动物造模,沙门氏菌 z-bio 2022-03-14 350

　　Objective: To establish a loop-mediated isothermal amplification detection method for Salmonella in tree shrew feces, and to make a preliminary application of the method.

　　METHODS: Based on the conserved sequence of Salmonella species-specific gene invA (invasion protein gene A), specific LAMP primers were designed and synthesized. Ten reaction temperatures, namely 57 °C ~ 66 °C and ten reaction times, namely 24 ~ 42 min, were respectively set for optimization, and the specificity and sensitivity of this method were determined; at the same time, ordinary PCR experiments were carried out as the verification and comparison of this method. 91 loose stool samples of wild tree shrew were detected by the established LAMP method.

　　Results: The optimized reaction conditions were selected as the reaction temperature of 62 ℃ and the reaction time of 34 min. The sensitivity of this method to Salmonella reached 3.36 × 101 CFU/mL, which was 10 to 100 times higher than that of ordinary PCR methods. The intestinal bacteria were tested by LAMP, only Salmonella Enteritidis and Salmonella paratyphi B were positive, and the rest were negative; 91 wild tree shrew fecal samples were tested by LAMP, and the positive detection rate was 20.88%; All reactions can be completed within 40 min? Results can be directly judged by visual observation of color changes.

　　Conclusion: The established LAMP method has the characteristics of convenience, rapidity, sensitivity and specificity, and can be used for large-scale rapid detection of Salmonella in tree shrew feces.