摘要
该研究旨在建立散装即食食品中沙门氏菌、金黄色葡萄球菌和蜡样芽孢杆菌3种食源性致病菌的多重荧光定量PCR检测方法,根据沙门氏菌侵染上皮细胞表面蛋白的invA基因、金黄色葡萄球菌的耐热核酸酶(nuc)基因、蜡样芽孢杆菌的cer A基因分别设计3对特异性引物与探针,并对体系中引物探针的浓度以及退火温度等反应条件进行优化筛选,建立多重荧光定量PCR检测方法,同时评估其特异性、灵敏性及重复性,并应用该方法检测散装即食食品样本中致病菌,同时与国标法比对。结果表明,建立的多重荧光定量PCR检测方法能特异性地扩增沙门氏菌、金黄色葡萄球菌和蜡样芽孢杆菌3种目标菌,其他菌株均不扩增,灵敏度分别为4×10^(2)、3×10^(2)、2×10^(2)cfu/mL,且批内和批间变异系数均小于2%,重复性和稳定性良好。同时用国标法和多重荧光定量PCR法对100份散装即食食品样本进行检测比对,多重荧光定量PCR法的阳性检出率略高于传统国标法,且检测时间大幅缩短。综上所述,建立的多重荧光定量PCR检测方法特异性强、灵敏度高、快速准确,在食源性致病菌快速筛查方面具有良好的应用前景。
The aim of this study was to establish a multiplex fluorescence quantitative PCR method for the detection of three food-borne pathogens,Salmonella,Staphylococcus aureus and Bacillus cereus,in bulk ready-to-eat food.Three pairs of specific primers and probes were designed according to the invA gene of the infected epithelial cell surface protein of Salmonella,the nuc gene of Staphylococcus aureus,and the cerA gene of Bacillus cereus.The addition amount of primers and probes in the system and the annealing temperature were optimized and screened.To establish a multiplex fluorescence quantitative PcR assay,and evaluate its specificity,sensitivity and repeatability,and apply this assay to detect pathogenic bacteria in bulk ready-to-eat food samples and compare it with the national standard method.The results showed that the established multiplex fluorescence quantitative PCR assay could specifically amplify only Salmonella,Staphylococcus aureus and Bacillus cereus,with the sensitivity of 4×10^(2) cfu/mL,3×10^(2) cfu/mL and 2×10^(2) cfu/mL respectively.The intra-run and inter-run coefficients of variation were less than 2%,and the repeatability and stability were good.At the same time,100 bulk ready-to-eat food samples were detected and compared by national standard method and multiple fluorescence quantitative PCR method.The positive detection rate of multiple fluorescence quantitative PCR method was slightly higher than that of traditional national standard method,and the detection time was greatly shortened.In conclusion,the established multiplex fluorescence quantitative PCR method has strong specificity,high sensitivity,rapid and accurate,and has a good application prospect in the rapid screening of foodborne pathogens.
作者
崔洁
黄朱梁
严卓彦
孙瑛
林吉恒
夏瑛瑛
王萍亚
邓尚贵
CUI Jie;HUANG Zhuliang;YAN Zhuoyan;SUN Ying;LIN Jiheng;XIA Yingying;WANG Pingya;DENG Shanggui(College of Food and Pharmacy,Zhejiang Ocean University,Zhoushan 316000,China;Zhoushan Institute of Food&Drug Control,Zhoushan 316000,China)
出处
《食品科技》
CAS
北大核心
2023年第4期312-319,共8页
Food Science and Technology
基金
浙江省药品监管科技计划项目(2023010)
2021年度舟山市科技计划项目(2021C31003)
浙江省市场监督管理局青年科技项目(QN2023455)。
