Isolation and Identification of Pathogenic Yersinia enterocolitica and Development of a Triple PCR Detection Method

This paper aimed to identify the pathogenic bacterium that caused anal swelling and abdominal bleeding of Carassius auratus, and establish a rapid detection method for the pathogenic bacterium. In this study, the pathogenic bacterium was isolated from Carassius auratus, and identified by morphological, physical and chemical characteristics analysis and 16S rDNA sequence analysis. The virulence gene of the bacterium was detected by PCR method. The drug sensitivity of isolated strain was detected by disk agar diffusion method and its pathogenicity was discussed by artificial infection. Three specific primers of ail gene, inv gene and intB gene were designed for the isolated strain. A triple PCR method for detecting the isolated strain was established and this method was used to test samples of sick Carassius auratus. The result showed that a Yersinia enterocolitica strain named fsznc-10 was isolated from the heart of diseased Carassius auratus. Yersinia enterocolitica fsznc-10 had virulence genes of ail, ystB, virF and intB. Yersinia enterocolitica fsznc-10 was nonresistant to five antibiotics such as norfloxacin and gentamicin, and it had certain pathogenicity to Carassius auratus. Triple PCR method could accurately amplify three target genes of ail, inv and intB of Yersinia enterocolitica, while other strains had not amplified the target genes. The minimum template concentration for the detection of Yersinia enterocolitica was 1.704 × 10⁻⁶ ng/μL. The positive rate of the triple PCR method for detecting heart samples of sick fish was about 86.67%, and its detection coincidence rate with the 16S rDNA sequence analysis method was 100%. The triple PCR detection method has the advantages of strong specificity, high sensitivity, simple operation and low cost. The present paper research provides scientific data reference for the prevention, control and detection of Yersinia enterocolitica in clinical practice.