-LFD method was 2.816×10
4 copies·μL
−1. The detection time of the RAA-LFD method was 15 min, and the color development was relatively stable and the specificity was strong. The application results show that the positive detection rates of qPCR and RAA-LFD were 87.50% and 85.00%, respectively, which was significantly higher than that of the common PCR method. The established qPCR method could accurately measure the bacterial load in the tissues of PA infected hosts. The highest bacterial load was found in the kidney (3.533×10
7 copies·ng
−1). Both methods can be used for rapid detection of early PA infection. The established qPCR can also be used to quantitatively analyze the bacterial load in infected hosts.