一种高效稳定的微生物总RNA提取方法  

A high-efficiency and stable method for extracting total RNA from micro-organisms

作　　者：吴思琪[1] 宋静颐 秦倩[2] 张策[3] 金君华[1] 谢远红[1] 刘慧[1] 张红星[1] 

机构地区：[1]北京农学院食品科学与工程学院食品质量与安全北京实验室/农产品有害微生物及农残安全检测与控制北京市重点实验室/微生态制剂关键技术开发北京市工程实验室/北京市食品安全免疫快速检测工程技术研究中心,北京102206 [2]中国青年政治学院,北京100089 [3]北京市经济管理学校,北京100142

出　　处：《江苏农业学报》2017年第3期517-523,共7页Jiangsu Journal of Agricultural Sciences

基　　金：北京市教委科研计划支持项目(KM201610020015);北京市属高等学校高层次人才引进与培养长城学者计划(CIT&TGD20140315);杨胜先生门生社群项目(C2016028)

摘　　要：本研究通过对玻璃珠-酚仿RNA提取方法进行改进优化,实现对革兰氏阳性菌、革兰氏阴性菌和真菌这3类微生物RNA高效稳定的提取。结果表明,玻璃珠-酚仿提取法能有效提取的RNA最低菌体细胞量为1×107个,远低于试验所选商业化试剂盒所需细胞量。以1×107个细胞量为例,利用Beadbeater仪破碎30 s,使用直径100μm的玻璃珠破碎提取得到的细菌RNA浓度显著高于使用直径500μm的玻璃珠提取的。使用2种直径的玻璃珠所得酵母菌RNA浓度无显著性差异。在直径100μm的条件下,破碎30 s即可保证RNA的高效提取,对于长双歧杆菌、大肠杆菌和酵母菌,破碎时间长达150 s也不会引起RNA降解。本法所提RNA的A260/A280为1.96~2.10,A260/A230为2.03~2.44,浓度和纯度均优于试验所选商业化试剂盒所提的RNA。In this study, the glass bead-phenol/chloroform RNA extraction method was optimized to realize the high-ly efficient and stable extraction of RNA from three kinds of microorganisms, gram positive bacteria, gram negative bacteria and fungus. The minimum thalli cell number that could be extracted by glass bead-phenol RNA extraction was 1＆#215;107, far less than that extracted by commercialized kit. The concentration of bacterial RNA extracted by glass beads withΦ= 100μm from 1＆#215;107 cells was significantly higher than that with Φ= 500 μm. For the yeast, there was no significant difference in RNA concentration extracted by glass beads with different diameters. Glass beads with 100μm diameter breaking for 30 s could ensure the efficient extraction of RNA, and the degradation of RNA did not happen even broken for 150 s for Bifidobacterium longum, Escherichia coli and yeast. The concentration and purity of the RNA extracted by optimized method were better than those by commercialized kit.

关 键 词：微生物 RNA提取 玻璃珠-酚仿 

分 类 号：Q522[生物学—生物化学]