Rapid and Quantitative Detection of Salmonella in Food by mini-MPN-qLAMP Based on ttr Gene
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Abstract:
In this study, the 3-tube mini most probable number (mini-MPN) counting method was used to establish a fluorescence quantitative loop-mediated isothermal amplification (qLAMP) method for rapid and quantitative detection of food-borne Salmonella. According to the ttr gene sequence of Salmonella, the primers for qLAMP and quantitative polymerase chain reaction (qPCR) were designed, and in combination with the 5h BPW amplification and MPN counting, a rapid and quantitative detection method by mini-MPN-qLAMP for Salmonella was established, and the consistency between the analysis results obtained by different assays was compared using the Bland-Altman analysis. The mini-MPN-qLAMP method was validated using two artificially contaminated samples, The results showed that the established qLAMP method and the qPCR method had good specificity. The pure culture experiments revealed the detection limit of qLAMP of 500 CFU/mL. The Bland-Altman analysis revealed that in the quick-frozen black rice, the established mini-MPN-qLAMP method had a relatively high consistency with the mini-MPN-qPCR, the mini-MPN counting and plate counting method, with the R2 not lower than 0.994, and the detection limit being -0.44 lg MPN/mL. In quick-frozen chicken breast, the mini-MPN-qLAMP method had the highest consistency with the mini-MPN-qPCR, with the R2 being 0.990 and detection limit being -0.64 lg MPN/mL. The spoilage bacteria in the meat products could affect the mini-MPN counting and plate counting results, whilst the mini-MPN-qLAMP method could eliminate the interference of spoilage bacteria in meat products. Accordingly, the mini-MPN-qLAMP method established in this study is simple, easy to be used and accurate, thus can be used for rapid and quantitative detection of Salmonella in food.