文章摘要
3种粪便标本保存方法对肠道菌群检测结果影响的比较研究
Comparison of Three Methods of Stool Samples Preservation Affecting Detection Results of Enteric Microbial Population
  
DOI：doi:10.3969/j.issn.1005-7021.2020.03.012
中文关键词: 肠道菌群  粪便标本  保存方法  高通量测序
英文关键词: enteric microbial population  stool samples  preservation method  high-throughput sequencing
基金项目:江苏省产学研合作项目（BY2018179）
作者单位
贠航 苏州大学 医学部护理学院，江苏 苏州 215006 
胡闭月 苏州大学 医学部护理学院，江苏 苏州 215006 
王健 苏州硒泰克生物科技有限公司研究中心，江苏 苏州 215123 
郭仁妹 苏州硒泰克生物科技有限公司研究中心，江苏 苏州 215123 
王丽 苏州大学 医学部护理学院，江苏 苏州 215006 
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中文摘要:
      比较3种粪便标本保存方法对肠道菌群检测结果的影响，为采集粪便标本后短期保存方法的选择提供参考。收集10名志愿者的新鲜粪便标本，对每份标本分别采用4 ℃、室温（25 ℃）、无水乙醇保存4 h，然后统一放入-80 ℃低温冰箱冻存。随后进行细菌DNA提取，并对16S rRNA V4区基因进行扩增，应用 Illumina MiSeq平台进行高通量测序，比较3种保存方法下肠道菌群多样性和组成差异。结果表明，3种保存方法下，肠道菌群在Alpha多样性方面无统计学差异（P>0.05）。肠道菌群属水平比较，毛螺旋菌属（Lachnospira）在无水乙醇组中相对丰度高于4 ℃组和室温组（P<0.05），萨特菌属（Sutterella）在无水乙醇组中相对丰度高于4 ℃组（P<0.05），而真杆菌属（Eubacterium）在无水乙醇组中相对丰度低于室温组（P<0.05）。PCoA分析发现，当UniFrac距离加权后，4 ℃和无水乙醇保存同一受试者粪便标本，菌群结果较为接近，而室温保存会导致个别受试者菌群出现离散现象。无水乙醇短期保存粪便标本，肠道菌群多样性、组成以及结构保持稳定，能满足不同研究的需要。此外，无水乙醇具备价格低廉、便于携带等优点。因此，在新鲜粪便标本不能立即低温冻存时，无水乙醇保存可以作为首要选择。
英文摘要:
      Effects of three stool samples preservation methods on the detection results of enteric microbial population were compared in order to provide reference for the selection of short term preservation methods after collecting stool sample. Fresh stool samples from 10 volunteers were collected, each one was stored at 4 ℃, room temperature (25 ℃) and anhydrous ethanol for 4 hours, and then, all of them were put into cryogenic refrigerator at -80 ℃ for freeze preservation. Subsequently, bacterial DNA was extracted, and then amplified their 16S rRNA V4 region genes, then, Illumina Miseq platform was applied for high throughput sequencing to compare the diversity and composition differences of enteric microbial population by the three preservation methods. The results showed that there was no significant statistic differences in Alpha diversity of enteric microbial population among the three preservation methods (P>0.05). Comparison of enteric microbial population at genera level showed that the relative abundance of Lachnospira in the anhydrous ethanol group was higher than that in the 4 ℃ group and the room temperature group (P<0.05), and the relative abundance of Sutterella in the anhydrous ethanol group was higher than that in the 4 ℃ group (P<0.05). Moreover, the relative abundance of Eubacterium in the anhydrous ethanol group was lower than that in the room temperature group (P<0.05). PCoA analysis showed that when UniFrac distance after weighted, the stool samples of the same subjects stored at 4 ℃ and anhydrous ethanol, and the results of enteric microbial population were relatively close, while room temperature preservation may lead to the dispersal of enteric microbial population in individual subjects. Anhydrous ethanol could preserve stool samples for a short time, and keep the diversity, composition and structure of enteric microbial population remain stable, and could meet the needs of different researches. In addition, anhydrous ethanol has the advantages of low cost and portability. Therefore, when fresh stool samples could not be frozen immediately, anhydrous ethanol preservation could be taken as a primary choice.
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