Rapid detection of Salmonella in raw milk by visual and real-time fluorescence loop-mediated isothermal amplification-based methods

Abstract:

[Background] Quality control of raw milk is a key part in the production process of dairy products. Salmonella is one of the main pathogenic bacteria that can contaminate raw milk. With the development of biological detection technology, it is of great significance to establish a practical method for rapid detection of Salmonella in raw milk. [Objective] To evaluate practical application effect of rapid Salmonella detection in raw milk by visual loop-mediated isothermal amplification (V-LAMP) and real-time fluorescence loop-mediated isothermal amplification (RF-LAMP), respectively. [Methods] We established the LAMP reaction system and optimized the reaction conditions, then tested and compared the specificity, sensitivity, stability and effectiveness in practical samples detection by using V-LAMP and RF-LAMP methods, respectively. [Results] Through the rapid detection of V-LAMP and RF-LAMP, all Salmonella standard strains were detected positive, while all non-Salmonella standard strains were negative. The limit of detection for the two methods was calculated to 13 CFU/mL. The detection accordance rate of the two methods were both 100%. The results obtained by detecting Salmonella in 10 artificial contaminated samples were highly consistent with the actual results. With respect to the result obtained by conventional detection method in the 73 real samples, the relative specificity of the two methods were respectively 94.12% and 92.65%, the relative sensitivity were respectively 100% and 100%, the relative accuracy were respectively 94.52% and 93.15%, and the χ2 value for consistency analysis were respectively 2.25 and 3.20, showing that no significant difference was found in the results above by the three mentioned methods (P>0.05). [Conclusion] V-LAMP and RF-LAMP methods show good specificity, high sensitivity, and high consistency, which makes them applicable for rapid detection of Salmonella in raw milk.