| Citation: |
WANG Yilin, AI Mingqi, JIANG Qibin, PENG Kun, ZHOU Keyu, FAN Wei, OUYANG Ping, CHEN Defang, HUANG Xiaoli, GENG Yi. Establishment and application of qPCR and RAA-LFD based on recA gene for detection of Pseudomonas anguilliseptica[J]. South China Fisheries Science, 2025, 21(2): 138-148. DOI: 10.12131/20240206
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For rapid diagnosis of early infection with Pseudomonas anguilliseptica (PA), we established SYBR Green I real-time quantitative PCR 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 2.816×102 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 2.816×104 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 were 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 (3.533×107 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.
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