恒温隔绝式PCR快速检测蜡样芽孢杆菌方法的建立及应用
Development of an Insulated Isothermal PCR-based Method for Rapid Detection of Bacillus cereus
寇肖迪 1孟含 1苏雅航 1汤承 2胡瑜 3唐俊妮4
作者信息
- 1. 西南民族大学食品科学与技术学院,四川成都 610225
- 2. 青藏高原动物遗传资源保护与利用教育部重点实验室,四川成都 610041
- 3. 康美包(苏州)有限公司,江苏苏州 215021
- 4. 西南民族大学食品科学与技术学院,四川成都 610225;青藏高原动物遗传资源保护与利用教育部重点实验室,四川成都 610041
- 折叠
摘要
该研究以编码旋转酶B亚基的gyrB基因为靶基因,利用恒温热隔绝式PCR(Insulatedisothermal PCR,IiPCR),建立一种快速检测蜡样芽孢杆菌的方法.同时,将建立的方法与聚合酶链式反应(Polymerase Chain Reaction,PCR)和实时荧光定量PCR(SYBER GreenⅠ荧光染料法和TaqMan探针法)检测方法相比较,并应用于不同类型零售食品中的蜡样芽孢杆菌检测.结果表明建立的检测方法能在40 min内迅速判定出结果(+、-、?);与大肠杆菌、金黄色葡萄球菌、单增李斯特菌、肠炎沙门氏菌等均无交叉反应,显示出良好的特异性;方法的最低检出限为 1.5×102 CFU/mL,优于普通PCR,与实时荧光定量PCR检出限相当;对 42 份零售食品进行检测,共发现45.23%的样本被蜡样芽胞杆菌污染(19/42).这一结果与常规PCR检测结果一致,但检测时间至少节省了 4 h以上.该研究为蜡样芽孢杆菌提供了一种快速、便捷、特异性强、灵敏度高、适用于现场实时检测的检测方法.
Abstract
Specific primers and probe were designed for the gyr B gene of Bacillus cereus encoding the gyrase B subunit,and a method based on insulated isothermal polymerase chain reaction(iiPCR)was established for the rapid detection of B.cereus and subsequently validated.The efficacy of this method was compared with that of traditional PCR and real-time fluorescent quantitative PCR(SYBER Green Ⅰ fluorescent dye and TaqMan probe methods)detection methods,and applied in the detection of B.cereus in different types of retail food.The results revealed that the newly developed method facilitated the rapid detection of B.cereus within 40 min.Moreover,there was no cross reaction with other bacteria,including Escherichia coli,Staphylococcus aureus,Listeria monocytogenes,and Salmonella enteritidis,thereby indicating good specificity.The minimum detection limit of the method was 1.5×102 CFU/mL,which was significantly superior to that of the traditional PCR method,and equivalent to that of quantitative PCR.Of the 42 retail food samples assessed,19(45.23％)were found to be contaminated with B.cereus.The results obtained for method validated were completely consistent with those obtained using traditional PCR detection,although test results were available within a considerably shorter timeframe,saving at least 4 h.The method developed in this study provides a rapid,convenient,specific,and sensitive procedure that is suitable for the real-time on-site detection of B.cereus.
关键词
蜡样芽孢杆菌/恒温隔绝式PCR/gyrB基因Key words
Bacillus cereus/insulated isothermal PCR/gyrB gene引用本文复制引用
基金项目
国家重点研发计划重点专项(2018YFD0500500)
西南民族大学研究生创新型科研项目(ZD2022257)
出版年
2024
国家科技期刊平台
NSTL
万方数据