基于ttr基因的mini-MPN-qLAMP法快速定量检测食品中沙门氏菌

Rapid and Quantitative Detection of Salmonella in Food by mini-MPN-qLAMP Based on ttr Gene

摘要:

该研究通过3管型微量MPN计数法（Mini Most Probable Number，mini-MPN）建立了一种快速定量检测食源性沙门氏菌的荧光定量环介导等温扩增（Quantitative Loop-Mediated Isothermal Amplification，qLAMP）方法。依据沙门菌属ttr基因设计了qLAMP和荧光定量PCR（Quantitative Polymerase Chain Reaction，qPCR）引物，结合5 h BPW增菌和MPN计数法建立了mini-MPN-qLAMP沙门氏菌快速定量检测方法；使用两种人工污染样品对mini-MPN-qLAMP法进行验证，使用Bland-Altman分析比较不同检测方法检测结果的一致性。结果表明，建立的qLAMP法与qPCR法反应特异性均良好，纯培养时qLAMP法检出限为500 CFU/mL。通过Bland-Altman分析表明所建立的mini-MPN-qLAMP法在速冻乌米饭中检测结果与mini-MPN-qPCR、mini-MPN计数法、平板计数法相比均具有较高的一致性，r2≥0.994，检出限为-0.44 lg MPN/mL；而在速冻鸡胸肉中该法检测结果与mini-MPN-qPCR结果一致性最佳，r2=0.990，检出限为-0.64 lg MPN/mL。肉制品中腐败杂菌会影响mini-MPN计数和平板计数结果，mini-MPN-qLAMP可排除肉制品中腐败杂菌对检测结果的影响。该研究所建立的mini-MPN-qLAMP法简单易行，准确度高，可用于食品中沙门氏菌的快速定量检测。

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.