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被你嫌弃的它,却为科学打开了“一扇门”

生信草堂  · 公众号  ·  · 2017-07-08 18:00

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现在国际上最具有影响力的微生物组研究项目有:Human Microbiome Project (HMP),Metagenomics of Human Intestinal Tract (MetaHIT),Earth Microbiome Project (EMP)

  

随着HMP,MetaHIT,EMP的开展,科学家们积累了越来越多微生物组学的研究经验,也使得更多科研工作者投入到该领域的研究中。在人体中,肠道微生物结构最为复杂,是人类微生物组研究的焦点。基于临床资料和文献报道,本文重点讨论开展肠道微生物组研究最重要的环节,即样本采集。


1. 样本筛选

1.1 基本信息

(1)年龄:在18到65岁

(2)饮食:1周内未食用酸奶

(3)人群:该地区常住人口

(4)非孕妇,哺乳期妇女

1.2 药物

两个月内无抗生素,益生菌、益生元、合生元,铋碱式水杨酸盐药物的使用;无药物及酒精滥用

1.3 疾病

(1)胃肠道疾病:无炎症性肠病(IBD),肠易激综合症(IBS),胃肠炎、结肠炎或胃炎,胃肠道出血、溃疡,胃肠道息肉或包块或发育不良,其他原因的慢性或持续性便秘或者腹泻

(2)慢性及代谢性疾病:无高血压,糖尿病,心血管疾病,糖尿病,肥胖,肝硬化,脂肪肝,癌症

(3)感染:无急慢性细菌、真菌、病毒感染

(4)手术:无较大的胃肠道手术

【参考:1,11-14】


2. 材料准备

表1:耗材和仪器列表


产品

公司

货号

价格

便盆

Fisherbrand™ Commode Specimen Collection System

Thermo

02-544-208

$216.09 / Case of 60


采集管

Faeces container 76x20mmlabel, sterile

Sarstedt

80.734.311

¥4.4 / Case of 1

分装管

Glass Bead Tubes, 0.1mm

QIAGEN

13118-50

$145 / Case of 50

试剂盒

DNeasy PowerLyzer PowerSoil Kit


QIAGEN

12855-50

12855-100

$274 / Case of 50

$514   / Case of 100

匀浆器

PowerLyzer 24 Bench Top Bead-Based Homogenizer

QIAGEN

13155

$9,949

其他

冰,冰盒,称量仪器,分装管,自封袋,无菌牙签,-80度冰箱,一次性手套,干冰

【备注】

1. MOBIO在2017年已被QIAGEN收购

2. 试剂盒选择参考:

(1)大量的肠道微生物组学研究使用【参考:1-10】

(2)HMP,EMP protocol引用


3. 采集方法

3.1 标记

医生标记病人信息(姓名,编号,采样时间等)

3.2 准备

病人取便盆,粪便采集管,一次性手套,自封袋

3.3 粪便采集

(1)嘱咐病人先排尽尿,再用干净的便盆收集粪便,如:

【注意】避免尿液,厕所里的其他液体,纸等污染样本

(2)用粪便收集器的刮勺,取粪便内部葡萄或核桃大小(>2.5g)的粪便到收集器,如:

(3)拧上盖子,丢弃并冲走粪便,丢掉塑料袋,手套,洗手,尽快把样本交给医生

【注意】尽量减少粪便暴露在空气的时间(减少外界微生物污染,样本细菌扩增和DNA降解造成检测结果不准);采样过程在15分钟之内完成

3.4 分装

(1)用无菌牙签挑取内部粪便到分装管,6-10管,每管0.25g

(2)完成后立即放到-80度保存

【注意】整个分装过程在冰上完成;整个采样,分装过程在30分钟之内完成

3.5 保存和转移

-80度保存,转移用干冰


4. DNA提取

4.1 DNA提取

(1)向含样本的Glass Bead Tubes加Bead Solution,用涡旋仪或匀浆器均质化样本

(2)根据试剂盒说明书提取DNA

4.3 核酸定量及保存

(1)DNA浓度测定(A260/A280:1.8-2.0),凝胶电泳鉴定

(2)20/-80度保存


参考文献:

[HMP] Product Information and Hyperlink Updates for the Human Microbiome Project (HMP) Manual of Procedures (MOP) 19 July 2012

[HMP] Manual of Procedures for Human Microbiome Project Core Microbiome Sampling Protocol A HMP Protocol # 07-001 Version Number: 12.0 29 Jul 2010

[EMP] DNA Extraction Protocol (EMP, 2011); http://www.earthmicrobiome.org/empstandard-protocols/dna-extraction-protocol/

[1] Gut microbiome remodeling induces depressive-like behaviors through a pathway mediated by the host’s metabolism. Molecular Psychiatry (2016)

[2] Synthetic long-read sequencing reveals intraspecies diversity in the human microbiome. Nat Biotechnol. 2016

[3] Capturing One of the Human Gut Microbiome's Most Wanted: Reconstructing the Genome of a Novel Butyrate-Producing, Clostridial Scavenger from Metagenomic Sequence Data. Front Microbiol. 2016

[5] Microbiota Transfer Therapy alters gut ecosystem and improves gastrointestinal and autism symptoms: an open-label study. Microbiome (2017)

[6] Structure and function of the healthy pre-adolescent pediatric gut microbiome. Microbiome. 2015

[7] Influence of early life exposure, host genetics and diet on the mouse gut microbiome and metabolome. NATURE MICROBIOLOGY 2016

[8] Dynamics of the human gut microbiome in inflammatory bowel disease. Nat Microbiol. 2017

[9] Genetic and environmental control of host-gut microbiota interactions. Genome Research 2015

[10] The severity of nonalcoholic fatty liver disease is associated with gut dysbiosis and shift in the metabolic function of the gut microbiota. Hepatology. 2016

[11] Alterations of the human gut microbiome in liver cirrhosis. Nature, 2014

[12] Metformin alters the gut microbiome of individuals with treatment-naive type 2 diabetes, contributing to the therapeutic effects of the drug. Nat Med, 2017.

[13] The Human Microbiome Project strategy for comprehensive sampling of the human microbiome and why it matters. The FASEB Journal • Research Communication 2012

[14] Altered fecal microbiota composition in patients with major depressive disorder. Brain Behav Immun, 2015