Chen Xingxing, Wu Xiaoli, Gan Min, Xu Feng, He Lihua, Yang Dong, Xu Hengyi, Shah Nagendra P, Wei Hua
State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, 330047, China.
School of Basic Medical Science, Jiangxi University of Chinese Traditional Medicine, Nanchang, Jiangxi, 330000, China.
J Dairy Sci. 2015 Mar;98(3):1563-70. doi: 10.3168/jds.2014-8828. Epub 2014 Dec 26.
Staphylococcus aureus is one of the main pathogens in dairy and meat products; therefore, developing a highly sensitive and rapid method for its detection is necessary. In this study, a quantitative detection method for Staph. aureus was developed using silica-coated magnetic nanoparticles and thermophilic helicase-dependent isothermal amplification. First, genomic DNA was extracted from lysed bacteria using silica-coated magnetic nanoparticles and amplified using thermophilic helicase-dependent isothermal amplification. After adding the nucleic-acid dye SYBR Green I to the amplicons, the fluorescence intensity was observed using a UV lamp or recorded using a fluorescence spectrophotometer. This detection system had a detection limit of 5×10(0) cfu/mL in pure culture and milk-powder samples and 5×10(1) cfu/mL in pork samples using a UV light in less than 2h. In addition, a good linear relationship was obtained between fluorescence intensity and bacterial concentrations ranging from 10(2) to 10(4) cfu/mL under optimal conditions. Furthermore, the results from contaminated milk powder and pork samples suggested that the detection system could be used for the quantitative analysis of Staph. aureus and applied potentially to the food industry for the detection of this pathogen.
金黄色葡萄球菌是乳制品和肉制品中的主要病原菌之一；因此，开发一种高灵敏度、快速的检测方法很有必要。在本研究中，利用二氧化硅包覆的磁性纳米颗粒和嗜热解旋酶依赖性等温扩增技术，建立了一种金黄色葡萄球菌的定量检测方法。首先，使用二氧化硅包覆的磁性纳米颗粒从裂解的细菌中提取基因组DNA，并通过嗜热解旋酶依赖性等温扩增技术进行扩增。向扩增产物中加入核酸染料SYBR Green I后，用紫外灯观察荧光强度或用荧光分光光度计记录。该检测系统在纯培养物和奶粉样品中的检测限为5×10(0) cfu/mL，在猪肉样品中使用紫外光在不到2小时内的检测限为5×10(1) cfu/mL。此外，在最佳条件下，荧光强度与细菌浓度在10(2)至10(4) cfu/mL之间呈现良好的线性关系。此外，受污染奶粉和猪肉样品的检测结果表明，该检测系统可用于金黄色葡萄球菌的定量分析，并有可能应用于食品工业中该病原菌的检测。
