用于熟制食品中耐药基因检测的DNA制备技术  

DNA preparation techniques for the detection of antibiotic resistance genes from cooked foodstuffs

作　　者：李娟[1] 段佳丽[1] 陈忠辉[1] LI Juan;DUAN Jiali;CHEN Zhonghui(Beijing Center for Disease Prevention and Control,Beijing 100013,China)

机构地区：[1]北京市疾病预防控制中心,100013

出　　处：《首都公共卫生》2024年第6期323-329,共7页Capital Journal of Public Health

基　　金：国家自然科学基金面上项目(编号:21976020);国家自然科学基金青年基金项目(编号:21607007);北京市委组织部优秀人才资助项目(编号:2016000021469G182);北京市疾病预防控制中心科研培育专项(编号:2020-BJYJ-01)。

摘　　要：目的改进细菌总DNA制备技术,用于熟制食品中抗生素耐药基因(antibiotic resistance genes,ARGs)的检测及进一步分析。方法经过清蒸、水煮、爆炒等三种传统中式烹饪加工后的蔬菜、肉、水产品等食品作为研究对象,针对样品的不同特性,通过改善离心条件、提升DNA吸附和洗脱,以及优化DNA萃取缓冲液用量等方式,改良商业试剂盒中的具体步骤,不经细菌分离培养等过程,直接提取熟制食品细菌的总DNA。通过DNA纯度和浓度的测量,以及16S rRNA扩增、高通量测序、SmartChip高通量实时定量PCR(high-throughput quantitative real-time PCR,HT-qPCR)等分子生物学操作,以验证本研究改进的细菌总DNA制备技术能够用于后续ARGs分析。结果本文制备的DNA,OD_(260)/OD_(230)和OD_(260)/OD_(280)的值分别在2.00~2.13和1.70~2.00,浓度均在50 ng/μL以上。经分子生物学技术验证,本文提取的DNA能够确保后续PCR、HT-qPCR等方法用于ARGs的检测,以及细菌群落结构分析的顺利进行。结论本文对于细菌总DNA制备技术的改进,能够为后续熟制食品中ARGs的检测及进一步分析提供技术支持。Objective This study aims to improve DNA extraction for the detection of antibiotic resistance genes(ARGs)in cooked foodstuffs.Methods In this study,based on the current commercial kit,specific steps were improved,such as improving centrifugation conditions,enhancing DNA adsorption and elution,and optimizing amount of DNA extraction buffer.The microbial genome DNA was extracted from the cooked foodstuffs(including vegetables,meat,aquatic products)with steaming,boiling and stir-frying,without the bacteria culture.The DNA purity and concentration were measured,and method such as 16S rRNA amplification,high-throughput sequencing,and SmartChip high-throughput quantitative real-time PCR(HT-qPCR)were used to verify the suitability of the improved DNA preparation method for subsequent ARGs analysis.Results The prepared DNA had OD_(260)/OD_(230)and OD_(260)/OD_(280)values ranging from 2.00-2.13 and 1.70-2.00,respectively,with a concentration above 50 ng/μL.After verification by molecular biology technology,the DNA extracted in this study can be used for microbial community analysis and ARGs detection in cooked foodstuffs.Conclusions The DNA extraction method improved in this study would provide technical support for ARGs detection and sequent analysis in cooked foodstuffs.

关 键 词：熟制食品 细菌群落结构 ARGs DNA制备技术 HT-qPCR 

分 类 号：TS251.6[轻工技术与工程—农产品加工及贮藏工程] R155.5[轻工技术与工程—食品科学与工程]