A one-step reaction for the rapid identification of Lactobacillus mindensis, Lactobacillus panis, Lactobacillus paralimentarius, Lactobacillus pontis and Lactobacillus frumenti using oligonucleotide primers designed from the 16S-23S rRNA intergenic sequences

Aims:  Species-specific primers targeting the 16S–23S ribosomal DNA (rDNA) intergenic spacer region (ISR) were designed to rapidly discriminate between Lactobacillus mindensis , Lactobacillus panis , Lactobacillus paralimentarius , Lactobacillus pontis and Lactobacillus frumenti species recently isolated from French sourdough.
Methods and Results:  The 16S–23S ISRs were amplified using primers 16S/p2 and 23S/p7, which anneal to positions 1388–1406 of the 16S rRNA gene and to positions 207–189 of the 23S rRNA gene respectively, Escherichia coli numbering (GenBank accession number V00331 ). Clone libraries of the resulting amplicons were constructed using a pCR2·1 TA cloning kit and sequenced. Species-specific primers were designed based on the sequences obtained and were used to amplify the 16S–23S ISR in the Lactobacillus species considered. For all of them, two PCR amplicons, designated as small ISR (S-ISR) and large ISR (L-ISR), were obtained. The L-ISR is composed of the corresponding S-ISR, interrupted by a sequence containing tRNA^Ile and tRNA^Ala genes. Based on these sequences, species-specific primers were designed and proved to identify accurately the species considered among 30 reference Lactobacillus species tested.
Conclusions:  Designed species-specific primers enable a rapid and accurate identification of L. mindensis , L. paralimentarius , L. panis , L. pontis and L. frumenti species among other lactobacilli.
Significance and Impact of the Study:  The proposed method provides a powerful and convenient means of rapidly identifying some sourdough lactobacilli, which could be of help in large starter culture surveys.     

Fluorescent oligonucleotide rDNA probes for specific detection of methane oxidising bacteria

Oligonucleotide probes targeting the 16S rRNA of distinct phylogenetic groups of methanotrophs were designed for the in situ detection of these organisms. A probe, MG-64, detected specifically type I methanotrophs, while probes MA-221 and MA-621, detected type II methanotrophs in whole cell hybridisations. A probe Mc1029 was also designed which targeted only organisms from the Methylococcus genus after whole cell hybridisations. All probes were labelled with the fluorochrome Cy3 and optimum conditions for hybridisation were determined. Non-specific target sites of the type I (MG-64) and type II (MA-621) probes to non-methanotrophic organisms are highlighted. The probes are however used in studying enrichment cultures and environments where selective pressure favours the growth of methanotrophs over other organisms. The application of these probes was demonstrated in the detection of type I methanotrophs with the MG-64 probe in an enrichment culture from an estuarine sample demonstrating methane oxidation. The detection of type I methanotrophs was confirmed by a 16S rDNA molecular analysis of the estuarine enrichment culture which demonstrated that the most abundant bacterial clone type in the 16S rDNA library was most closely related to Methylobacter sp. strain BB5.1, a type I methanotroph also isolated from an estuarine environment.     

Design of species-specific oligonucleotide probes for the detection of Bacteroides and Parabacteroides by fluorescence in situ hybridization and their application to the analysis of mouse caecal Bacteroides-Parabacteroides microbiota

Aims: To develop species‐specific monitoring techniques for rapid detection of Bacteroides and Parabacteroides inhabiting the mouse intestine by fluorescence in situ hybridization. Methods and Results: The specificity of oligonucleotide probes was evaluated by fluorescence whole‐cell hybridization. Oligonucleotide probes specific for each species hybridized only with the target bacteria. Using these probes, caecal Bacteroides–Parabacteroides microbiota of conventional mice and specific pathogen‐free (SPF) mice from three different breeders were analysed. It was shown that Bacteroides acidifaciens Group‐1, Group‐2 and Group‐3 were dominant in conventional mice and SPF mice from two out of three breeders. Bacteroides vulgatus and Parabacteroides distasonis were detected in one of these two SPF breeding colonies in addition to Bact. acidifaciens. SPF mice of the remaining breeder harboured characteristic Bacteroides–Parabacteroides microbiota, consisting of Bacteroides sp. ASF519 and Bacteroides caccae. Conclusions: Bacteroides acidifaciens is the dominant and most typical species in the mouse Bacteroides–Parabacteroides microbiota. The Group‐3 was identified as a novel group and revealed to occupy a major niche together with Bact. acidifaciens Group‐1 and Group‐2. Significance and Impact of the Study: The species‐specific probe set developed in this study was the efficient tool for rapid detection of target bacterial groups inhabiting the mouse intestine. The results of this study provide important new information on the mouse Bacteroides–Parabacteroides community.     

设计乳杆菌特异性引物并运用菌落PCR技术快速检出和鉴定四川泡菜中的乳杆菌

乳杆菌(Lactobacillus)是益生菌, 也是当前的研究热点之一。研究泡菜等样品中的乳杆菌需要快速的检出方法。根据已完成全基因组测序的14种乳杆菌的16S rDNA序列, 设计一对乳杆菌特异性引物。PCR检测结果表明该引物对乳杆菌和明串珠菌能扩增出800 bp的片段, 对表皮葡萄球菌、乳酸乳球菌和枯草芽胞杆菌却没有扩增条带, 具有一定的乳杆菌特异性。结合MRS乳杆菌半选择培养基和革兰氏染色, 运用菌落PCR技术, 可以快速高效地检出四川泡菜中的乳杆菌。再通过对PCR扩增片段测序, 可以将乳杆菌鉴定到种。从16份四川泡菜样品中检出了15株乳杆菌, 其中14株被鉴定为植物乳杆菌, 1株需进一步鉴定才能确定种。该方法可以检出乳杆菌新种。     

Identification of Lactobacillus alimentarius and Lactobacillus farciminis with 16S-23S rDNA intergenic spacer region polymorphism and PCR amplification using species-specific oligonucleotide

AIMS: The restriction fragment length polymorphism (RFLP) method was used to differentiate Lactobacillus species having closely related identities in the 16S-23S rDNA intergenic spacer region (ISR). Species-specific primers for Lact. farciminis and Lact. alimentarius were designed and allowed rapid identification of these species. METHODS AND RESULTS: The 16S-23S rDNA spacer region was amplified by primers tAla and 23S/p10, then digested by HinfI and TaqI enzymes and analysed by electrophoresis. Digestion by HinfI was not sufficient to differentiate Lact. sakei, Lact. curvatus, Lact. farciminis, Lact. alimentarius, Lact. plantarum and Lact. paraplantarum. In contrast, digestion carried out by TaqI revealed five different patterns allowing these species to be distinguished, except for Lact. plantarum from Lact. paraplantarum. The 16S-23S rDNA spacer region of Lact. farciminis and Lact. alimentarius were amplified and then cloned into vector pCR(R)2.1 and sequenced. The DNA sequences obtained were analysed and species-specific primers were designed from these sequences. The specificity of these primers was positively demonstrated as no response was obtained for 14 other species tested. RESULTS AND CONCLUSIONS: The species-specific primers for Lact. farciminis and Lact. alimentarius were shown to be useful for identifying these species among other lactobacilli. The RFLP profile obtained upon digestion with HinfI and TaqI enzymes can be used to discriminate Lact. farciminis, Lact. alimentarius, Lact. sakei, Lact. curvatus and Lact. plantarum. SIGNIFICANCE AND IMPACT OF THE STUDY: In this paper, we have established the first species-specific primer for PCR identification of Lact. farciminis and Lact. alimentarius. Both species-specific primer and RFLP, could be used as tools for rapid identification of lactobacilli up to species level.     

16S rRNA PCR鉴定嗜酸乳杆菌鸡源分离株

用自行设计的嗜酸乳杆菌16S rRNA的特异性引物和建立的PCR方法对初步鉴定为嗜酸乳杆菌的鸡源分离株进行检测。PCR检测结果表明分离株和嗜酸乳杆菌参考株扩增出大小一致的目的片段,从分子水平对鸡源分离菌进行了鉴定。     

Development of molecular methods for identification of Streptococcus bovis from human and ruminal origins

Streptococcus bovis has been identified as a causative agent in humans for a variety of diseases, including endocarditis, meningitis, and septicemia. Identification of S. bovis strains of human origin in clinical settings has been problematic due to variations in biochemical tests as compared to ruminal strains of S. bovis, and other streptococcal species. DNA-DNA hybridization with chromosomal DNA from various S. bovis strains indicates that strains of human origin are different from those of ruminal origin. Specific probes have been designed from S. bovis 16S rDNA gene sequences that differentiate strains of human and ruminal origin by direct hybridization and PCR analyses. These techniques now allow for rapid identification of S. bovis strains for clinical and other scientific investigations.     

A rapid PCR procedure for the specific identification of Lactobacillus sanfranciscensis, based on the 16S-23S intergenic spacer regions

AIMS: The organization of ribosomal RNA (rrn) operons in Lactobacillus sanfranciscensis was studied in order to establish an easy-to-perform method for identification of L. sanfranciscensis strains, based on the length and sequence polymorphism of the 16S-23S rDNA intergenic spacer region (ISR). METHODS AND RESULTS: PCR amplification of the 16S-23S rDNA ISRs of L. sanfranciscensis gave three products distinguishing this micro-organism from the remaining Lactobacillus species. Sequence analysis revealed that two of the rrn operons were organized as in previously reported lactobacilli: large spacer (L-ISR), containing tRNA(Ile) and tRNA(Ala) genes; small spacer (S-ISR) without tRNA genes. The third described spacer (medium, M-ISR), original for L. sanfranciscensis, harboured a tRNA-like structure. An oligonucleotide sequence targeting the variable region between tDNA(Ile) and tDNA(Ala) of L. sanfranciscensis L-ISR was approved to be suitable in species-specific identification procedure. Analysis by pulse-field gel electrophoresis of the chromosomal digest with the enzyme I-CeuI showed the presence of seven rrn clusters. Lactobacillus sanfranciscensis genome size was estimated at c. 1.3 Mb. CONCLUSIONS: Direct amplification of 16S-23S ISRs or PCR with specific primer derived from L-ISR showed to be useful for specific typing of L. sanfranciscensis. This was due to the specific rrn operon organization of L. sanfranciscensis strains. SIGNIFICANCE AND IMPACT OF THE STUDY: In this paper, we have reported a rapid procedure for L. sanfranciscensis identification based on specific structures found in its rrn operon.     

乳杆菌喂食大鼠后目标菌克隆筛选与确认

目的确认乳杆菌能否顺利通过胃酸屏障并在肠道内定植为进一步研究乳杆菌对大鼠的生理代谢影响做基础,为乳杆菌菌株的应用提供有效依据。方法采用浓度梯度药物平板筛选利福平耐受菌株,用耐利福平（15 mg/L）乳杆菌菌株喂食SD雄性大鼠并采集其新鲜粪便,在耐药平板上筛选出能在大鼠结肠内定植良好的耐药菌株,并得到16S rDNA的鉴定结果,利用16S rDNA序列同源性分析对乳酸菌进行分类鉴定。结果粪便中检测出的乳杆菌耐药菌株经纯化鉴定后与所喂食的乳杆菌同源。结论实验所筛选出的耐利福平乳杆菌在大鼠结肠内成功定植。     

以23S rDNA一个多变区作为分子标记对致病杆菌属细菌进行分类鉴定

【目的】致病杆菌属(Xenorhabdus)细菌是一类重要的生物杀虫剂,斯氏属昆虫病原线虫的共生菌,建立快速准确的分类鉴定方法,对研究开发这类细菌至关重要。【方法】本研究PCR扩增测序了本室保藏的26株,含20种已定名致病杆菌属细菌的一段845 bp的23S rDNA序列,构建了基于这段序列的致病杆菌属系统树并与基于几乎全长16S rDNA序列的相应系统树进行比较,分析了两者作为致病杆菌属细菌分类鉴定分子标记的优缺点。【结果】结果表明,与全长16S rDNA序列相比,所选择的23S rDNA序列片段所含可变位点、简约信息位点比例更高,遗传距离数值跨度大。【结论】上述结果显示该序列片段可用于致病杆菌属细菌进行分类鉴定,特别适用于对野外资源调查中采集到的大量菌株进行快速鉴定。     

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,明确大理乳扇制品中乳杆菌的多样性及优势种群分布,为科学利用奠定基础。方法采用表型鉴定及16S r RNA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 r RNA鉴定为植物乳杆菌和马酒乳杆菌,06422株与植物乳杆菌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 r RNA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。     

高抑菌活性乳酸杆菌的筛选及性质

从自酿酸奶中分离得到1株高抑菌活性菌株,经16S rDNA测序后鉴定为Lactobacillus sp.FSZ。以大肠杆菌、金黄色葡萄球菌为指示菌,取得良好抑菌效果。经组分分析及蛋白酶降解,抑菌活性物质确定为蛋白物质,推测其由一些高分子的蛋白类物质和低分子的多肽类物质组成。抑菌活性物质在酸性条件下显示出良好的抑菌活性,发酵液经60℃处理30 min后,活性基本没有下降,经100℃处理30 min仍保留83.9%的活性,表现出良好的热稳定性。     

Differentiation of Lactobacillus isolates from infant faeces by SDS-PAGE and rRNA-targeted oligonucleotide probes

Isolates of lactobacilli from infant faeces phenotypically characterized as Lactobacillus paracasei subsp. paracasei (six strains), Lact. rhamnosus (six strains), Lact. gasseri (three strains), Lact. acidophilus (one strain) and Lact. fermentum/reuteri (three strains) according to recent classification systems were subjected to SDS-PAGE of whole cell proteins and rRNA-targeted oligonucleotide probe hybridization, in order to confirm the phenotypic characterization and elucidate the exact taxonomic position of the three strains that had properties between fermentum and reuteri. Results suggested a good agreement between the phenotypic characterization, SDS-PAGE and rRNA-targeted oligonucleotide probe hybridization for strains of all species except for the Lact. fermentum/reuteri strains. Results obtained by rRNA probes suggested a possible phylogenetic relatedness of the strains to Lact. reuteri. Isolates from infant faeces with interesting probiotic properties could be used as components of fermented milk products.     

黄土高原地区大豆根瘤菌的遗传多样性和系统发育

【目的】研究黄土高原地区大豆根瘤菌的遗传多样性和系统发育。【方法】采用BOX-PCR、16S rDNAPCR-RFLP、16S-23S IGS PCR-RFLP和16S rRNA基因序列分析方法对分离自我国黄土高原地区4个省的15个地区的130株大豆根瘤菌及部分参比菌株进行了遗传多样性和系统发育分析。【结果】BOX-PCR反映的菌株多样性最丰富,形成的遗传群最多,16S rDNA PCR-RFLP方法在属、种水平上聚群较好,16S-23S IGSPCR RFLP反映的多样性介于BOX-PCR和16S rDNA PCR-RFLP之间,能够较好地反映出属、种和亲缘关系很近的菌株间的差异,3种方法聚类分析结果基本一致,可将所有供试菌株分为两大类群,中华根瘤菌属(Sinorhizobium)和慢生根瘤菌属(Bradyrhizobium)。从系统发育来看,供试的快生大豆根瘤菌为费氏中华根瘤菌(Sinorhizobium fredii),慢生大豆根瘤菌为日本慢生大豆根瘤菌(Bradyrhizobium japonicum)和辽宁慢生根瘤菌(Bradyrhizobium liaoningense)。【结论】我国黄土高原地区大豆根瘤菌具有较丰富的遗传多样性,S.fredii优势种,慢生大豆根瘤菌仅占10%,同时,分离到2株B.liaoningense。     

PCR-mediated detection of the chemolithotrophic bacterium Thiobacillus cuprinus using 23S rDNA- and 16S/23S intergenic spacer region-targeted oligonucleotide primers

Abstract Bioleaching is carried out by chemolithotrophic microorganisms, most of them belonging to the genera Thiobacillus and Leptospirillum . The role of the mixotrophic species T. cuprinus in this process is controversial, since its ecological study applying classical detection techniques to natural or industrial environments is very difficult. For this reason, we have developed an alternative method based on PCR-mediated detection using specific oligonucleotide primers that target variable regions of the 23S rRNA coding gene and of the 16S/23S intergenic spacer region. Specificity and sensitivity of PCR amplifications performed with both kinds of primers were studied.     

珠江口沉积物好氧反硝化细菌的筛选及鉴定

研究首次于珠江口沉积物中分离出多株好氧反硝化细菌,从中筛选出一株反硝化性能最强的菌株A14-1。综合其生理生化及分子生物学鉴定的结果确定此菌株为红球菌属Rhodococcus aetherivorar。此菌株可在48 h内将培养基中的硝酸盐含量从157.91mg·L^-1降低至32.07mg·L^-1,反硝化效率高达26.20 mg·L^-1·h^-1,且不会产生亚硝酸盐的明显积累。以细菌总基因组DNA为模板成功扩增出亚硝酸还原酶基因nirS,说明亚硝酸还原酶可能参与了此菌株的好氧反硝化过程,将亚硝酸盐进一步还原,从而不会造成水体亚硝酸盐的积累。菌株A14-1在珠江口多个站点均有分布,环境适应能力强,且不会对环境造成危害,因此有望应用于污水的生物脱氮处理中。     

嗜盐古生菌br基因的遗传分析

从新疆阿尔金山地区阿乌拉仔盐湖分离纯化到几株极端嗜盐古生菌AJ11, AJ12和AJ13, 采用PCR技术分别扩增了其16S rRNA基因(16S rDNA)和编码螺旋C至螺旋G的细菌视紫红质(bacteriorhodopsin, BR)蛋白基因片段, 测定了基因的核苷酸序列。基于16S rDNA序列的同源性比较以及系统发育学研究表明, 分离到的菌株是Natrinema属中成员, 并构成一个独立的微生物种群。随后的遗传分析, 包括GC含量、转换与颠换的比率、同义突变率分析, 表明br基因间具有较高的遗传分歧程度, 并面临着净化选择和偏倚突变压的双重抑制。研究为物种资源及BR蛋白资源的进一步利用打下基础。     

微囊藻毒素降解菌的筛选、鉴定及其降解活性研究

采用从巢湖水华蓝藻细胞中提取、提纯的藻毒素（Microcystins,MCs）为微生物生长的唯一碳源和氮源,通过平板分离纯化,从巢湖底泥中分离出5株能够降解藻毒素的菌株,并对其中降解活性较高的一株进行分子鉴定。应用PCR技术克隆到16S rDNA片段,核苷酸序列分析结果表明,该菌的16S rDNA的全序列与吉氏库特菌kurthia gib-soniistrain HC050630C-1的相似性达99%。微囊藻毒素降解实验结果表明,用15mg/L乙醇作为外加碳源时可显著提高菌株M9降解MCs的能力,在48h内对初始浓度分别为17.1mg/L的MC-RR和11.3mg/L的MC-LR的降解率分别达到70.0%和81.6%。而葡萄糖对菌株M9的生长有明显抑制作用。     

Genotypic and phenotypic diversity of Lactobacillus rossiae strains isolated from sourdough

AIM: To characterize the genetic and phenotypic diversity of 33 strains of Lactobacillus rossiae. METHODS AND RESULTS: Genotypic identification was carried out by partial 16S rRNA gene sequence analysis. Genetic diversity was evaluated by RAPD-PCR analysis. Phenotypic diversity was evaluated through fermentative profile by Biolog system, proteinase and peptidase activities using synthetic substrates, and acidification capacity and amino acid profile during sourdough fermentation. The genetic analyses excluded clonal relatedness among the strains used. A large phenotypic diversity was found. It mainly concerned the capacity to use carbon sources available in sourdough during fermentation, the quotient of fermentation and the peptidase activities, especially towards proline containing synthetic substrates. The free amino acid profiles differed either for the total concentration or for the type of amino acids. With a few exceptions, proteinase activity towards wheat albumins and globulins was weak. CONCLUSIONS: Overall, no relationships between genetic and physiological analyses were found, and the strains examined showed a marked genetic and phenotypic heterogeneity. L. rossiae strains had interesting properties for application in sourdough fermentation. Although some strains combined several technological traits, the association of more strains seemed to be a requisite to get optimal sourdough characteristics. SIGNIFICANCE AND IMPACT OF THE STUDY: It represents the first characterization of the diversity within the L. rossiae species. Besides, it may represent an example of computerized analysis of genotypic and phenotypic information to select strains for improving sourdough characteristics.