仔猪常见12种致病菌的23S rRNA基因序列分析

以107条从NCBI下载和测序的12种仔猪常见致病菌的23S rRNA基因序列为研究对象,运用DNASTAR软件进行系统发育分析.结果显示:以各种细菌的23S rRNA基因代表序列所进行的种间序列分析结果与伯杰氏细菌分类手册和http://www.wiki.cn/wiki/细菌分类表的分类结果一致,也与这12种菌的16S rRNA基因序列分类结果一致. 因此,在23S rRNA基因序列保守区设计通用引物,在其变异区设计各种细菌特异性探针,用PCR捕捉被检样品中未知细菌的23S rDNA片段并与种特异性23S rRNA基因探针进行杂交,有可能将未知细菌鉴定到种.     

利用RFLP分型方法鉴定牛源性链球菌和肠球菌

为分离及种属鉴定引起牛乳腺炎相关的链球菌和肠球菌,本研究于兰州及周边地区采集疑似奶牛乳腺炎乳样382份,通过THB(Todd-Hewitt Broth)固体选择培养基初步分离到67株疑似链球菌或疑似肠球菌。参照已发表文献合成链球菌属16S r RNA和16S~23S r RNA间隔区基因引物序列,扩增分离菌株16S~23S r RNA间隔区序列,产物分别利用AluⅠ和RsaⅠ单酶切消化,并以参考菌株的16S~23S r RNA酶切图谱为参考,对分离株进行限制性片段多态性(RFLP)分类分析;再选取各RFLP类群的任一菌株,扩增其16S r RNA基因并测序,并经NCBI核酸数据库进行比对,结果显示67株疑似链球菌中有53株为粪肠球菌(79.1%)、3株为屎肠球菌(4.5%)、3株为肠道肠球菌(4.5%)、8株为无乳链球菌(11.9%)。结果表明肠球菌属细菌(粪肠球菌、肠道肠球菌、屎肠球菌)与兰州市及周边地区奶牛乳腺炎的发病紧密相关,肠球菌属细菌与链球菌属细菌16S r RNA基因同源性很高,但可以通过分子生物学方法准确区分。     

16S rRNA、23S rRNA及16S～23S rRNA基因在细菌分离与鉴定中的应用

在细菌的进化过程中,rRNA结构既具保守性又具高变性.保守性反映生物物种的亲缘关系,高变性则揭示生物物种的特征核酸序列,rRNA是属种鉴定的分子基础.因此,可以利用保守区设计通用引物扩增细菌的相应靶序列,再利用可变区的差异鉴别菌种,文章就16S rRNA、 23s rRNA和16S～23S rRNA基因在细菌分离与鉴定中的应用做以下综述.     

大肠埃希菌PCR检测方法的建立与应用

为建立一种快速检测动物大肠杆菌病的方法,基于大肠埃希菌16S~23S rRNA基因序列分析,利用Premier 5.0软件设计并合成了一对特异性引物,建立了可检测16S~23S rRNA基因的PCR检测方法,通过反复试验确定了最佳PCR反应体系和反应条件,并评估了此方法的灵敏性和特异性。同时应用该方法对临床病料进行检测,检测结果与生化试验鉴定结果的符合率为100%。说明该方法具有良好的特异性和敏感性,与细菌分离培养、生化鉴定方法相比较有效降低了检测时间,提高了检测效率。     

采用PCR-SSCP技术分析蚯蚓粪便的微生物群落结构

通过SDS/蛋白酶的方法抽提蚯蚓粪便微生物总DNA,以总DNA为模板,采用通用引物分别成功扩增细菌16S rRNA基因的V4-V5可变区,真菌18S rRNA基因的V8-V9可变区,应用单链构象多态性技术（SSCP）分析了蚯蚓粪便中不同空间层次的微生物种群的多样性。结果表明,在粪便的不同空间层次上呈现出明显的空间分布多样性,并且与纯蚯蚓粪微生物种群的多样性有很大的差别。为进一步的蚯蚓粪生态学功能研究提供了有益的指导。     

16S-23S rRNA基因间隔序列PCR及RFLP对奇异变形杆菌分离株的鉴别与系统发育分析

根据临床常见致病菌16S-23S rRNA基因间隔序列(ISR)两端的16S及23S rRNA保守序列设计PCR扩增的通用引物,对9株奇异变形杆菌和6株相近菌株应用通用引物PCR扩增16S-23S rRNA ISR序列.通过PCR长度多态性比较、RFLP分析以及部分序列测序比较,分析鉴别奇异变形杆菌.结果显示,PCR长度多态性可以将奇异变形杆菌同其余菌种进行区分;RFLP分析可以将所有试验菌种进行区分;部分序列测序可以对奇异变形杆菌进行分型.由此表明,16S 23S rRNA ISR序列PCR及RFLP分析可以简单、快速、准确的鉴定奇异变形杆菌.     

流产奶牛胎儿肺炎克雷伯氏菌的分离与鉴定

从流产奶牛胎儿组织中分离到1株革兰氏阴性短杆菌,采用细菌分离纯化、生化试验、16S rRNA和16S～23S rRNA测序、药敏试验和小鼠致死性试验对其进行了鉴定。结果显示,该分离菌与GenBank中肺炎克雷伯氏菌16S rRNA和16S～23S rRNA基因序列一致性分别高达99%和96%,16S～23S rRNA基因被扩增出3条不同长度条带;该菌生化特性均符合肺炎克雷伯氏菌的生化反应特性;对头孢曲松、阿米卡星、多粘菌素等敏感,对多西环素、四环素、卡拉霉素等中度敏感,对青霉素、红霉素、米诺环素等耐药;对小鼠有很强的致病性。以上结果表明,该分离细菌为肺炎克雷伯氏菌。     

赣南地区柑橘黄龙病菌遗传多样性分析

为探究赣南地区柑橘黄龙病菌的遗传多样性。从赣南16个县市区采集32个具典型柑橘黄龙病症状的脐橙样品，运用PCR-RFLP(聚合酶链式反应-限制性长度多态性分析)技术研究了柑橘黄龙病菌16S rDNA序列、16S/23S rDNA间隔区序列和核糖体蛋白基因的遗传多态性；并对三个基因片段进行测序，分析其序列的碱基含量、转换与颠换、同源性、核苷酸遗传距离和系统进化特征。RFLP结果表明：赣南16个县市区柑橘黄龙病菌XbaⅠ酶切图谱均与亚洲韧皮部杆菌（Candidatus Liberibacter asiaticus, CLas）的一致；进一步分析显示，三个基因片段各自之间的RFLP指纹图谱一致，并未表现多态性。序列分析结果表明：32个分离物三个基因片段各自的同源性均在98%~100%，与CLas的同源性最大，且与CLas的遗传距离最小。系统进化树展示赣南地区柑橘黄龙病菌与CLas处于同一分支，结合酶切图谱、序列同源性及系统进化分析结果表明： 赣南地区柑橘黄龙病菌均为CLas。结论：赣南地区柑橘黄龙病菌的16S rDNA序列、16S/23S rDNA间隔区序列和核糖体蛋白基因未表现遗传多样性，表明赣南地区柑橘黄龙病菌间遗传多样性不显著。 关键词：柑橘黄龙病；16S rDNA序列；核糖体蛋白基因；16S/23S rDNA间隔区；RFLP；序列分析     

16S-23S rDNA间隔区在奶牛乳房炎诊断中的应用

以16S-23S rDNA间隔区为扩增靶序列的PCR技术是近年来发展的细菌分类和鉴定新方法，具有快速、敏感和特异性高的优点。作者综述了细菌16S-23S rDNA间隔区序列的特性及其在奶牛乳房炎病原诊断中的应用等研究进展。     

16S rRNA基因在细菌菌种鉴定中的应用

本文综述了应用16S rRNA基因作为靶基因对细菌进行鉴定的各种分子生物学技术，并指出应用16S rRNA基因在乳酸菌鉴定方面的研究进展。     

瑞士乳酸杆菌HZ521株的分子鉴定及其部分生物学特性研究

根据GenBank中乳酸杆菌16 S～23 S rRNA基因间沉默区序列设计引物,进一步鏊定猪源乳酸杆菌分离株HZ521;并通过体外试验,以嗜酸乳酸杆菌ATCC 4356为参考菌株,探讨HZ521株的益生素作用.部分16 S～23 S rRNA基因序列同源性分析结果表明,该分离株属于瑞士乳酸杆菌.HZ521株具有较强的产酸能力,能耐受强酸,对Hela细胞的黏附率为87.2%,显著高于ATCC 4356株(P<0.01).表明瑞士乳酸杆菌HZ521株具有益生素特性,可作为肠道益生素的候选菌株.     

随机扩增多态性DNA技术对绿色气球菌的相关性分析

为考察恩诺沙星与乙酰甲喹的联合抗菌活性,采用肉汤稀释棋盘法测定了恩诺沙星与乙酰甲喹单用及联用对大肠杆菌、沙门氏菌临床菌株的体外抗菌活性。结果显示:恩诺沙星与乙酰甲喹单独使用对大肠杆菌的最小抑菌浓度为8μg/m L和32μg/m L,对沙门氏菌的最小抑菌浓度为16μg/m L和256μg/m L。两药联用对大肠杆菌和沙门氏菌的FIC指数为0.5和0.75,分别表现为协同作用和相加作用。试验表明,恩诺沙星与乙酰甲喹联用可以增强对大肠杆菌和沙门氏菌感染的治疗效果。     

Genetic diversity of Streptococcus equi subsp. zooepidemicus isolated from horses

Streptococcus equi subsp. zooepidemicus (SEZ) is an opportunistic and zoonotic pathogen of horses. In this study, genetic intraspecies variability of SEZ obtained mainly from respiratory and genital samples of horses was investigated by analysis of the 16S–23S rRNA intergenic spacer region (ISR) and of the 16S rRNA gene. 16S–23S ISR rRNA type A1 was predominant, although a high rate of multiple products (30.5%) was obtained. Phylogenetic analysis of the 16S rRNA gene detected three genogroups (I, II and III). 16S rRNA variable regions V1 and V2 are the most important regions for evaluating SEZ intraspecies variability, but at least V1-V5 regions should be considered to avoid mistakes. Analysis of all 16S rRNA sequences available in databases assigned human SEZ to groups I and III but not to group II. These results show a high genetic variability in SEZ collected from different specimens of horses from various regions of Italy.     

Examination of the 16S-23S rRNA intergenic spacer sequences of 'Candidatus Mycoplasma haemobos' and Mycoplasma haemofelis

The intergenic spacer region between the 16S and 23S rRNA genes of mycoplasmas has been used for a genetic marker for identification of the species. Here we show the intergenic spacer regions of two hemotropic mycoplasmas, Mycoplasma haemofelis and 'Candidatus Mycoplasma haemobos (synonym: 'C. M. haemobovis')' are also useful for classification of this particular group of mycoplasms. The spacer region of M. haemofelis and `C. M. haemobos' consisted of 209 and 210 base pairs, respectively, and both lacked the spacer tRNA genes. Phylogenetic analysis suggested a monophyletic relationship among hemoplasmas and M. fastidiosum. A hypothetical secondary structure predicted in the spacer regions tentatively assigned the boxA and boxB motifs peculiar to the members of the genus Mycoplasma. M. haemofelis and 'C. M. haemobos' possessed a stem-loop structure in common, despite the presence of a palindromic nucleotide substitution in the stem region.     

Characterization of the 16S-23S rRNA intergenic spacer regions among strains of the Fusobacterium necrophorum cluster

The 16S-23S rRNA intergenic spacer regions (ISRs) of Fusobacterium necrophorum subsp. necrophorum and F. necrophorum subsp. funduliforme were characterized. Products of two sizes, about 360 bp (small) and 530 bp (large), were generated by PCR amplification from the 16S-23S rRNA ISR of all the strains tested. The large and small 16S-23S rRNA ISRs of F. necrophorum exhibited a level of sequence similarity of 93.9% to 99.7% and 94.2% to 98.6% homologies within the species, respectively. Only the large spacer regions in these bacteria contained one or two tRNA genes. F. necrophorum subsp. necrophorum contains the isoleucine and alanine tRNA gene, whereas F. necrophorum subsp. funduliforme contains the isoleucine tRNA gene.     

Comparison of the 16S-23S rRNA intergenic spacer regions between Fusobacterium varium and "Fusobacterium pseudonecrophorum" strains

The 16S-23S rRNA intergenic spacer regions (ISRs) of five strains of "Fusobacterium pseudonecrophorum" which had been proposed as a new species, were compared with those of F. varium ATCC 8501T. All the strains of "F. pseudonecrophorum" exhibited of sequence similarities of 97.7% to 100% to the strain of F. varium in their 16S-23S rRNA ISR sequences. This indicates that the strains of "F. pseudonecrophorum" and the type strain of F. varium are identical at the species level.     

Development of a polymerase chain reaction for the detection of Mycoplasma felis in domestic cats

Mycoplasma felis is associated with conjunctivitis and respiratory disease in domestic cats. Currently no rapid diagnostic test is available for the detection of M. felis in clinical samples that does not rely on prior cultivation of the organism. The objective of this study was to determine whether a polymerase chain reaction (PCR) based upon the 16S/23S rRNA intergenic spacer sequence is suitable for the identification of M. felis directly in feline clinical samples. The high conservation between the 16S/23S rRNA intergenic spacers (IGS) of differing isolates of M. felis was established by sequence analysis and a PCR was developed to this region by comparison to IGS of other mycoplasmas. The PCR was found to be highly specific for M. felis and further PCR analysis on clinical samples showed the PCR to be highly sensitive and more rapid than the other methods of identification currently available.     

Molecular identification of Arcanobacterium bialowiezense and Arcanobacterium bonasi based on 16S-23S rRNA intergenic spacer region sequences

In the present study, the 16S-23S rDNA intergenic spacer region (ISR) of Arcanobacterium (A.) bialowiezense DSM 17162, A. bonasi DSM 17163, A. bernardiae DSM 9152, A. haemolyticum DSM 20595, A. hippocoleae DSM 15539, A. phocae DSM 10002, A. pluranimalium DSM 13483 and A. pyogenes DSM 20630 was amplified, sequenced and compared with the corresponding 16S rRNA gene sequences yielding comparable phylogenetic relationships. The ISR sequence of A. bialowiezense and A. bonasi allowed the design of species-specific oligonucleotide primers which could successfully be used for PCR-mediated identification of previously characterized A. bialowiezense and A. bonasi isolated from infections of the European bison. The presented molecular identification might help to improve a future diagnosis of both newly described bacterial pathogens.     

The Mycoplasma gallisepticum 16S-23S rRNA intergenic spacer region sequence as a novel tool for epizootiological studies

Mycoplasma gallisepticum (MG) contains two sets of rRNA genes (5S, 16S and 23S) in its genome, but only one of the two is organized in an operon cluster and contains a unique 660-nucleotide intergenic spacer region (IGSR) between the 16S and the 23S rRNA genes. We designed a polymerase chain reaction (PCR) for the specific amplification of the complete MG IGSR segment. The MG IGSR PCR was tested on 18 avian mollicute species and was confirmed as MG specific. The reaction sensitivity was demonstrated by comparing it to the well-established MG mgc2 PCR. The MG IGSR sequence was found to be highly variable (discrimination [D] index of 0.950) among a variety of MG laboratory strains, vaccine strains, and field isolates. The sequencing of the MG IGSR appears to be a valuable single-locus sequence typing (SLST) tool for MG isolate differentiation in diagnostic cases and epizootiological studies.