ESTABLISHMENT OF QPCR DETECTION ASSAY AND PHYLOGENETIC ANALYSIS BASED ON NOCARDIA SERIOLAE SECA GENE

Nocardia seriolae is one of the most common pathogenic bacteria in aquaculture animals, and the nocardiosis caused by it has brought huge economic losses to the global aquaculture industry. In order to perform diagnosis of N. seriolae infection quickly and early, a TaqMan qPCR detection method for N. seriolae was established. The primers and probes were designed with the housekeeping gene SecA of N. seriolae, and the reaction conditions were optimized, and standard curves was developed. Then the established qPCR method was tested for sensitivity, reproducibility and specificity, and applied for the detection of clinical diseased fish samples. The assay of qPCR showed high linearity in the range of 9.85×10¹⁰—9.85×10⁰ copies with a sensitivity of up to 9.85 copies under the optimal primers concentration (0.3 μmol/L), the optimal probe concentration (0.1 μmol/L), and the optimal annealing extension temperature (60℃). The results of the repeatability experiment showed that the coefficient of variation between groups and within groups was less than 1%. The specificity showed that there was no amplification curve for 13 pathogens such as S. agalactiae, C. freundii and A. veronii. The test results of the 42 diseased M. salmoides samples showed that the positive rate of qPCR was 14.24% higher than that of normal PCR, and the content of nocardia in the nodules was significantly higher than that in the non-nodal site of tissues, with a maximum difference of 1000-fold. The results of SecA gene analysis showed that SecA gene was highly conserved in passage and within species, and has certain discreteness among species, so it was a good target gene for Nocardia species identification. The qPCR method established in this study exhibits remarkable sensitivity, specificity and repeatability, and can be used in early diagnosis and quantitative detection of N. seriolae, and provides an effective tool for early diagnosis and treatment of nocardiosis.