昆明犬肠道中乳杆菌的分离鉴定及16S rRNA同源性分析

目的 为了进一步开发动物微生态资源.方法 用生理生化表型及16SrRNA同源性分析,鉴定从昆明犬肠道内容物中分离到的7株乳杆菌,分别命名为LD1、LD2、LD3、LD4、LD5、LD6和LD7.结果 LD1、LD2、LD4为路氏乳杆菌(Lactobacillus reuteri),LD3、LD5、LD6、LD7为鼠乳杆菌(Lactobacillus murinus).结论 该研究为犬用微生态制剂及益生菌的开发应用奠定基础.     

16S rRNA PCR鉴定脆弱类杆菌

目的:应用16SrRNA序列设计PCR引物鉴别脆弱类杆菌。方法:通过脆弱类杆菌16SrRNA序列特异性位点设计引物,对4株脆弱类杆菌及大肠杆菌、乳酸杆菌、嗜热链球菌等进行PCR扩增。应用琼脂糖电泳法对PCR扩增产物进行特异性检测。结果:脆弱类杆菌在176bp左右出现特异性条带,而其他细菌均未出现特异性条带。结论:通过16SrRNA序列中特异位点设计引物进行PCR,可特异性鉴定脆弱类杆菌。     

耐热嗜盐菌的分离及16S rRNA基因序列分析

从位于西藏自治区澜沧江边一个47℃的盐井中分离筛选到一株耐热嗜盐菌菌株YJ0238, 对其进行了生理生化特性研究, 采用PCR方法扩增其16S rRNA基因序列, 并进行了测定。基于生理生化特性和16S rRNA基因序列的同源性比较, 以及系统发育分析, 发现菌株YJ0238是Idiomarina属中成员zobellii的一个亚种, 其16S rRNA基因序列已被GenBank数据库收录, 序列号为EF693953。迄今为止, 国内极少有关高温、高盐环境中微生物研究的报道, 本研究可为今后研究同类极端环境中新的物种资源以及微生物多样性提供素材和参考。     

乳扇制品中乳酸杆菌的分离鉴定及16S rRNA序列同源性分析

目的 乳扇是云南大理白族的一种传统乳制品,为明确大理乳扇制品中乳杆菌的多样性及优势种群分布,为科学利用奠定了基础。方法 本研究采用表型鉴定及16S rRNA鉴定方法,对10个家庭作坊的大理乳扇制品中的乳杆菌进行了分离鉴定。结果 共分离到50株乳杆菌,通过表型鉴定为8个种,包括植物乳杆菌10株、德氏乳杆菌7株、发酵乳杆菌6株、干酪乳杆菌6株、棒状乳杆菌4株、鼠乳杆菌2株、弯曲乳杆菌3株和食果糖乳杆菌2株;06422和06430两株表型鉴定未能定种,进一步通过16S rRNA鉴定为植物乳杆菌和马酒乳杆菌,06422株其与植物乳杆菌L.arizonensin、L.pentosus和L.plantarum P158的同源性分别是100%、100%和99.9%,与乳杆菌属其它种的同源性为83.4%（L.gallinarum）至93.5%（L.brevis）,06430株与L.kefiranofaciens.subsp.Kefirgranum的16S rRNA同源性是99.9%,与乳杆菌属其它种的同源性为83.7%（L.plantarum P158）至96.3%（L. acidophilus）。结论 大理乳扇制品中有9种乳杆菌,其优势种群为植物乳杆菌、德氏乳杆菌、发酵乳杆菌和干酪乳杆菌等四种。     

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。     

一株芽孢杆菌的分离和鉴定

从中国农业科学院北京畜牧兽医研究所鸡舍附近土壤中分离到一株芽孢杆菌P-25,并进行了分子鉴定。通过形态鉴定、革兰氏染色、生理生化测定、16SrRNA序列分析和系统发育树构建,确定该菌株为蜡状芽孢杆菌(Bacillus cereus),其16SrRNAGenBank登录号为GU271135。     

嗜盐菌HBCC-2的16S rRNA基因测序分析及其培养特性

从连云港台南盐场海盐生产区中分离纯化到一株嗜盐古菌HBCC-2,该菌株经PCR扩增后,测定其16S rRNA基因序列,采用BLAST软件对基因库中基因序列进行同源性比较,选取其相似性序列,采用Clustalx1.8和MEGA3.1软件对其16S rDNA序列进行了系统发育分析研究,结果表明HBCC-2菌株与菌株Halorubrum sp.GSL5.48的相似性达99%,结合其形态观察及生理生化反应特性,初步确定该菌株属于嗜盐红菌属(Halorubrum),菌株HBCC-2的16S rDNA序列已登陆到GenBank,其序列号为EF687739.通过比较不同NaCl浓度、pH和培养温度对该菌株生长的影响情况,研究了该菌株的生长特性,结果表明NaCl浓度为4mol/L、温度为35℃和pH为7.0的培养条件下其生长最佳.     

极端嗜盐菌 16S rDNA的PCR扩增

四株嗜盐菌Haloarcula vallismortis(EM201)、Haloferax denitrificans(EM303)、A_5和B_2已通过一对特定引物用PCR技术从总DNA中扩增出各自的16SrDNA片段,分子大小在1.47kd左右.DNA杂交也表明这些PCR产物具有嗜盐菌的同源性.     

一株高毒力致病杆菌CB6的鉴定

从北京郊区果园采集的小卷蛾斯氏线虫（Steinernema carpocapsae）肠道内分离到一株具有较强杀虫和抑菌活性的致病杆菌菌株CB6。形态特征及生理生化特征测定结果表明,CB6菌株与致病杆菌属（Xenorhabdus）中的嗜线虫致病杆菌（X. nematophila）种的特征基本一致。测定了该菌株的16S rRNA序列并根据16S rRNA序列构建了系统发育树;在系统发育树中,CB6菌株与嗜线虫致病杆菌其他4个菌株形成一个类群,序列同源性大于99%。但CB6菌株的酪氨酸酶、脂酶（蛋黄）的产生、核糖产酸等生化特征与嗜线虫致病杆菌种内的其他菌株存在一定的差异,且具有更强的杀虫和抑菌活性。因此认为CB6菌株是嗜线虫致病杆菌的一个变种,命名为嗜线虫致病杆菌北京变种（X. nematophila var. pekingensis）。     

双歧杆菌四联活菌片中嗜酸乳杆菌的分离及生长动力学研究

目的以双歧杆菌四联活菌片为实验材料,利用酸化的MRS培养基筛选分离得到嗜酸乳杆菌,对其进行进一步的生长动力学研究,确定嗜酸乳杆菌的生长数学模型。方法通过浓度梯度稀释法利用改良MRS培养基对双歧杆菌四联活菌片中的嗜酸乳杆菌进行分离,利用分光光度仪和平板菌落计数两种不同的方法测定嗜酸乳杆菌在发酵过程中不同发酵时间的细胞浓度的动态变化,经软件处理后拟合出嗜酸乳杆菌细胞生长的Logistic数学模型。结果Logistic方程能很好地拟合嗜酸乳杆菌细胞生长的动态变化,并得到嗜酸乳杆菌在本实验条件下的数学模型,为进一步研究、利用嗜酸乳杆菌生长能力、产酸能力和产香能力等具有重要的理论指导意义。     

The PCR amplification of non-tuberculous mycobacterial 16S rRNA sequences from soil

Non-tuberculous mycobacteria are free living saprophytic organisms commonly found in soil and water. Some are major causes of opportunistic infection, particularly in immuno-compromised patients, and may influence the efficacy of bacille Calmette-Guérin vaccinations. Many of these organisms are not amenable to culture, so information about their distribution is limited. PCR primers designed to amplify part of the mycobacterial 16S rRNA gene were applied to DNA extracted from cultured organisms and soil. The PCR products from soil contained sequences with similarity to slow growing mycobacteria similar to Mycobacterium lentiflavum, and to fast growing mycobacteria such as the xenobiotic degraders PYR-I and RJGII.     

用细菌16S rRNA荧光定量PCR法检测肠道菌群的变化

目的应用细菌的16SrRNA序列设计双歧杆菌、大肠埃希菌及乳酸杆菌的引物并对肠道的3种细菌进行定量测定。方法收集轮状病毒肠炎患儿及正常对照组的粪便标本提取DNA。取准确定量的3种细菌经系列稀释后抽提细菌的DNA做荧光定量PCR,制作出标准曲线。待测样品同时进行PCR反应并和标准曲线进行比较,获得各样品中3种细菌的量。结果患儿肠道中双歧杆菌和乳酸杆菌的数量较正常儿童明显减低,而大肠埃希菌的数量差异无显著性。与其他文献报道的用细菌培养的方法所得结果一致。结论荧光定量PCR是一种特异性高、敏感性强的定量方法。可正确定量肠道中的细菌数量。     

一株耐盐细菌的16S rRNA基因序列分析

从舟山群岛滩涂土壤中获得了海水和底泥样品,并从中提取到了一株耐盐细菌,通过用不同盐浓度的培养基培养,挑取单菌落,反复划线纯化,得到了耐盐菌的单菌落。通过菌株基因组DNA的提取、菌株的抗性实验、质粒的提取、16S rRNA的PCR扩增及克隆、16S rRNA的全序列分析等手段,对该菌株的16S rRNA的基因序列进行了研究。     

Detection of Laribacter hongkongensis using species-specific duplex PCR assays targeting the 16S rRNA gene and the 16S-23S rRNA intergenic spacer region (ISR)

Aims: For the rapid detection of Laribacter hongkongensis, which is associated with human community‐acquired gastroenteritis and traveller’s diarrhoea, we developed a duplex species‐specific PCR assay. Methods and Results: Full‐length of the 16S–23S rRNA intergenic spacer region (ISR) sequences of 52 L. hongkongensis isolates were obtained by PCR‐based sequencing. Two species‐specific primer pairs targeting 16S rRNA gene and ISR were designed for duplex PCR detection of L. hongkongensis. The L. hongkongensis species‐specific duplex PCR assay showed 100% specificity, and the minimum detectable level was 2·1 × 10^?2 ng μl^?1 genomic DNA which corresponds to 5000 CFU ml^?1. Conclusions: The high specificity and sensitivity of the assay make it suitable for rapid detection of L. hongkongensis. Significance and Impact of the Study: This species‐specific duplex PCR method provides a rapid, simple, and reliable alternative to conventional methods to identify L. hongkongensis and may have applications in both clinical and environmental microbiology.     

利用16S rRNA基因同源性分析鉴定两株明串珠菌

从酸马奶中分离出2株明串珠菌KLDS 5.0301和KLDS 5.0302,对2株菌的16S rRNA基因经PCR扩增测序,将测序结果同该属内菌株的16S rRNA序列作多序列比较,并建立明串珠菌属的系统发育树.结果表明,KLDS 5.0301的16S rRNA序列同L. garlicum的同源性百分比为100%.KLDS 5.0302的16S rRNA序列同L.mesenteroides LM2菌株的16S rRNA序列的同源性百分比为99.9%.根据系统发育树的结果,将KLDS5.0301鉴定为L.garlicum,KLDS 5.0302鉴定为L.mesenteroides.菌株KLDS 5.0301和KLDS 5.0302的16SrRNA序列已经在GeneBank申请国际序列注册号,分别为DQ239691和DQ297412.     

PCR detection and 16S rRNA sequence-based phylogeny of a novel Propionibacterium acidipropionici applicable for enhanced fermentation of high moisture corn

AIMS: The aims of this study were to develop a sensitive and more rapid detection of Propionibacterium acidipropionici DH42 in silage and rumen fluid samples, and to explore its 16S rRNA sequence-based phylogeny. METHODS AND RESULTS: Nested polymerase chain reaction (PCR) was used with DH42-specific primers dhb1 and dhb2 for the secondary amplification of a 1267-bp fragment of 16S rRNA encoding gene. Using the established protocols for PCR amplification, as low as 10(2) and 10(3) CFU ml(-1) of strain DH42 in silage extracts and rumen fluid, respectively, were detected. To determine phylogenetic relationships between DH42 and other representatives of Propionibacterineae, a 1529-bp fragment of its 16S rRNA was amplified by PCR and sequenced. The propionibacterium DH42 formed a cluster with Eubacterium combesii, P. acidipropionici and P. microaerophilus. CONCLUSIONS: 16S rRNA-based PCR detection technique was developed for DH42 in silage and rumen fluid samples. The 16S rRNA sequence confirmed the earlier identification of strain DH42 as P. acidipropionici. However, variable nucleotide positions were revealed. SIGNIFICANCE AND IMPACT OF THE STUDY: Variability of 16S rRNA sequence within the species P. acidipropionici, determined in this study, poses the need of re-sequencing for some species of the suborder Propionibacterineae for a more reliable classification.     

Rapid identification of 11 human intestinal Lactobacillus species by multiplex PCR assays using group- and species-specific primers derived from the 16S-23S rRNA intergenic spacer region and its flanking 23S rRNA

Rapid and reliable two-step multiplex polymerase chain reaction (PCR) assays were established to identify human intestinal lactobacilli; a multiplex PCR was used for grouping of lactobacilli with a mixture of group-specific primers followed by four multiplex PCR assays with four sorts of species-specific primer mixtures for identification at the species level. Primers used were designed from nucleotide sequences of the 16S-23S rRNA intergenic spacer region and its flanking 23S rRNA gene of members of the genus Lactobacillus which are commonly isolated from human stool specimens: Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus delbrueckii (ssp. bulgaricus and ssp. lactis), Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus jensenii, Lactobacillus paracasei (ssp. paracasei and ssp. tolerans), Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus and Lactobacillus salivarius (ssp. salicinius and ssp. salivarius). The established two-step multiplex PCR assays were applied to the identification of 84 Lactobacillus strains isolated from human stool specimens and the PCR results were consistent with the results from the DNA-DNA hybridization assay. These results suggest that the multiplex PCR system established in this study is a simple, rapid and reliable method for the identification of common Lactobacillus isolates from human stool samples.     

鼠三毛滴虫新疆灰仓鼠分离株形态学观察及其16SrRNA基因分析

目的：对从新疆实验动物研究中心饲养的封闭群灰仓鼠体内分离到的1株鞭毛虫进行形态学鉴定及基因鉴定。方法取灰仓鼠回盲部内容物进行直接涂片和常规姬姆萨染色后镜检观察,提取虫体总DNA,PCR扩增该鞭毛虫的16S rRNA基因,测序后与国外已报道的鞭毛虫进行核酸同源性分析,并应用MEGA5.22软件绘制系统发育进化树。结果形态学观察表明分离到的鞭毛虫为鼠三毛滴虫。测序后核酸同源性分析表明鼠三毛滴虫新疆灰仓鼠分离株16S rRNA序列与国外已报道的三毛滴虫高度同源。系统发育进化树表明鼠三毛滴虫新疆灰仓鼠分离株序列与已报道的鼠三毛滴虫16S rRNA（序列号AY886846.1）位于同一进化分支,与其他相关三毛滴虫亲缘关系较远。结论形态学鉴定和16 S rRNA基因分析表明,此次从新疆实验动物研究中心饲养的封闭群灰仓鼠体内分离到的鞭毛虫为鼠三毛滴虫。     

双歧杆菌地高辛标记寡核苷酸基因探针制备和应用研究

目的探讨地高辛标记寡核苷酸基因探针应用于微生态研究的可行性和实用性。方法制备双歧杆菌属和部分种的地高辛标记16S rRNA寡核苷酸探针,初步应用于微生态制剂鉴定和临床肠道微生态检测,评价寡核苷酸探针杂交在肠道微生态研究和检测中的应用价值。结果地高辛标记寡核苷酸探针具有较好的特异性与灵敏度：地高辛标记的双歧杆菌属和种的共6种寡核苷酸基因探针与标准菌株杂交后灵敏度和特异度分别为属探针95％、75％,青春双歧87．5％、90％,两歧双歧87．5％、87．5％,短双歧87．5％、92．5％,婴儿双歧75％、95％,长双歧75％、100％。结论寡核苷酸基因探针用于肠道细菌的鉴定显示出一定前景,加大探针的种类与扩大调查范围有可能使该技术替代现有细菌培养技术。