文章摘要
杨金玉;杨焕蝶;陈相艳;王易芬;朱丽萍;陈蕾蕾;.食品检测用4种病原菌基因组试剂盒提取方法的比较与优化[J].中国食品学报,2020,20(4):246-253
食品检测用4种病原菌基因组试剂盒提取方法的比较与优化
Comparison and Optimization of Genomic DNAs Extraction of Four Pathogenic Bacteria by Genomic DNA Extraction Kit for Foodborne Microbial Detection
  
DOI:
中文关键词: 食源性病原菌  基因组提取  方法优化  特征毒力基因
英文关键词: foodborne pathogen  genomic DNA extraction  method optimization  characteristic virulence genes
基金项目:科技部“食品安全关键技术研发”重点专项项目(2017YFC1601400); 国家自然科学基金青年科学基金项目(31801608); 山东省外专双百计划项目(WST2017004); 山东省重点研发计划项目(2019GNC106154); 山东省农业科学院创新工程项目(CXGC2017B06,CXGC2017A01); 泰山学者工程专项
作者单位
杨金玉;杨焕蝶;陈相艳;王易芬;朱丽萍;陈蕾蕾; 山东省农业科学院农产品研究所山东省农产品精深加工技术重点实验室农业农村部新食品资源加工重点实验室
山东师范大学生命科学学院
齐鲁工业大学(山东省科学院)生物工程学院山东省微生物工程重点实验室
 
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中文摘要:
      目的:采用国产的天根细菌基因组提取试剂盒,制备具有自主知识产权的、达到国家核酸标准物质要求的食源性病原菌的基因组样品。方法:以4种食源性病原菌大肠埃希氏菌O157:H7、单增李斯特氏菌、肠炎沙门氏菌和金黄色葡萄球菌为研究对象,首先使用进口OMEGA细菌基因组提取试剂盒和国产天根细菌基因组提取试剂盒提取4种病原菌的基因组,然后对国产试剂盒提取的基因组浓度和纯度较低的大肠埃希氏菌O157:H7、肠炎沙门氏菌和金黄色葡萄球菌的基因组提取方法进行优化。最后对4种病原菌基因组的完整性和纯度进行琼脂糖凝胶电泳检测,并分别对4种病原菌的特征毒力基因进行PCR扩增验证。结果:通过方法优化,使用国产天根细菌基因组提取试剂盒提取的4种病原菌的基因组质量浓度均在50 ng/μL以上,A260/A280和A260/A230的值在1.7~2.0之间,基因组DNA条带完整,无RNA和蛋白污染,各菌株的特征毒力基因均为阳性。结论:通过基因组提取方法的优化,得到符合国家核酸标准样品要求的4种病原菌基因组样品,为后续大量制备核酸标准样品奠定了基础。
英文摘要:
      Objective: This study describes preparing genomic DNA samples of foodborne pathogens which meet the requirements of national nucleic acid reference materials with independent intellectual property rights by using the domestic bacterial genomic DNA extraction kit. Methods: Four foodborne pathogens, Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enteritidis and Staphylococcus aureus were used in this study. The imported OMEGA bacterial genomic DNA extraction kit and the domestic TIANGEN bacterial genomic DNA extraction kit were used to extract the genomic DNAs of the four pathogens. And the genomic DNA extraction methods of E. coli O157:H7, S. enteritidis and S. aureus with low concentration and purity extracted from domestic TIANGEN kit were optimized. Finally, the integrity and purity of the genomic DNAs of the four pathogens were detected by agarose gel electrophoresis, and the characteristic virulence genes of the four pathogens were verified by PCR amplification. Results: The genomic DNAs’ mass concentrations of the four pathogens extracted by the domestic TIANGEN bacterial genomic DNA extraction kit were greater than or equal to 50 ng/μL, and the values of A260/A280 and A260/A230 were between 1.7-2.0. The bands of the genomic DNAs are clear, free of RNA and protein contamination, and the characteristic virulence genes of each strain are positive. Conclusion: Through the optimization of genomic DNA extraction method, four pathogen genomic samples conforming to the requirements of national nucleic acid standard materials were obtained, which laid a foundation for the subsequent preparation of large quantities of nucleic acid standard samples.
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