成都地区母乳双歧杆菌对婴儿肠道双歧杆菌构建的影响

目的 观察不同喂养方式婴儿肠道双歧杆菌的构建规律，初步探索母乳双歧杆菌与婴儿肠道双歧杆菌之间的相关性。方法 在四川大学华西第二医院纳入57对母婴志愿者，根据喂养方式分为母乳（BF）组（n=31）和混合喂养（MF）组（1月龄内添加奶粉时间>8 d,n=26），收集产妇产后0天、第30天的母乳，以及婴儿出生后0天、第7天、第30天的粪便。采用实时荧光定量PCR测定母乳和婴儿粪便中双歧杆菌属、角双歧杆菌（B. angulatum）、链双歧杆菌（B. catnulatum）、齿双歧杆菌（B. dentium）、短双歧杆菌（B.breve）、两歧双歧杆菌（B. bifidum）、长双歧杆菌长亚种（B. longum subsp. longum）、长双歧杆菌婴儿亚种（B.longum subsp. infantis）及青春双歧杆菌（B. adolescentis）的丰度。观察2组婴儿肠道双歧杆菌0―7―30天的构建规律；分析母乳双歧杆菌与婴儿肠道双歧杆菌之间的相关性。结果 0―7―30天2组婴儿肠道双歧杆菌构建过程存在差异。母乳双歧杆菌与婴儿肠道双歧杆菌存在相关：0 d时，BF组母乳B. a...     

哈尔滨城市和乡村青年居民肠道菌群多样性研究

【目的】分析居住于哈尔滨城市和乡村的青年居民肠道菌群多样性的异同。【方法】采用PCR和DGGE技术相结合的方法对生活于哈尔滨城市和乡村的青年志愿者肠道菌群多样性进行研究。基于DGGE指纹图谱,分别使用聚类和PCA分析对志愿者肠道微生物相似性进行分析,使用Shannon-Weine多样性指数(H′)、丰度(S)和均匀度(EH)对志愿者肠道微生物多样性进行分析,对图谱中具有代表性的共性和特异性条带进行胶回收和克隆测序以分析志愿者肠道微生物组成。基于PCR技术在种水平上对城乡志愿者肠道内乳杆菌属和双歧杆菌属多样性进行定性分析。【结果】相似性分析显示,城乡青年居民间肠道微生物群落结构存在分开趋势,相似性小于城市或乡村青年居民内部;多样性分析显示,城乡青年居民肠道微生物多样性差异不显著;测序结果表明,城乡居民肠道微生物组成在门水平上相同,但是在种属水平上存在差异。PCR定性分析显示Lactobacillus plantarum、L.casei和L.salivarius在哈尔滨城乡青年居民肠道内检出率接近100%,Bifidobacterium longum和B.breve的检测率约90%,在哈尔滨城乡青年居民肠道内普遍存在;乳杆菌属和双歧杆菌属各细菌种在城乡居民肠道中的检出频率差异不显著。【结论】哈尔滨城市和乡村青年居民肠道微生物多样性差异不显著。     

双歧杆菌属特异性测序引物筛选及优化

【背景】目前双歧杆菌的益生功能被普遍认可,越来越多的研究开始关注肠道中双歧杆菌的生物多样性。然而双歧杆菌是肠道中的低丰度物种,现有技术尚难以深入研究其多样性。【目的】基于双歧杆菌16SrRNA基因序列筛选一对适用于分析粪便样品中低丰度双歧杆菌属多样性的特异性引物。【方法】依据已有引物的相对位置及其与双歧杆菌属16S rRNA基因序列的匹配率,将引物重组优化得到扩增片段800 bp的双歧杆菌属特异性引物;通过PCR扩增和琼脂糖凝胶电泳对引物进行实验筛选和特异性验证;以细菌通用引物(27f/1492r)为参照,通过单分子实时(Single-molecule real-time,SMRT)测序技术对不同引物的3份粪便样品中细菌的DNA扩增子进行测序,在种水平上分析比较不同引物的优劣。【结果】对文献中已有的9对双歧杆菌特异性引物进行重组并优化,其中2对引物的理论特异性较好且扩增产物大于800 bp,它们分别为Bif164-f/Pbi R2和Pbi F1/Pbi R2。PCR扩增和琼脂糖凝胶电泳实验发现,Bif164-f/Pbi R2的扩增条带明亮且无拖尾。此外,利用SMRT测序平台对引物27f/1492r和Bif164-f/Pbi R2的3份粪便样品中细菌的DNA扩增子进行测序并分析。27f/1492r扩增子的分析结果显示,3份样品依次分别含1、3、4个双歧杆菌种且双歧杆菌的平均相对含量为0.34%;而Bif164-f/Pbi R2扩增子的分析结果显示,3份样品依次分别含2、6和8个双歧杆菌种且双歧杆菌的平均相对含量为98.72%。上述结果表明,Bif164-f/Pbi R2可在种水平上特异地检出粪便中低丰度的双歧杆菌,进而实现样品中双歧杆菌的多样性分析。【结论】筛选出一对双歧杆菌特异性引物Bif164-f/PbiR2,可在种水平上分析粪便样品中低丰度双歧杆菌的生物多样性,同时也验证了理论结合实验进行引物筛选这种方法的可行性。     

健康老年人肠道双歧杆菌和乳杆菌种群多样性分析

从31个60岁以上的符合中华医学会老年医学分会健康老年人标准的健康老人中随机选取4例作为研究对象,使用分子生物学方法,对他们的肠道双歧杆菌和乳杆菌种群多样性进行分析.研究结果表明,健康老人肠道中双歧杆菌的优势种群为长双歧杆菌（Bifi∥dobacterium longum）和假小链双歧杆菌（Bifidobacterium pseudocatenulatum）,分别占双歧杆菌种群的55%和45%;而肠道乳杆菌则有唾液乳杆菌（Lactobacillus salivarius）50%、Lactobacillus mocosae 31.1%、口腔乳杆菌（Lactobacillus oris）6.3%、鼠李糖乳杆菌（Lactobacillus rhamnosus）6.3%和瑞士乳杆菌（Lactobacillus helveticus）6.3%,其中唾液乳杆菌、Lactobacillus mocosae为健康老人肠道优势乳杆菌种群.     

属特异性T-RFLP技术在双歧杆菌群落分析中的应用

【目的】以双歧杆菌标准菌株为材料,构建双歧杆菌属特异性末端限制性片段长度多态性分析(T-RFLP)技术,用于微生物群落中双歧杆菌的特异性分析。【方法】采用16S rRNA基因的双歧杆菌属特异性引物,5′-端用HEX荧光标记,结合通用引物1510r进行双歧杆菌特异性PCR扩增,软件模拟酶切后选取Hae III和Alu I进行限制性酶切,对酶切消化产物的荧光标记末端测序得到T-RFLP峰谱图。同时将该技术与实验室已建立的乳酸杆菌属特异性T-RFLP技术相结合,建立多相T-RFLP技术应用于对市面上益生菌产品的时效性检测。【结果】建立的方法能够快速准确地对不同种的双歧杆菌及合生元产品中的益生菌进行定性或半定量分析。【结论】据此,成功搭建T-RFLP技术用于微生态环境中双歧杆菌的检测,并成功将多相T-RFLP技术用于市售益生菌产品的时效性检测。     

分子信标-实时 PCR法快速检测双歧杆菌的研究

为建立双歧制品中双歧杆菌快速、敏感、特异的检测方法,根据双歧杆菌16SrRNA/16SrDNA基因设计合成了双歧杆菌属特异性引物和分子信标探针,建立了快速检测双歧杆菌的分子信标-实时PCR检测方法,并对反应条件进行优化。检测方法重复性好,批内和批间变异系数均小于5%;特异性强,扩增曲线呈现明显的S型,无非特异性扩增;灵敏度高,是普通PCR的100倍,对纯双歧杆菌DNA的检出限为5.7fg/PCR反应体系,纯双歧杆菌菌液的检出限为2×103CFU/mL;线形范围宽,起始模板数在2×108CFU/mL～2×104CFU/mL之间具有良好的线性关系,相关系数大于97%。该方法具有灵敏、特异、简便和快速的特点,可用于对双歧杆菌原位菌数的定量检测。     

基于16S rRNA基因检测双歧杆菌的方法

本文综述了基于16S rRNA序列(16S rDNA)检测、鉴定双歧杆菌的方法。包括基因探针法、荧光原位杂交法、PCR扩特异性片段法、多重PCR法、荧光定量PCR法和PCR-DGGE/TGGE法等。     

青年人肠道菌群分布及关键益生菌群落结构分析

以30例20～25岁中国青年人的肠道菌群为研究对象,采用纯培养方法和16S rDNA测序等分子生物学技术并结合代谢产物β_半乳糖苷酶和短链脂肪酸的测定,分析了此年龄段健康人群肠道菌群分布和益生菌群落结构。实验表明,高厌氧菌水平和高B/E值（175.66）反映出此年龄段人群良好的肠道环境;肠道关键益生菌群落结构分析表明,双歧杆菌由1～4种菌种组成,青春双歧杆菌(检出率93.3%,占总双歧杆菌数量百分比>85%)、长双歧杆菌(检出率86.7%,占总双歧杆菌数量百分比10%)为肠道优势双歧杆菌,其余菌种在不同人肠道中的差异数量只占不到5％,此年龄段青年人肠道具有呈现稳定的双歧杆菌群落结构,肠道双歧杆菌群落结构与膳食结构、生活环境等因素相关性不大,主要与人的生理年龄紧密相关;卷曲乳杆菌和唾液乳杆菌为青年人肠道优势乳杆菌(检出率分别为86.7%和93.3%,两者之和占总乳杆菌的数量百分比为60%～75%),但其余乳杆菌在不同人肠道中的之间的差异数量达到20%～30％,肠道乳杆菌群落结构与饮食结构、生活环境等因素紧密相关,不同个体具有各自独特的乳杆菌群落组成。     

感染微生态研究进展——肠道菌群对机体代谢影响

1肠道菌群的组成 在人体,肠道菌群是一个复杂的微生态系统,由于细菌培养手段的局限性,我们对其结构和组成知之甚少。近年来,随着分子生物学技术在感染微生态学上的应用,根据细菌16S rRNA序列的分类,发现肠道内寄住的微生物群的种类超过800种,主要有拟杆菌属、梭菌、乳杆菌、大肠埃希菌属和双歧杆菌属等;     

一名肥胖儿童营养干预后肠道内假小链双歧杆菌碳水化合物利用能力的探究

双歧杆菌被认为是肠道内的有益菌,可以利用膳食中的多种植物多糖或寡糖,能够被人体无法消化的复杂碳水化合物选择性富集。本研究采用序列引导的分离策略,于厌氧条件下采用MRS琼脂培养基,得到了一名肥胖儿童营养干预后肠道内优势的双歧杆菌。采用ERIC指纹图谱技术对分离物进行基因水平上的分型,并通过16S r RNA基因测序鉴定分离株的分类地位。73个双歧杆菌分离物分为5种ERIC类型,均为假小链双歧杆菌。与Bacteroides thetaiotaomicron ATCC 29148、Eschrichia coli D45和Anaerostipes hadrus BPB5这些肠道中常见共生菌的代表菌株相比,丰度最高的ERIC类型代表菌株Bifidobacterium pseudocatenulatum C95利用复杂碳水化合物的能力更强。Bifidobacterium pseudocatenulatum C95被膳食中的复杂碳水化合物和益生元显著富集,成为该儿童营养干预后肠道内的优势菌。菌株自身较强的碳水化合物利用能力可能是其生长优于肠道中其他共生细菌的原因。     

Molecular profiling of Bacteroides spp. in human feces by PCR-temperature gradient gel electrophoresis

A group-specific PCR-based temperature gradient gel electrophoresis (TGGE) method was developed to study the population composition of genus Bacteroides in human gut. Highly reproducible and well-separated bands in TGGE fingerprints of ten unrelated human fecal samples showed complex and host-specific Bacteroides species composition. Dynamic monitoring over 22 months of samples from one healthy 10-year-old boy indicated a relatively stable population profile of Bacteroides. The species identity of each band in TGGE gel of this boy was also resolved via comigration analysis of sequenced inserts in a Bacteroides group-specific clone library. This work provides a rapid and effective technique for analyzing the species composition of Bacteroides in human gut.     

婴幼儿粪便中双歧杆菌的分离及其菌株特性研究

为筛选具有潜在益生功能的双歧杆菌,本研究采用改良MRS培养基,从婴幼儿粪便中分离到5株菌株:AR499,AR667,AR668,AR669和AR610。通过16S r DNA测序分别鉴定为两歧双歧杆菌,短双歧杆菌,动物双歧杆菌,长双歧杆菌和假小链双歧杆菌。对其耐酸耐胆盐能力和黏附性能进行测试,结果表明,菌株AR499耐酸性较好,菌株AR610有较强的耐胆盐能力,菌株AR499的自动聚集能力和细胞表面疏水性都较高。实验表明,菌株AR499可作为一株耐受性和黏附性能较好的益生菌进一步深入研究,以期应用于优良双歧杆菌产品的开发。     

Species specific identification of nine human Bifidobacterium spp. in feces

Based on the 16S rDNA sequences, species specific primers were designed for the rapid identification by DNA amplification of nine human Bifidobacterium spp., namely B. adolescentis, B. angulatum, B. bifidum, B. breve, B. catenulatum, B. dentium, B. infantis, B. longum, B. pseudocatenulatum. B. lactis currently included in dairy products was added to the series. The primers were designed to target different positions of the 16S rDNA, allowing the simultaneous identification of these ten species of Bifidobacterium using two mixtures of primers. The identification procedure described in this paper was validated by establishing a correlation with an AluI restriction pattern of the different full length amplified 16S rDNA. This multiple primer DNA amplification technique was applied for the identification of pure colonies of Bifidobacterium spp. or directly from total bacteria recovered from human fecal samples. The technique was shown to be useful to detect dominant species and, when primers were used in separate reactions, underrepresented species could be identified as well.     

Comparison of intestinal microflora in healthy infants and infants with allergic colitis

Twenty-eight exclusively breast-fed healthy infants and 16 infants also exclusively breast-fed with allergic colitis (aged 85 +/- 60 and 98 +/- 58 d, respectively) were screened for differences in fecal flora. Bifidobacteria were detected in 23 healthy infants and only in 4 fecal samples of infants with allergic colitis. All bifidobacteria-free infants possessed Gram-positive regular rods as a major group of their fecal flora. These bacteria were identified as clostridia using genus-specific FISH probe. Infants with allergy colitis possessed significantly lower counts of bifidobacteria and total anaerobes and significantly higher counts of clostridia in their feces. In healthy infants, Bifidobacterium longum was the most frequently found species (54.5% of the samples), followed by B. adolescentis (20.0), B. breve (18.2), B. bifidum (16.4), B. dentium (10.9) and B. pseudocatenulatum (1.80). Bifidobacterial isolates from two babies with allergic colitis were identified as B. longum, one child from patients group contained species B. dentium and one baby B. adolescentis. Our results suggest that there are significantly lower counts of bifidobacteria in infants with allergic colitis than in healthy infants.     

儿童肠道双歧杆菌和乳杆菌种群结构分析

以21例2～5岁中国儿童的肠道菌群为研究对象,利用传统培养计数法和分子生物学技术,对此年龄段健康儿童的肠道菌群分布及其中关键益生菌的种群结构进行了定量研究。实验表明,儿童肠道厌氧菌的数量高达109CFUg(湿重),其肠道菌群的定植抗力(平均BE=2.38)较强;不同的个体之间所能检测到的关键益生菌的种类有所不同,一般能检测到其中的1～4种双歧杆菌和1～5种乳杆菌;长双歧杆菌和假小链双歧杆菌的平均数量多达107CFUg(湿重),检出率分别为90.48%和85.71%,为儿童肠道内双歧杆菌的优势菌种;L.mucosae和发酵乳杆菌的数量较多,平均为3.68log10CFUg(湿重)和3.97log10CFUg(湿重),检出率分别为71.43%和52.38%,为稳定定植于儿童肠道内的优势乳杆菌;不同种类的益生菌在不同样本之间的数量组成均存在有很大差异,双歧杆菌的样本差异为1.86～3.85,乳杆菌的为2.43～4.07。     

Evaluation of the PCR method for identification of Bifidobacterium species

AIMS: Bifidobacterium species are known for their beneficial effects on health and their wide use as probiotics. Although various polymerase chain reaction (PCR) methods for the identification of Bifidobacterium species have been published, the reliability of these methods remains open to question. METHODS AND RESULTS: In this study, we evaluated 37 previously reported PCR primer sets designed to amplify 16S rDNA, 23S rDNA, intergenic spacer regions, or repetitive DNA sequences of various Bifidobacterium species. CONCLUSIONS: Ten of 37 experimental primer sets showed specificity for B. adolescentis, B. angulatum, B. pseudocatenulatum, B. breve, B. bifidum, B. longum, B. longum biovar infantis and B. dentium. SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggest that published Bifidobacterium primer sets should be re-evaluated for both reproducibility and specificity for the identification of Bifidobacterium species using PCR. Improvement of existing PCR methods will be needed to facilitate identification of other Bifidobacterium strains, such as B. animalis, B. catenulatum, B. thermophilum and B. subtile.     

Identification, detection, and enumeration of human bifidobacterium species by PCR targeting the transaldolase gene

Methods that enabled the identification, detection, and enumeration of Bifidobacterium species by PCR targeting the transaldolase gene were tested. Bifidobacterial species isolated from the feces of human adults and babies were identified by PCR amplification of a 301-bp transaldolase gene sequence and comparison of the relative migrations of the DNA fragments in denaturing gradient gel electrophoresis (DGGE). Two subtypes of Bifidobacterium longum, five subtypes of Bifidobacterium adolescentis, and two subtypes of Bifidobacterium pseudocatenulatum could be differentiated using PCR-DGGE. Bifidobacterium angulatum and B. catenulatum type cultures could not be differentiated from each other. Bifidobacterial species were also detected directly in fecal samples by this combination of PCR and DGGE. The number of species detected was less than that detected by PCR using species-specific primers targeting 16S ribosomal DNA (rDNA). Real-time quantitative PCR targeting a 110-bp transaldolase gene sequence was used to enumerate bifidobacteria in fecal samples. Real-time quantitative PCR measurements of bifidobacteria in fecal samples from adults correlated well with results obtained by culture when either a 16S rDNA sequence or the transaldolase gene sequence was targeted. In the case of samples from infants, 16S rDNA-targeted PCR was superior to PCR targeting the transaldolase gene for the quantification of bifidobacterial populations.     

Distribution of bifidobacterial species in human intestinal microflora examined with 16S rRNA-gene-targeted species-specific primers.

In order to clarify the distribution of bifidobacterial species in the human intestinal tract, a 16S rRNA-gene-targeted species-specific PCR technique was developed and used with DNAs extracted from fecal samples obtained from 48 healthy adults and 27 breast-fed infants. To cover all of the bifidobacterial species that have been isolated from and identified in the human intestinal tract, species-specific primers for Bifidobacterium longum, B. infantis, B. dentium, and B. gallicum were developed and used with primers for B. adolescentis, B. angulatum, B. bifidum, B. breve, and the B. catenulatum group (B. catenulatum and B. pseudocatenulatum) that were developed in a previous study (T. Matsuki, K. Watanabe, R. Tanaka, and H. Oyaizu, FEMS Microbiol. Lett. 167:113-121, 1998). The specificity of the nine primers was confirmed by PCR, and the species-specific PCR method was found to be a useful means for identifying Bifidobacterium strains isolated from human feces. The results of an examination of bifidobacterial species distribution showed that the B. catenulatum group was the most commonly found taxon (detected in 44 of 48 samples [92%]), followed by B. longum and B. adolescentis, in the adult intestinal bifidobacterial flora and that B. breve, B. infantis, and B. longum were frequently found in the intestinal tracts of infants. The present study demonstrated that qualitative detection of the bifidobacterial species present in human feces can be accomplished rapidly and accurately.     

PCR-ELISA II: Analysis of Bifidobacterium populations in human faecal samples from a consumption trial with Bifidobacterium lactis Bb-12 and a galacto-oligosaccharide preparation

A PCR-ELISA method was extended for detection of most common Bifidobacterium species in humans and applied to a feeding trial including administration of Bifidobacterium lactis Bb-12 and galacto-oligosaccharide (GOS)-containing syrup as probiotic and prebiotic preparations, respectively. For PCR-ELISA, oligonucleotide probes based on 16S rDNA sequences were designed and tested for specificity and sensitivity with nine different bifidobacterial species followed by analysis of faecal samples. Bifidobacteria were monitored for their fluctuations during and after the feeding trial. Bifidobacterium longum was the most common species found in the faecal samples, followed by B. adolescentis and B. bifidum. During ingestion of the probiotic B. lactis Bb-12, the strain appeared in the faeces but was absent again one week after finishing of the trial. The species that were observed in the faecal samples taken prior to the feeding experiments persisted also in samples derived from the pre-feeding and feeding periods. The most consistent change observed was the decrease in the relative amount of B. longum in the test group ingesting either B. lactis Bb-12 alone or in combination with GOS-syrup. Since the amounts of B. longum increased again in the post-feeding sample with these subjects, it may suggest that to some extent B. lactis Bb-12 is able to transiently replace B. longum.     

Simultaneous identification of five bifidobacterium species isolated from human beings using multiple PCR primers

On the basis of 16S rRNA sequences, 5 species-specific forward primers were designed for the identification of 5 Bifidobacterium species isolated from human intestine, namely B. bifidum, B. adolescentis, B. infantis, B. breve and B. longum. As the 5 primers targeted at different sites of 16S rDNA, by using their mixture and a genus-specific reversed primer, the 5 Bifidobacterium species can be simultaneously identified in individual or in mixed culture through PCR amplification. The specificity of the primers was confirmed by the use of genomic DNAs from type strains of all 32 Bifidobacterium species and 6 other relatives. The 5-primer mixture was also applied to the identification of Bifidobacterium strains used commercially. The results turned out to be in accordance with those from conventional identification. This multiple-primer method provides a useful tool for rapid identification of the 5 Bifidobacterium species indicated.