基于菌株水平肠道内植物乳杆菌定性定量研究方法的建立  

Establishment of Qualitative and Quantitative Method for Intestinal Lactobacillus plantarum at the Strain Level

作　　者：姜帅铭 马臣臣 游政凯 张家超[1] JIANG Shuaiming;MA Chenchen;YOU Zhengkai;ZHANG Jiachao(College of Food Science and Engineering,Hainan University,Haikou 570228,China)

机构地区：[1]海南大学食品科学与工程学院,海南海口570228

出　　处：《食品科学》2020年第14期82-87,共6页Food Science

基　　金：国家自然科学基金青年科学基金项目(31701577)。

摘　　要：以1株具有潜在益生功效的Lactobacillus plantarum HNU082为例,采用传统选择性分离培养和基因组学技术相结合的研究方法,从菌株定性研究角度,首先基于L. plantarum HNU082全基因组序列通过比较基因组学方法筛选获得该菌株的特异性片段,设计引物与其他L. plantarum菌株共扩增进行菌株特异性引物的筛选和验证。其次,基于菌株全基因组抗性基因的注释选取相应抗生素进行验证,使其更容易从宿主肠内容物中实现分离。通过选择性培养基的分离和特异性引物的验证完成菌株的定性分析。同时,基于菌株特异性引物,通过实时聚合酶链式反应技术完成菌株拷贝数的定量研究。最后,通过1例大鼠实验验证了本方法的可行性和准确性。基于此,建立了L. plantarum HNU082菌株在肠道内的定性定量研究方法。本研究建立完善的宿主肠道内L. plantarum HNU082定性定量研究方法,将为益生乳杆菌的体内研究提供重要方法学支持。In this study, we aimed to establish a qualitative and quantitative method for the potential probiotic strain of Lactobacillus plantarum HNU082 using traditional selective isolation and culture method and genomic technology. For the purpose of qualitative analysis, the specific DNA fragment of the strain was selected by comparative genomics based on its whole genome sequence. Primers were designed targeting this DNA fragment and validated by using it to co-amplify L. plantarum HNU082 with other strains of L. plantarum. Subsequently, based on the annotation of antibiotic resistance genes from the whole genome of strain HNU082, the corresponding antibiotics were selected for verification, which made it easier to isolate L. plantarum HNU082 from the intestinal contents of the host. The qualitative analysis of the strain was accomplished by bacterial isolation using a selective medium and verification of the specific primers. Besides, using the specific primers of the strain, the quantitative analysis of the copy number of the strain was performed by quantitative realtime PCR. Finally, we validated the feasibility and accuracy of this method through a rat experiment. The qualitative and quantitative method will provide important methodological support for the in vivo study of priobiotic Lactobacillus.

关 键 词：植物乳杆菌 肠道 抗性基因 特异性基因序列 实时聚合酶链式反应 

分 类 号：Q93-3[生物学—微生物学]