用于蔬菜残留耐药基因检测的DNA制备技术

摘要

目的 建立用于蔬菜残留耐药基因(antibiotic resistance genes,ARGs)检测的DNA制备技术.方法 以樱桃萝卜、香菜为研究对象,对商业试剂盒PowerWater?DNA Isolation kit和PowerPlant?Pro DNA Isolation kit中的具体步骤进行改良,不经细菌分离培养等过程,提取其可食用部分残留微生物基因组DNA.利用NanoDrop?微量紫外-可见光分光光度计,检测DNA的纯度和产量,并以DNA为模板,对16S rRNA、ARGs(sulⅠ、tetM、mef)进行PCR扩增,以验证本研究建立的DNA制备方法对于后续ARGs检测抑制物的去除情况.结果 本研究所制备的DNA(n=5),OD260/OD280值均在1.7～1.9之间,浓度均为10 ng/μL以上.经电泳检测后,目的基因的PCR扩增条带清晰,且经过序列分析比对证实,目的基因与以往文献报道的同源性均达到97％以上.结论 本研究建立的DNA制备方法,无需细菌分离培养,简单、省时,所获得的DNA纯净,为后续蔬菜残留ARGs检测等研究提供技术支持.%Objective To establish a method of DNA extraction for detection of antibiotic resistance genes (ARGs) from vegetables. Methods The specific steps of PowerWater? DNA Isolation kit and PowerPlant? Pro DNA Isolation kit were improved, and the microbial genome DNA was extracted from the fresh cherry radish and coriander samples without bacteria culture. The purity and yield of DNA were determined by NanoDrop-UV visible spectrophotometer, and the DNA was used as template to amplify the 16S rRNA and ARGs (sul ,Ⅰ tetM, mef) in order to verify the removal of inhibitor in the subsequent ARGs detection. Results The values of OD260/OD280 were ranged from 1.7 to 1.9, and the yields of DNA were more than 10 ng/μL (n=5). After electrophoresis, the PCR amplification bands of the 16S rRNA and ARGs (sul ,Ⅰ tetM, mef) were clear. After comparing with GenBank sequences for the target genes using the BLAST alignment tool (http://www.ncbi.nlm.nih.gov/blast/), the identity were all more than 97％. Conclusion The established method is simple and rapid without bacteria culture, and the extracted DNA is pure, which is suitable for subsequent ARGs detection from vegetables.