Rapid detection of Salmonella based on loop-mediated isothermal amplification.

Abstract

Salmonella enterica is a zoonotic pathogen of substantial concern to human and animal health and is a leading cause of morbidity and mortality in people worldwide. Loop-mediated isothermal amplification (LAMP) technology is a new type of nucleic acid amplification technology, which has the characteristics of high specificity, high sensitivity, simple operation, convenience, and low cost. This study aims to establish a rapid detection method for Salmonella based on LAMP technology. Primers were designed for Salmonella's specific conservative invA gene. Through primer screening and optimization of reaction conditions, and a LAMP method for detecting Salmonella with real-time fluorescence and visual observation results was established. The sensitivity and specificity of the method were assessed, and the accuracy was evaluated through the testing of Salmonella-contaminated and non-contaminated clinical samples. The optimal reaction temperature of LAMP was 60-65 °C, and the optimal reaction time was 25-30 minutes. The detection limits of real-time fluorescence and visual observation were both 1.4 pg/µL. There was no cross-reactivity observed with 22 non-Salmonella species, and the specificity was 100%. Additionally, 30 samples contaminated with Salmonella, 30 samples not contaminated with Salmonella, and 8 clinical samples identified as positive by bacterial culture and microbial mass spectrometry were tested. The positive coincidence rate of the detection system was 97.4% by real-time fluorescence and 89.5% by visual observation, the negative coincidence rate was 100%, and the total coincidence rate was 98.5% and 94.1%, respectively. In the scene of infection, primary hospital, disaster area treatment and other scenarios, the conditions of environment, equipment and personnel was limited, therefore, the established real-time fluorescence and visual lamp method can provide a powerful means for the rapid detection of Salmonella.