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

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(P0.05)。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(P0.05),萨特菌属(Sutterella)在无水乙醇组中相对丰度高于4℃组(P0.05),而真杆菌属(Eubacterium)在无水乙醇组中相对丰度低于室温组(P0.05)。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。     

粪便样本不同保存方式对肠道菌群测序结果的影响

[目的]本文探究了3种室温保存剂和-80℃C冷冻保存对粪便样本中菌群结构的影响,为大规模、标准化的采样提供参考.[方法]本研究采集了5名健康志愿者的新鲜粪便作为测试样本,采用4种不同的保存方式保存:DETs室温保存、GITC室温保存、RNAlater室温保存和-80℃冷冻保存,在保存0、1、3、7、14、28 d后,采...     

三种粪便总DNA提取方法的比较

目的比较不同粪便总DNA提取方法对肠道菌群多样性研究的影响。方法采用Bead beating法、化学裂解法和QIAamp DNA Stool Mini Kit提取同一份人粪便样品的总DNA,对比3种方法的DNA得率和16S rRNA基因V3区的变性梯度凝胶电泳（DGGE）图谱。结果Bead beating法的DNA得率约是其他2种方法的2倍;3种方法得到的DGGE图谱的Dice相似性为60％～70％,2条优势条带只出现在Bead beating法图谱中。在2～5min的Bead beating法击打时间里,DNA得率随击打时间的延长有一定的增加,但DGGE图谱无显著变化。结论不同的DNA提取方法会影响菌群的多样性分析。比较其他2种方法,Bead beating的裂解效率更高,能够检测到更多种类的细菌,更合适肠道菌群组成的分子研究。     

肉鸭养殖过程中发酵床垫料菌群结构变化的研究

【目的】探究发酵床使用时间和肉鸭粪便微生物对发酵床垫料菌群结构、总菌和大肠杆菌数量的影响。【方法】采集江苏某肉鸭发酵床养殖场内刚制作完成的发酵床垫料样品,及其饲养4批次、8批次后的垫料样品,同时采集各批次34日龄肉鸭粪便样品,采用变性梯度凝胶电泳技术(Denaturing gradient gel electrophoresis,DGGE)、16S rRNA基因序列分析和实时荧光定量PCR (Quantitative real-time PCR)技术对发酵床使用过程中垫料菌群结构进行定性和定量研究。【结果】0批次(D0)与4批次(D4)、8批次(D8)垫料菌群相似性分别为68.81%、70.82%,而4批次与8批次间垫料菌群的相似性则达81.93%,显著高于D4、D8与D0间相似性(P<0.05)。条带6、8 (最相似菌分别为Leqionella tunisiensis、Pedobacte bauzanensis)在3个时间点垫料菌群中均表现优势,且含量较为稳定;条带10 (最相似菌为Rummeliibacillus suwonesis)仅在2个重复使用垫料菌群中表现优势;条带12、13 (最相似菌分别是Psychrobacter sp. PRwf-1、Iamia majanohamensis)共同存在于垫料样和粪便样。肉鸭粪便中大肠杆菌的数量显著高于4批次、8批次垫料中的数量(P<0.05),与0批次垫料间差异不显著(P>0.05)。【结论】使用时间和肉鸭粪便微生物共同影响了发酵床垫料菌群结构和数量,菌群结构随使用时间的延长而趋于稳定。     

ERIC-PCR指纹图谱技术分析糖尿病小鼠肠道细菌群落变化

目的通过比较1型糖尿病模型组和空白对照组雄性小鼠肠道菌群结构的变化,探索糖尿病造模与肠道菌群的关系。方法收集造模2周后空白对照组(n=5)、STZ造模成功组(n=5)和造模不成功组(n=3)ICR小鼠的新鲜粪便样品,提取粪便样品的总DNA,ERIC-PCR扩增形成DNA指纹图谱,借助多变量统计分析方法研究各组样品肠道菌群结构上的异同。结果ERIC-PCR指纹图谱结合偏最小二乘法(PLS-DA)分析表明造模成功组和造模不成功组小鼠的肠道菌群结构显著区别于空白对照组,而造模不成功组小鼠的肠道菌群结构与造模成功组仍有一定的区别。结论STZ诱导的1型糖尿病会造成小鼠的肠道菌群结构的变化,而部分小鼠造模失败可能与这些小鼠的肠道菌群结构有关。     

四氯化碳一过性处理对巴马香猪肠道菌群的影响

目的探讨四氯化碳（CCl4）一过性处理对巴马香猪肠道菌群的影响,评估CCl4诱导处理的肝损伤动物模型能否用于肠道微生态研究。方法选取3头雄性巴马香猪,一次性按每千克体重腹腔注射0.25 ml[0.25ml/（kg·bw）]40%CCl4橄榄油溶液。于注射12 h之后的第1、2、3、4、7和10天连续采集粪便,以注射CCl4溶液之前所采集粪便样作对照。分别提取粪样总DNA,采用变性梯度凝胶电泳技术（DGGE）对CCl4一过性处理后巴马香猪肠道菌群的多样性变化进行动态监测。结果PCR-DGGE法分析表明,巴马香猪肠道菌群组成在注射CCl4前后差异无显著性（P〉0.05）。相似性聚类分析结果表明,菌群组成在注射CCl4前后相似度在65%以上。结论CCl4一过性处理后巴马香猪肠道菌群组成未发生显著变化。表明CCl4处理诱导的肝损伤动物模型可以用来研究相关疾病与肠道菌群之间的关系。     

高通量 16S rRNA 标签测序法比较人与不同动物肠道微生物组多样性

收集人、大猪、小猪、大鼠、小鼠以及鸡五个不同个体的粪便样品 , 每个个体取两个平行样 , 提取总 DNA;PCR 扩增 , 获得 16S rRNA V6 标签片段 ; Illumina 测序 ; 经 BIPES 以及 QIIME 分析并比较菌群结构及多样性。研究结果发现 , 不同物种之间肠道菌群差异较大。五类物种肠道菌群均以厚壁菌门、拟杆菌门、以及变形菌门为主 , 但鸡的厚壁菌门显著减少 , 而变形菌门显著增加。从α多样性角度来看 , 猪肠道菌群种属丰富度及 Shannon 指数均显著高于人及鸡肠道菌群。从β多样性角度 , 尽管不同人之间的肠道菌群相似度较低 , 但不同物种之间相比较 , 小猪与大鼠肠道菌群与人相似性高于小鼠和鸡肠道菌群。与人类相比 , 小猪的肠道微生物组最相近 , 而鸡的肠道菌群相似度最低。     

基于PCR DGGE的小鼠肠道菌群基因组提取方法的建立

通过比较4种小鼠粪便细菌总DNA提取方法对基于PCR-DGGE检测的肠道菌群多样性分析的影响,旨在建立适于PCR—DGGE的小鼠肠道微生物宏基因组提取的稳定、经济、快捷的方法。采用SDS裂解法、某国产市售粪便DNA提取试剂盒、改进的化学裂解法、改进的溶菌酶法4种方法提取小鼠粪便细菌总DNA,通过琼脂糖凝胶电泳、紫外分光光度法、细菌16S rRNAV3区PCR扩增结合DGGE对提取结果进行比较分析。SDS裂解法和国产市售试剂盒2种方法提取粪便细菌总DNA均未得到理想结果,另2种方法均能够检测到粪便中20种左右的细菌。改进的化学裂解法和改进的溶菌酶提取法的建立为基于PCR—DGGE进行肠道菌群结构的定量及定性分析提供了可靠的前提基础和实验保障。     

造血干细胞移植术对肠道菌群结构的影响

目的监测造血干细胞移植术（Hematopoietic stem cell transplantation,HSCT）前后肠道菌群结构的动态变化。方法收集3例造血干细胞移植患者手术前后8个时间点的粪便样品,提取样品总DNA进行16S rRNA基因的V3区的bar coded 454焦磷酸测序,并用MANOVA、聚类分析、Pearson相关等统计方法对菌群结构的变化进行动态分析。结果 HSCT移植前,经过放、化疗及预防性抗生素治疗,患者的肠道菌群结构和组成发生显著的改变,多样性明显减少;移植4周后,菌群多样性有恢复的趋势,但菌群结构和组成与治疗前仍有明显的差异。整个HSCT过程中,Escherichia/Shigella及Enterococcus属变成肠道中最优势的细菌类群。结论肠道菌群结构在HSCT术前已发生显著的改变,机会致病菌Escherichia/Shigella及Enterococcus属成为HSCT患者肠道中最优势的细菌类群。     

老龄小鼠术后认知功能障碍和肠道菌群失调的关系

摘要 目的：评价肠道菌群失调和老龄小鼠术后认知功能障碍的潜在作用机制和关系。方法：清洁级健康雄性老龄小鼠 C57BL/6J 20只,16-17月龄,体重28-35ｇ,采用随机数字表法分为两组：对照组（A=10只）和手术组（AS=10只）。AS组小鼠行剖腹探查术。术后48小时采集小鼠粪便和血清标本,16S rDNA测序检测粪便肠道细菌丰度变化,Elisa法检测血清的IL-1β、IL-6和TNF-α含量。术后3天行Morris水迷宫实验。结果：与A组比较,AS组小鼠术后逃避潜伏期延长,空间探索试验在目标象限所花的时间减少（P＜0.05）;α多样性与β多样性指数分析两组小鼠粪便肠道菌群的丰富度和多样性发生差异性改变,AS组的肠道菌群结构中门（6种）和属（15种）水平相对丰度的差异较显著（P＜0.05）;两组小鼠血清中IL-1β、IL-6和TNF-α水平表达对比差异无统计学意义（P＞0.05）。结论：肠道菌群失调改变可能与老龄小鼠术后认知功能障碍的发病机制有关。     

ERIC-PCR分子杂交技术分析大熊猫肠道菌群结构

目的了解大熊猫肠道微生物区系结构的相似性和稳定性,并找出大熊猫肠道微生物群落结构的变化与健康状况的关系。方法对上海动物园及上海野生动物园所饲养的3只大熊猫2次采集的粪便样品进行微生物群落总DNA的抽提,并以此为模板获得反映肠道微生物群落结构特征的ERIC—PCR和Southern杂交指纹图谱,比较各DNA样品指纹图谱的相似性指数。结果除国庆(大熊猫)的第1次采集的样品(当时处于腹泻状态),其他各DNA样品的ERIC-PCR及Southern杂交指纹图谱的相似性都达到85％～100％;佳斯及川川(大熊猫)2个个体2次采集的样品之间ERIC指纹图谱的相似性分别为93％和87％,而国庆腹泻时的样品与健康时的样品之间则为71％。结论大熊猫不同个体之间肠道微生物群落结构比较相似,而且同一个体在不同时期表现出比较高的稳定性,但当个体的健康出现问题时肠道优势菌菌群结构有一定波动。所采用的DNA提取方法、ERIC—PCR和Southern杂交指纹图谱的高度重复性证明了之一分子生态学技术在大熊猫肠道微生物区系动态监测中的可行性。     

Characterization of the Fecal Microbiota Using High-Throughput Sequencing Reveals a Stable Microbial Community during Storage

The handling and treatment of biological samples is critical when characterizing the composition of the intestinal microbiota between different ecological niches or diseases. Specifically, exposure of fecal samples to room temperature or long term storage in deep freezing conditions may alter the composition of the microbiota. Thus, we stored fecal samples at room temperature and monitored the stability of the microbiota over twenty four hours. We also investigated the stability of the microbiota in fecal samples during a six month storage period at −80°C. As the stability of the fecal microbiota may be affected by intestinal disease, we analyzed two healthy controls and two patients with irritable bowel syndrome (IBS). We used high-throughput pyrosequencing of the 16S rRNA gene to characterize the microbiota in fecal samples stored at room temperature or −80°C at six and seven time points, respectively. The composition of microbial communities in IBS patients and healthy controls were determined and compared using the Quantitative Insights Into Microbial Ecology (QIIME) pipeline. The composition of the microbiota in fecal samples stored for different lengths of time at room temperature or −80°C clustered strongly based on the host each sample originated from. Our data demonstrates that fecal samples exposed to room or deep freezing temperatures for up to twenty four hours and six months, respectively, exhibit a microbial composition and diversity that shares more identity with its host of origin than any other sample.     

The Effect of Sampling and Storage on the Fecal Microbiota Composition in Healthy and Diseased Subjects

Large-scale cohort studies are currently being designed to investigate the human microbiome in health and disease. Adequate sampling strategies are required to limit bias due to shifts in microbial communities during sampling and storage. Therefore, we examined the impact of different sampling and storage conditions on the stability of fecal microbial communities in healthy and diseased subjects. Fecal samples from 10 healthy controls, 10 irritable bowel syndrome and 8 inflammatory bowel disease patients were collected on site, aliquoted immediately after defecation and stored at -80°C, -20°C for 1 week, at +4°C or room temperature for 24 hours. Fecal transport swabs (FecalSwab, Copan) were collected and stored for 48-72 hours at room temperature. We used pyrosequencing of the 16S gene to investigate the stability of microbial communities. Alpha diversity did not differ between all storage methods and -80°C, except for the fecal swabs. UPGMA clustering and principal coordinate analysis showed significant clustering by test subject (p<0.001) but not by storage method. Bray-Curtis dissimilarity and (un)weighted UniFrac showed a significant higher distance between fecal swabs and -80°C versus the other methods and -80°C samples (p<0.009). The relative abundance of Ruminococcus and Enterobacteriaceae did not differ between the storage methods versus -80°C, but was higher in fecal swabs (p<0.05). Storage up to 24 hours (at +4°C or room temperature) or freezing at -20°C did not significantly alter the fecal microbial community structure compared to direct freezing of samples from healthy subjects and patients with gastrointestinal disorders.     

Fecal collection, ambient preservation, and DNA extraction for PCR amplification of bacterial and human markers from human feces

Feces contain intestinal bacteria and exfoliated epithelial cells that may provide useful information concerning gastrointestinal tract health. Intestinal bacteria that synthesize or metabolize potential carcinogens and produce anti-tumorigenic products may have relevance to colorectal cancer, the second most common cause of cancer deaths in the USA. To facilitate epidemiological studies relating bacterial and epithelial cell DNA and RNA markers, preservative/extraction methods suitable for self-collection and shipping of fecal samples at room temperature were tested. Purification and PCR amplification of fecal DNA were compared after preservation of stool samples in RNAlater (R) or Paxgene (P), or after drying over silica gel (S) or on Whatman FTA cards (W). Comparisons were made to samples frozen in liquid nitrogen (N2). DNA purification methods included Whatman (accompanying FTA cards), Mo-Bio Fecal (MB), Qiagen Stool (QS), and others. Extraction methods were compared for amount of DNA extracted, DNA amplifiable in a real-time SYBR-Green quantitative PCR format, and the presence of PCR inhibitors. DNA can be extracted after room temperature storage for five days from W, R, S and P, and from N2 frozen samples. High amounts of total DNA and PCR-amplifiable Bacteroides spp. DNA (34%+/-9% of total DNA) with relatively little PCR inhibition were especially obtained with QS extraction applied to R preserved samples (method QS-R). DNA for human reduced folate carrier (SLC19A1) genomic sequence was also detected in 90% of the QS-R extracts. Thus, fecal DNA is well preserved by methods suitable for self-collection that may be useful in future molecular epidemiological studies of intestinal bacteria and human cancer markers.     

Optimization of terminal restriction fragment polymorphism (TRFLP) analysis of human gut microbiota

Some compounds originating from the human gut microbial metabolism of exogenous and endogenous substrates may have properties that profoundly affect the host's physiological processes. The influence of these metabolites on differences in disease risk among individuals could be mediated by metabolism specific to the gut microbial community composition. In this study, we evaluated the effectiveness of terminal restriction fragment polymorphism (TRFLP) as a biomarker of the fecal microbial community (as a surrogate of gut microbiota) for application in human population-based studies. We tested the effects of experimental conditions on DNA quality, DNA quantity, and TRFLP patterns derived from gut bacterial communities. Genomic DNA was extracted from fecal slurries and the bacterial 16S rDNA genes were amplified and analyzed by TRFLP. We found that the composition of the TRFLP fingerprints varied by different extraction procedure. The best quality and quantity of community DNA extracted from fecal material was obtained by using the QIAamp DNA stool minikit (Qiagen, Valencia, CA) with 95 degrees C incubation and moderate bead beating treatment during the cell-lysis step. Homogenization of fecal samples reduced variation among replicates. Once the TRFLP procedure was optimized, we assessed the methodological and inter-individual variation in gut microbial community fingerprints. The methodological variation ranged from 4.5-8.1% and inter-individual variation was 50.3% for common peaks. In conclusion, standardized TRFLP is a robust, reproducible, and high-throughput method that will provide a useful biomarker for characterizing gut microbiota in human fecal samples.     

Long-term stability of the human gut microbiota in two different rat strains

Human microbiota associated rats are frequently used as a model to study host microbe interactions. This study investigated the long-term stability of the bacterial community in such rats. Following the association of two strains of germ-free rats (12 male animals each) with fecal bacteria from a human donor the development of the microbiota was monitored for 12 months by PCR-denaturing gradient gel electrophoresis. During this time the Dice similarity coefficient (Cs) for the fecal microbial community of the rats associated with a human microbiota in comparison to the donor sample ranged between 73% +/- 8 and 74% +/- 3 for the Wistar and the Fischer 344 rats, respectively. After 12 months the similarity coefficients were 78% +/- 9 and 76% +/- 7, respectively, while the similarity coefficients for rat sample replicates ranged from 77% +/- 7 to 88% +/- 5; the similarity coefficient of the donor sample replicates was 78% +/- 9. DNA sequences of bands observed in the different denaturing gradient gel electrophoresis profiles exhibited the highest degree of identity to uncultured bacteria previously found in samples of human, mouse or pig intestinal origin. The results of this study suggest that the dominant human fecal microbiota can be maintained in the human microbiota associated rat model for at least one year.     

16S rDNA技术研究新生腹泻仔猪粪样细菌区系的多样性变化

用PCR/DGGE技术跟踪一窝5头新生腹泻仔猪自然康复、补饲、断奶过程中粪样细菌区系的演变,构建3头仔猪42日龄粪样的16S rDNA克隆库,分析匹配于DGGE优势谱带23个克隆的16S rDNA序列。结果表明,DGGE图谱由简单(2日龄)到复杂(10日龄),再回复简单(16日龄)到复杂(断奶),最后趋于稳定。2、16日龄DGGE图谱最简单、相似,最优势谱带为大肠杆菌;10日龄(补饲后3天)图谱复杂,大肠杆菌存在但不是最优势谱带,补饲前后图谱的相似性低,补饲导致了粪样细菌区系结构的显著变化;断奶前(27日龄)和后(35、42日龄)图谱复杂,优势谱带、图谱相似性均趋向稳定。序列分析表明,23个克隆中除5个与未知细菌最相似外,其余最相似菌分属于肠球菌(Enterococcus),链球菌(Streptococcus),梭菌(Clostridium),消化链球菌(Peptostreptococcus)和乳酸杆菌(Lactobacillus)。