生物技术通报 ›› 2016, Vol. 32 ›› Issue (11): 137-143.doi: 10.13560/j.cnki.biotech.bull.1985.2016.11.016

• 技术与方法 • 上一篇    下一篇

宏基因组靶向测序分析皮肤表面微生物群落方法优化

姚雪1, 刘文丽2, 裴广倩2, 童贻刚2, 罗亚平1   

  1. 1. 中国人民公安大学刑事科学技术学院,北京 100038;
    2. 军事医学科学院微生物流行病研究所,北京 100071
  • 收稿日期:2016-03-28 出版日期:2016-11-25 发布日期:2016-11-11
  • 作者简介:姚雪,女,博士研究生,研究方向:刑事科学技术;E-mail:yaoxue1986@163.com
  • 基金资助:
    北京市科技新星计划交叉学科合作课题(xxhz201510),上海市刑事科学技术研究院现场物证重点实验室开放课题(2014XCWZK14)

Optimization of Method for Analyzing Microbial Community on Human Skin by Targeted Sequencing of Metagenomics

YAO Xue1, LIU Wen-li2, PEI Guang-qian2, TONG Yi-gang2, LUO Ya-ping1   

  1. 1. School of Criminal Science and Technology,People's Public Security University of China, Beijing 100038;
    2. Institute of Microbiology and Epidemiology,the Academy of Military Medical Sciences,Beijing 100071
  • Received:2016-03-28 Online:2016-11-25 Published:2016-11-11

摘要: 通过实验设计和数据分析,筛选出尽可能真实反映含量较低的皮肤微生物群落的高通量测序分析方法。使用多因素多水平完全随机实验设计,分析DNA提取、PCR扩增、样品采集、测序过程中各种实验因素对微生物群落组成特征分析结果的影响。结果显示,在样品采集方面,筛选出了最佳的采集拭子与采集液体;在DNA提取方面,筛选出了最佳的前处理方法及试剂盒种类;在PCR反应方面,筛选出了最佳的模板浓度及退火温度;在测序方面,筛选出了合适的测序深度。通过一系列的实验条件筛选,确定了适合分析低含量细菌群落结构组成特征的方法,并成功地应用于人类手掌面皮肤。

关键词: 皮肤表面微生物群落, 宏基因组靶向测序, 16S rRNA基因

Abstract: Based on the experimental design and data analysis, we screened a high-throughput sequencing method which may measure the community of microorganisms in a low level on the human skin as real as possible. By completely random design of the experiments with multi-factor multi-level, we analyzed the effects of varied factors in the DNA extraction, PCR amplification, sample collection and sequencing on the composition characteristics of microbial community. The results showed that regarding the sample collection, the optimal collection swab and extraction liquid were screened out. In the aspect of DNA, the optimal pretreatment method and reagent box type were selected; on PCR reaction, the optimal template concentration and annealing temperature were determined; and on the sequencing aspect, the suitable sequencing depth was also chosen. In conclusion, through above series of experiments to screen the conditions, the appropriate method for analyzing the composition characteristics of community structure of bacteria in a low level was determined, and successfully applied to human palm skin.

Key words: microbial community on human skin, targeted sequencing of metagenomics, 16S rRNA gene