细菌分类与鉴定的新热点：16S—23S rDNA间区

随着分子生物学的迅速发展,细菌的分类鉴定亦从传统的表型分类进入到各种基因型分类水平、如（G＋C）mol%、DNA杂交、rDNA指纹图、质粒图谱和16S rDNA序列分析等。rRNA存在于所有细菌中,rRNA基因由保守区和可变区组成,在细菌中高度保守。rRNA基因包含5‘端到3‘端的若干种成分,分别是16S rDNA、间区、23S rDNA、间区和 5S rDNA。16S－23S rDNA间区近年来在细菌系统发育学,特别是相近种和菌 区分和鉴定方面倍受关注。作为细菌分类和鉴定中的一个热点,本文将就16S－23S rDNA间区的一些特性及其胡细菌分类鉴定方面的作用做一简要的介绍。     

棕囊藻北部湾株的18S rDNA分子鉴定

为研究北部湾棕囊藻(Phaeocystis)藻华的成因,采用PCR克隆了棕囊藻北部湾株核糖体18S r DNA序列。结果表明,棕囊藻北部湾株具有游动单细胞与群体结构两种形态;其18S r DNA序列和NCBI基因库中球形棕囊藻(Phaeocystis globosa)的同源性为99%~100%,在系统进化树上与不同海域来源的球形棕囊藻聚在一大分支上,且与球形棕囊藻间的遗传距离均小于其他种。首次从分子生物学上确定棕囊藻北部湾株为球形棕囊藻。     

16S～23S RDNA间区在链球菌和流感嗜血杆菌分类中的应用

利用16S~23S rDNA间区（intergenic spacer regions,ISR）在不同细菌中拷贝数、碱基排列、序列长度及所含tRNA基因种类和数目的差异,对15株链球菌和流感嗜血杆菌进行属、种、型和株系的分类鉴定。在16S rDNA的3′端和23S rDNA的5′端的保守区中合成引物,PCR扩增16S~23S rDNA ISR序列,对多态片段切胶纯化直接测序。在GenBank上查找对应细菌的ISR序列。用DNAMAN软件进行系统进化分析。链球菌属为单拷贝16S~23Sr RNA ISR、有一个tRNAAla基因编码区、分子大小在269~446bp之间,序列分成4个保守区和4个可变区,可变区碱基排列方式和数目的不同是种分类的依据。7株链球菌的同源率在78%~88%。同种异株的差异反映在碱基的插入和缺失上。流感嗜血杆菌各生物型均为2个拷贝的ISR,小片段为514~519bp,编码1个tRNAGlu基因,有3个狭窄可变区。大片段富含A T碱基,在I、II和IV型中分别是868、848和856bp,编码一个tRNAIle基因和一个tRNAAla基因。不同生物型小分子ISR与标准菌株比较,同源性在97.3%~99.6 %之间。 ISR作为细菌分类的目的基因具有属、种、型和株特异性与灵敏性。简单的基因分离分析技术为认识病原微生物提供了更多的机会。 Abstract:To facilitate species level identification of bacteria without the requirement of presumptive identification,the paper describes a rapid identification method of bacteria by amplification and direct sequencing 16S~23S rDNA intergenic spacer regions (ISR) of the pathogens which cause the upper respiratory tract infective disease by Streptococcus and Haemophilus.Three pairs of primer targeting conserved sequences flanking the 3′ end of 16S and the 5′end of 23S rRNA were used to amplify 16S~23S rRNA ISR of 7 streptococcus strains and 8 Haemophilus strains.The PCR products were separated by 1% agarose gel electrophoresis and the polymorphisms fragments were purified with the Wizard PCR Min-Prep Kit (Promega) and Protocol-SK131(Sangon).The nucleotide sequences of ISR inserts were determined by using the XEQTM DTCS Kit——Terminator Cycle Sequencing and a CEQTM 2000XL DNA Analysis system (Backman Coulter) automatic DAN sequencer.Then those sequences were compared with known seqnences on the GenBank.The alignment of nucleotide sequence,evolutionary distances and phylogenetic tress were analyzed by software DANMAN version 4.0.The PCR products were showed polymorphism patterns with agarose gel.One band was contained in streptococcus genus.The significant variation was found among the spacer sequences of different species in Streptococcus with the lengths of the spacer varying from 269 to 446bp.All the ISR of the streptococcal species had a tRNA Ala gene in the spacer and the sequence identities varied from 78 to 88% within genera.It was found that some spacer sequence blocks were highly conserved between operons of a genome,whereas the presence of others was variable,three regions showed significant spatial variation.Most of the differences between the sequences came from several bases insertions/deletions and substitutions.There are two major bands in the Haemophilus biotypes(515 and 884bp),the small ISR amplicon contained one tDNA coding for tRNAGlu.In contrast to the large one contained two tRNA genes coding for tRANAla and tRNAIle.Two regions of repeating motifs with only A or T were present in higher copy numbers between tRANAla and tRNAIle.The phylogenetic trees varied from 97.5 to 98.8%.The PCR and direct sequencing of 16S~23S rRAN ISR were successful in the pathogen species identification.     

深海细菌的分子鉴定分类

从东太平洋海底 5,30 0m深的沉积物中分离到一批嗜冷细菌 ,克隆了其中 1 3株细菌的1 6SrRNA基因 ,通过其序列的测定和比较 ,对这 1 3株细菌进行了分子鉴定 ,结果表明它们分属于副球菌 ,假单胞菌 ,盐单胞菌和假交替单胞菌 4个不同的菌属 ,同时进行了系统发育分析。     

阪崎克罗诺杆菌的16S rDNA鉴定分型

阪崎克罗诺杆菌（Cronobacter sakazakii）是一种重要的食源性条件致病菌,易引发新生儿、早产儿、低体重新生儿和免疫力低下的婴幼儿产生严重的脑膜炎、菌血症和坏死性小肠结肠炎。本研究以分离自不同来源、通过生化鉴定的37株阪崎克罗诺杆菌作为研究对象,利用16S rDNA基因测序的方法对阪崎克罗诺杆菌进行鉴定和分型。结果表明：ES4并非为阪崎克罗诺杆菌,说明采用16S rDNA基因测序进行阪崎克罗诺杆菌的鉴定更为可靠;通过构建系统发育树分析,分离菌株位于同一系统发育分支;根据序列同源性比对,可将这一分支下的所有菌株分成7个子群,这表明16S rDNA基因测序可以对阪崎克罗诺杆菌进行分型,进一步揭示其系统发育关系。     

16S和23S rDNA基因序列分析分类鉴定中国衣原体流行株

通过分析比较部分16S/23S rDNA序列,对现有保存的9株国内衣原体流行株进行了分子遗传学鉴定。虽然这些分离株分离自不同的动物,但它们的16S/23S扩增部分完全相同,经16S/23S rDNA序列同源性比较可以一致鉴定国内流行株为鹦鹉热嗜衣原体。     

16S～23S rDNA间隔区序列在分枝杆菌分类鉴定中的应用研究

采用聚合酶链反应（ＰＣＲ）技术对分枝杆菌１６Ｓ￣２３Ｓ ｒＤＮＡ间隔区序列进行扩增,其产物经聚丙烯酰胺凝胶电泳（ＰＡＧＥ）,并通过计算机聚类分析,在基因水平上评价其对分枝杆菌分类与鉴定的意义。退火温度４５℃时,ＰＣＲ扩增的敏感性为５００ｆｇ／μＬ,而５０℃时的敏感性为５ｐｇ／μＬ。已通过对２２种分枝杆菌和９种非分枝杆菌的特异性实验,结果表明：扩增条带多集中在３００￣６００ｂｐ之间,多数受试快速生长     

拮抗放线菌B-20的种类鉴定及其16S rDNA序列分析

对分离自东北蔬菜保护地土壤的1株拮抗放线菌菌株B-20进行了形态特征、培养特征、生理生化、细胞壁组分分析及16S rDNA序列分析。基内菌丝无横隔、不断裂,气生菌丝多分枝;孢子丝波曲至螺旋形,孢子椭圆形,表面光滑。细胞壁化学组分Ⅰ型。以16s rDNA序列为基础构建了包括13株相关种属细菌在内的系统发育树。根据多相分类鉴定结果表明,放线菌B-20的上述特征与淡紫灰链霉菌(Streptomyces lavendulae)的高度一致。二者的16S rDNA序列的相似性达到了99%。因此,可将链霉菌B-20定名为淡紫灰链霉菌B-20 (S.lavendulae B-20)。     

分离自补骨脂等宿主根瘤的24株菌的数值分类和16S rDNA PCR-RFLP 研究

采用数值分类和16S rDNA PCR-RFLP对分离自云南省豆科植物补骨脂(Psoralea corylifolia)、葛藤(Pueraria lobata)、杭子梢(Campylotropis macrocarpa)等宿主的24株菌及10株根瘤菌参比菌株进行了研究。数值分类结果表明, 在84%相似性水平上, 所有的菌株可分为3群：群Ⅲ为未知菌群, 群Ⅰ为慢生菌群, 群Ⅱ为快生和中慢生菌群。从依据16S rDNA PCR-RFLP分析建立的树状图来看, 在70%相似性水平上, 所有的菌株可分为5个系统发育分支：分支Ⅰ和Ⅴ没有参比菌株, 为未知分支; 分支Ⅱ为Agrobacterium-Sinorhizobium-Rhizobium, 分支Ⅲ为Mesorhizo- bium, 分支Ⅳ为Bradyrhizobium。数值分类和16S rDNA PCR-RFLP的结果部分一致, 有2株菌与A. tumefaciens IAM13129T聚在一起。     

2株创伤弧菌的16S-23S rDNA间区的克隆、测序及分析

根据细菌的16SrDNA3’端和23SrDNA5’端的高度保守区设计引物,PCR扩增了2株创伤弧菌（Vibrio vulnificus）的16S-23SrDNA间区（Intergenic spacer,IGS）,克隆到pGEM-T载体上,测序。用BLAST和DNA star软件对16S-23SrDNA间区序列及其内的tRNA基因进行比较分析。结果表明,2株创伤弧菌共测出9条16S-23SrDNA间区序列,其中ZSU006测出5条,间区类型分别为：IGS^GLAV、IGS^GLV、IGS^LA、IGS^A和IGS^G.其中IGS^GLAv最大,包含tRNA^Glu、tRNA^Lys、tRNA^Ala。和tRNA^Val基因;IGS^GLV包含tRNA^Glu、tRNA^Lys。和tRNA^Val基因;IGS^LA,则包含tRNA^Ile和tRNA^Ala基因;IGS^G包含tRNA^Glu基因;而IGS^A仅包含tRNA^Ala基因。菌株CG021测出的16S-23SrDNA IGS序列有4条,除缺少IGS^A外,其余的IGS类型均与ZSU006的相同。与GenBank内的创伤弧菌ATCC27562的IGS序列比较,发现创伤弧菌所有类型的IGS的tRNA基因两端的非编码区具有较高的种内同源性。16S-23SrDNA间区结构的差异为建立一种新的创伤弧菌检测方法奠定了基础。     

PCR amplification of species specific sequences of 16S rDNA and 16S-23S rDNA intergenic spacer region for identification of Streptococcus phocae

Streptococcus phocae, a bacterial pathogen of seals, could reliably be identified by PCR amplification using oligonucleotide primers designed according to species specific segments of the previously sequenced 16S rRNA gene and the 16S-23S rDNA intergenic spacer region of this species. The PCR mediated assay allowed an identification of S. phocae isolated from harbor and gray seals and from Atlantic salmons. No cross-reaction could be observed with 13 different other streptococcal species and subspecies and with Lactococcus garvieae strains investigated for control purposes.     

Molecular characterization of Serratia marcescens strains by RFLP and sequencing of PCR-amplified 16S rDNA and 16S-23S rDNA intergenic spacer

AIMS: To establish the specific DNA patterns in 16S rDNA and 16S-23S rDNA intergenic spacer (IGS) regions from different kinds of Serratia marcescens strains using polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) and sequences analysis. METHODS AND RESULTS: Two pairs of primers based on the 16S rDNA and 16S-23S rDNA IGS were applied to amplify the rrn operons of two kinds of S. marcescens strains. About 1500 bp for 16S rDNA and four fragments of different sizes for 16S-23S rDNA IGS were obtained. PCR-amplified fragments were analysed by RFLP and sequence analysis. Two distinct restriction patterns revealing three to five bands between two kinds of strains were detected with each specific enzyme. According to the sequence analysis, two kinds of strains showed approximately 97% sequence homology of 16S rDNA. However, there was much difference in the sequences of IGS between the two kinds of strains. Intercistronic tRNA of strains H3010 and A3 demonstrated an order of tRNA of 5'-16S-tRNA(Ala)-tRNA(Ile)-23S-3', but strain B17 harboured the tRNA of 5'-16S-tRNA(Glu)-tRNA(Ile)-23S-3'. CONCLUSIONS: The method was specific, sensitive and accurate, providing a new technique for differentiating different strains from the same species. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper provided the first molecular characterization of 16S rDNA and 16S-23S rDNA IGS from S. marcescens strains.     

Rapid species identification and partial strain differentiation of Clostridium butyricum by PCR using 16S-23S rDNA intergenic spacer regions

Some Clostridium butyricum strains have been used as probiotics for both humans and animals. Strain-specific identification is necessary for the manufacturing process of probiotics. The aim of this study was to determine whether there are sufficient genetic variations in 16S-23S intergenic spacer regions (ISRs) to discriminate C. butyricum at the biovar level. We amplified ISRs from five reference strains, a probiotic strain (MIYAIRI 588) and 22 isolates, and we classified them into four groups on the basis of amplification patterns (type A through D). However, amplification of ISRs is not sufficient for discriminating strains. Moreover, we compared genetic structures of these ISRs. Sequence analysis revealed that the size variations of ISRs were generated by the insertion of tRNA genes and unique sequences into the internal portion, while the external portions were highly conserved. On the basis of the highly conserved nucleotide sequences within the ISRs, we developed a PCR primer set specific to C. butyricum. In addition, the PCR primer designed from the unique inserted sequence in type B strain was useful to differentiate probiotic strains at the biovar level.     

昆虫病原线虫共生细菌的分子生物学鉴定

对来自全国各地的23个未鉴定的共生菌菌株和本室保存的5个菌株的部分16S rDNA序列进行PCR扩增和测序,与GenBank中已知种的共生菌及4个其它肠杆菌的同源序列比较,利用DNAstar软件绘制系统树,初步确定了这28个共生菌的分类地位。其中品系0396Y与Xenorhabdus bovineii的相似值为100%,可能是该种的一个品系;品系0312-4与X.beddingii的相似值为100%,可能是该种的一个品系。     

Simple and rapid PCR method for identification of streptococcal species relevant to animal infections based on 23S rDNA sequence

A PCR identification system targeting 23S rDNA sequences for the identification of eight streptococcal species relevant to animal infections (Streptococcus agalactiae, S. bovis, S. canis, S. dysgalactiae, S. equi, S. porcinus, S. suis and S. uberis) was developed. This system consists of two PCR reactions, A and B, in which seven and eight primers, respectively, are used simultaneously, and was designed so that each amplification product indicates a species by its size. A total of 111 cultures, including the type strain of eight species, could be successfully identified and differentiated as individual species, except for the cross reactivity between S. bovis and S. equinus. The developed PCR system can complete the identification procedure for eight streptococcal species through two tube reactions per isolate, and, therefore, might provide a rapid, simple and accurate diagnostic tool for veterinary laboratories.     

基于23S／5SrDNA序列的柑橘黄龙病病原菌亚洲种系统发育研究

根据柑橘黄龙病亚洲种23S/5S的DNA序列设计一对引物对不同地理来源的6个柑橘黄龙病样品DNA进行扩增,扩增片段大小均为1 654 bp包括一个假定细胞壁水解酶假基因(putative cell wall hydrolase pseudogene)和5S rRNA 基因.序列同源性分析结果表明;6个柑橘黄龙病病原菌样品与柑橘黄龙病病原菌亚洲种Sihui样品的同源性为99%,然而与土壤杆菌,布鲁氏菌,根瘤菌,中华根瘤菌,巴通体菌和中慢生根瘤菌的同源性只有89%～95%,说明在23S/5S rDNA序列上黄龙病病原菌亚洲种与α变形菌纲根瘤菌目的其他病原菌相差较大.对黄龙病病原菌亚洲种种内的23S/5S rDNA序列进行比较分析,结果发现黄龙病病原菌亚洲种种内之间putative cell wall hydrolase pseudogene和5S rRNA的基因序列非常保守,但不同地理来源的柑橘黄龙病样品碱基序列间确实存在差异,差异的大小与地理的远近无关.利用简约法对黄龙病病原菌亚洲种及α变形菌纲其它病原菌的23S/5S rDNA序列构建的系统发育树显示黄龙病病原菌亚洲种单独聚为一类,其他细菌聚为另一类,该结果与基于rplJ基因及16S rRNA基因的DNA序列构建的分子系统进化树结果一致.     

慢生根瘤菌16S—23S rDNA IGS的RFLP分析

本文对慢生根瘤菌属（Bracyrhizobium）3个已知种及从10种豆科植物中分离的32株慢生根瘤菌进行了16S—23SrDNAIGS的RFLP分析。IGS的PCR产物电泳只出现一条rDNA片段,但表现在长度上菌株间有一定差异,大小在930～1050bp之间,可大致划分为IGSa、IGSb和IGSc3种。用4种四碱基识别序列的限制性内切酶AluI、HaeIII、HinfI和MspI酶解IGSrDNA,综合得到26种IGS－RFLP类型．每一种酶可产生6—12种不同的酶切图谱．结果表明这一方法能很好区分、鉴别慢生根瘤菌,也支持该技术是一种快速、简单、准确及重复性好的微生物鉴定手段．     

Genetic variation in 16S-23S rDNA internal transcribed spacer regions and the possible use of this genetic variation for molecular diagnosis of Bacteroides species

The structural variation in 16S-23S rDNA internal transcribed spacer regions (ITS) among Bacteroides species was assessed by PCR amplification and sequencing analysis, and its possible use for molecular diagnosis of these species was evaluated. Ninety strains of the genus Bacteroides, including the species B. distasonis, B. eggerthii, B. fragilis, B. ovatus, B. thetaiotaomicron, B. uniformis and B. vulgatus, produced one to three ITS amplification products with sizes ranging from 615 to 810 bp. Some Bacteroides strains could be differentiated at species level on the basis of ITS amplification patterns and restriction fragment length polymorphism (RFLP) analysis using a four-nucleotide-recognizing enzyme, Msp I. The results of sequence analysis of ITS amplification products revealed genes for Ile-tRNA and Ala-tRNA in all strains tested. The nucleotide sequence, except for that in tRNA-coding regions, was highly variable and characteristic for each species, but a common sequence among B. fragilis, B. thetaiotaomicron and B. ovatus was observed. A digoxigenin-labeled oligonucleotide probe (named FOT1), which was designed from this conserved sequence, specifically hybridized to the ITS amplification products from B. fragilis, B. thetaiotaomicron and B. ovatus. These results suggest that the ITS region is a useful target for the development of rapid and accurate techniques for identification of Bacteroides species.     

Species identification of clinically important Aeromonas spp. by restriction fragment length polymorphism of 16S rDNA

AIMS: The aim of the study was to characterize 16S rDNA of Aeromonas spp. to rapidly identify clinically important species of these bacteria. METHODS AND RESULTS: Sequence analysis of published 16S rDNA for unique restriction sites revealed prospect of species identification. Extraction of genomic DNA followed by amplification and step-by-step restriction endonuclease digestion of 16S rDNA was able to identify Aeromonas spp. of medical significance. Validation of the method was performed by subjecting 53 Aeromonas strains of multiple origin to similar treatment. Results of the study were in agreement with corresponding species of the isolates. CONCLUSIONS: The method developed offers an easily interpretable tool for the identification of Aeromonas spp. of clinical relevance. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed methodology should facilitate routine laboratory diagnosis of Aeromonas spp. from clinical cases to species level.     

Combining denaturing gradient gel electrophoresis of 16S rDNA V3 region and 16S-23S rDNA spacer region polymorphism analyses for the identification of staphylococci from Italian fermented sausages

Separation of amplified V3 region from 16S rDNA by denaturing gradient gel electrophoresis (PCR-DGGE) and 16S-23S rDNA intergenic spacer region polymorphism (ISR-PCR) analyses were tested as tool for differentiation of staphylococcal strains commonly isolated from fermented sausages. Variable V3 regions of 25 staphylococcal reference strains and 96 wild strains of species belonging to the genera Staphylococcus, Micrococcus and Kocuria were analyzed. PCR-DGGE profiles obtained were species-specific for S. sciuri, S. haemolyticus, S. hominis, S. auricularis, S. condimenti, S. kloosi, S. vitulus, S. succinus, S. pasteuri, S. capitis and S. (Macrococcus) caseolyticus. Moreover, 7 groups could be distinguished gathering the remaining species as result of the separation of the V3 rDNA amplicons in DGGE. Furthermore, the combination of the results obtained by PCR-DGGE and ISR-PCR analyses allowed a clear differentiation of all the staphylococcal species analysed, with exception of the pairs S. equorum-S. cohnii and S. carnosus-S. schleiferi. The suitability of both molecular techniques and of the combination their results for the identification of staphylococci was validated analysing partial nucleotide sequence of the 16S rDNA of a representative number of wild strains.