速冻面米制品中金黄色葡萄球菌和肠毒素A基因三重PMA-qPCR快检方法的建立  

Development of Triplex PMA-qPCR for Rapid Quantification of Viable Total and Sea-positive Staphylococcus aureus in Quick-Frozen Flour and Rice Products

作　　者：宋明辉 施春雷[2] 李琼琼[1] 史贤明[2] 杨美成[1] Song Minghui;Shi Chunlei;Li Qiongqiong;Shi Xianming;Yang Meicheng(Shanghai Institute for Food and Drug Control,NMPA Key Laboratory for Testing Technology of Pharmaceutical Microbiology,Shanghai 201203;School of Agriculture and Biology,Shanghai Jiao Tong University,Shanghai 200240)

机构地区：[1]上海市食品药品检验所国家药品监督管理局药品微生物检测技术重点实验室,上海201203 [2]上海交通大学农业与生物学院,上海200240

出　　处：《中国食品学报》2020年第7期244-252,共9页Journal of Chinese Institute Of Food Science and Technology

基　　金：国家重点研发计划项目(2018YFC1603900)。

摘　　要：为建立准确定量速冻面米制品中金黄色葡萄球菌活菌,筛查肠毒素sea基因并指示PCR反应假阴性的三重PMA-qPCR检测方法。针对金黄色葡萄球菌特异靶基因RpiR和肠毒素基因sea序列保守区,设计引物、探针和构建扩增内标,优化建立三重qPCR检测体系,建立PMA食品前处理方法去除死菌DNA,构建纯培养物和速冻面米制品中污染金黄色葡萄球菌的定量标准曲线,应用三重PMA-qPCR方法检测人工污染金黄色葡萄球菌的速冻面米制品。采用GB4789.10-2016中规定的选择平板计数法,评价三重PMA-qPCR方法的检测性能。结果表明,三重PMA-qPCR检测体系扩增效率高,特异性好。当食品中金黄色葡萄球菌污染量大于10^3CFU/g时,靶基因RpiR可获得有效扩增,污染量在10^3~10^7CFU/g之间时,扩增曲线线性良好(R^2=0.994),PMA-qPCR定量结果与平板计数结果一致性较好。当sea阳性菌株污染量大于10^3CFU/g时,应用PMA-qPCR方法可有效评估食品污染肠毒素A的风险。综上所述,本研究建立的三重PMA-qPCR检测方法操作简便,可快速定量检测速冻面米制品中金黄色葡萄球菌,评估食品污染肠毒素A的风险。The objective of this study is to develop a triplex PMA-qPCR method for rapid quantification of viable total and sea-positive Staphylococcus aureus in quick-frozen flour and rice products,simultaneously indicating the possible false negative detection results.Firstly,primers and probes targeting the conserved region of RpiR and sea gene were designed,and a competitive internal amplification control was constructed.Then triplex qPCR method was developed by optimizing reaction conditions and evaluating the specificity and detection limit.Food sample preparation combining propidium monoazide(PMA) was used to eliminate DNA of dead cells.The standard curves for quantification of viable total and sea-positive Staphylococcus aureus in pure culture and frozen foods were established,respectively.Artificial contamination assays and plate count methods were used to evaluate the accuracy of triplex PMA-qPCR method developed in this study.It was shown that triplex PMA-qPCR method had good amplification efficiency and specificity;when quickfrozen flour and rice products were contaminated above 10^3 CFU/g Staphylococcus aureus,the target gene RpiR could be effectively detected,especially,the contamination of Staphylococcus aureus was between 10^3 CFU/g and 10^7 CFU/g,the cycle threshold values(Ct) and the log number of bacterial concentration presented the satisfactory linear,the correlation coefficient R^2 approached 0.994,and the quantification result obtained by PMA-qPCR method showed good consistency with plate count methods;when quick-frozen flour and rice products were contaminated above 10^3 CFU/g sea-positive Staphylococcus aureus,the contamination risk of enterotoxin A could be effectively evaluated.Taken all together,triplex PMA-qPCR method developed in this study could accurately quantify viable Staphylococcus aureus and evaluate the contamination risk of enterotoxin A in quick-frozen flour and rice products.

关 键 词：速冻面米制品 金黄色葡萄球菌 肠毒素A基因 叠氮溴化丙啶 荧光定量聚合酶链式反应 

分 类 号：TS207.3[轻工技术与工程—食品科学]