实时荧光PCR鉴定婴幼儿配方食品中克罗诺杆菌的研究

Identification of Cronobacter in infant formula using real-time polymerase chain reaction

目的建立一种能够快速准确鉴定克罗诺杆菌的实时荧光聚合酶链式反应(PCR)方法,并对食品样品和人工污染样品进行克罗诺杆菌检验。方法根据克罗诺杆菌DNA旋转酶B亚基(gyrB)基因保守区序列设计引物和探针。通过特异性试验、绝对灵敏度、相对灵敏性试验、抗干扰试验对所建立方法进行方法学验证。采用人工污染样品增菌液进行方法灵敏度试验。结果本研究建立的方法能够特异性扩增7种克罗诺杆菌,但对与其亲缘关系较近的其他肠杆菌及食品中较为常见的其他致病菌均无扩增,表明本研究建立的方法具有很好的特异性和抗干扰能力。采用阪崎克罗诺杆菌验证绝对灵敏度达1~10 pg,相对灵敏度可以达到10^(3)CFU/mL。在基因组水平和培养物水平均具有很好的抗干扰能力。人工污染样品在36℃增菌24 h后检测灵敏度可以达到10^(0)CFU/mL。结论本研究所建立的实时荧光PCR方法对婴幼儿配方食品样品中的克罗诺杆菌的检测具有快速、特异、灵敏和稳定的特点,可以为传统婴幼儿配方食品中的克罗诺杆菌的检验提供技术参考。

Objective To establish a real-time quantitative polymerase chain reaction(PCR)method for rapid and accurate identification of Cronobacter spp.in food samples and artificially contaminated samples.Methods Primers and probes were designed based on the conserved region of gyrB of Cronobacter spp.DNA.The method was verified using a specificity test,absolute sensitivity test,relative sensitivity test,and anti-interference test.Detection sensitivity was determined using artificially contaminated samples.Results The method established in this study could specifically amplify seven kinds of Cronobacter spp.,but not the other Enterobacter species closely related to it and other pathogens common in food,suggesting that this method has good anti-interference ability.The absolute sensitivity was 1-10 pg and the relative sensitivity was 10^(3)CFU/mL using Cronobacter sakazakii.It had good anti-interference ability at the genome and culture level.The sensitivity of artificially contaminated samples could reach 10^(0)CFU/mL after incubation at 36℃for 24 h.Conclusion The real-time PCR method developed in this study is rapid,specific,sensitive,and stable for the detection of Cronobacter spp.in infant formula food samples and can provide technical reference for the detection of Cronobacter spp.in infant formula food.