mini-MPN-STE-qPCR法快速定量检测食品中沙门氏菌

Quantitative detection of Salmonella in food by mini-MPN-STE-qPCR

目的建立一种快速、简易、可定量检测食源性沙门氏菌定量PCR (quantitative PCR, qPCR)方法。方法依据沙门菌属invA基因序列设计染料法qPCR引物,建立沙门氏菌qPCR检测方法,并通过短时间增菌(short time enrichment, STE)的5管微型多管发酵计数法(mini-most probable number, mini-MPN)进行定量检测,构建mini-MPN-STE-qPCR法。使用人工污染的鸡肉混合液进行定量检测,检测结果与传统MPN计数法和平板计数法进行比较分析。结果建立的qPCR法特异性良好,方法灵敏度为50CFU/mL,结合48孔板min-MPN和4 h短时间增菌,建立的mini-MPN-STE-qPCR法可将整个检测流程缩短至7 h。人工添加沙门氏菌的鸡肉混合液检测结果表明方法检出限为-0.347logMPN/mL,Bland-Altman分析结果显示,此法与传统MPN计数法相关系数r2=0.994,与平板计数法相关系数r2=0.992。结论该方法准确、简单易行、成本低、灵敏度高,可用于食品中沙门菌的快速定量检测。

Objective To establish a fast, easy and quantitative detection method of Salmonella in food. Methods Primers based on the invA gene sequence of Salmonella published on Genbank were designed to establish a quantitative PCR(qPCR) method for the detection of Salmonella, 5 tubes mini-MPN method with short time enrichment(STE) was joined to establish a mini-MPN-STE-qPCR method. The artificially contaminated chicken breast mixture was used for quantitative detection, and the detection results were compared with the traditional MPN method and plate counting method. Results The qPCR method established had good specificity with the sensitivity of 50 CFU/mL. With min-MPN in 48-well plate and 4 h short time enrichment in buffered peptone water, the entire experiment process of mini-MPN-STE-qPCR was shortened to 7 h. The quantitive detection limit was-0.347 log MPN/mL in the artificial contaminated chicken breast mixture. The Bland-Altman analysis showed that the correlation coefficient r2 between this method and the traditional MPN counting method was 0.994, and the correlation coefficient r2 with the plate counting method was 0.992. Conclusion This method is accurate, reliable and low-cost, and can be used for rapid quantitative detection of Salmonella in food.