应用16S rRNA基因序列鉴定柞蚕空胴病病原菌

20世纪70年代末,采用形态分类学方法,将引起柞蚕空胴病的致病菌鉴定为柞蚕链球菌(Streptococcus pernyi sp.nov)。分别提取已分离柞蚕空胴病的5株病原菌株的基因组DNA,PCR扩增16S rRNA基因片段,经克隆、测序后,与GenBank中登录的相关肠球菌、链球菌菌株的16S rRNA基因序列进行同源性比对并构建系统进化树。结果表明,供试的5株菌株的16S rRNA基因序列相似性在99.5%～99.9%之间,相互之间存在着10个可变位点,推测5株菌株属于同一个菌种;5株菌株的16S rRNA基因序列与肠球菌属(Enterococcus)16S rRNA基因序列的相似性较高,在92.4%～99.8%之间,而与链球菌属(Streptococcus)16S rRNA基因序列的相似性相对较低,在87.3%～87.8%之间;5株菌株与肠球菌属在系统进化树上聚为一类。基于菌株的16S rRNA基因序列分析,鉴定柞蚕空胴病的病原菌应归属于肠球菌属。     

猪粪便中粪肠球菌的筛选及其在金针菇菌糠发酵中的应用

为了将金针菇菌糠转化成益生素饲料,试验采用MRS琼脂培养基从猪粪便中分离筛选出耐酸、耐胆汁的产酸菌株,然后将菌株的16S rRNA基因进行PCR扩增,进而对菌株的16S rRNA基因序列和分子进化树进行了分析。用分离得到的细菌进行金针菇菌糠发酵处理,30 d后检测活菌数及粗蛋白、氨基酸含量。结果表明:从猪粪便中分离得到1株存活率高的粪肠球菌。用该菌对金针菇菌糠进行发酵处理后,活菌数达45亿cfu/g,粗蛋白达17. 33%,所测18种氨基酸总量达11. 94%,有较高的饲用价值。说明可以采用粪肠球菌固态发酵金针菇菌糠,使其转化为益生素饲料。     

牛源马巴氏杆菌的分离鉴定

为了解引起牛肺炎的主要病原,试验从患有肺炎的牛支气管中分离获得一株巴氏杆菌,并对其进行生化鉴定、16S rRNA测序、特异性PCR鉴定及药敏试验。结果表明:分离菌株与马巴氏杆菌(Pasteurella caballi)的生化特性一致;16S rRNA序列与Pasteurella caballi参考菌株ATCC49197的同源性为100%;特异性PCR鉴定为Pasteurella caballi;分离菌株对诺氟沙星、头孢噻吩、四环素等多种抗生素敏感。说明分离菌株为Pasteurella caballi,该细菌同样可以感染牛。     

副猪嗜血杆菌黑龙江株的分离与鉴定

为确定黑龙江某猪场发病猪群的病原,本研究从疑似浆膜炎的病料中分离到一株革兰氏阴性细小杆菌,通过对其进行细菌形态学、培养特征、生化特性和16S rRNA基因PCR鉴定,结果表明该分离菌株为副猪嗜血杆菌(HLJ-1分离株)。16S rRNA基因序列分析结果显示:该分离菌株与参考株的基因同源性达99%;药敏试验结果表明HLJ-1分离株对头孢曲松、头孢噻肟等β-内酰胺类抗生素高度敏感;小白鼠致病性试验结果显示,该分离株具有较强致病性。     

鸡约氏乳杆菌的分离鉴定及进化分析

从3日龄健康小鸡肠道中分离到1株乳杆菌,用PCR方法从分离菌株扩增16S rRNA基因,获得大小为1340 bp的DNA片段,该片段的核酸序列已提交GenBank,登录号为EU290749。将分离株的16S rRNA基因核苷酸序列与GenBank上其它乳杆菌进行同源性分析并建立进化树。结果表明,分离株的16S rRNA基因核苷酸序列与NCBI公布的鼠约氏乳杆菌分离株(AB295648)的同源性为99.6%,因此该分离菌株被鉴定为鸡约氏乳杆菌(chicken Lactobacillus johnsonii),命名为HN/0711。     

副猪嗜血杆菌的分离鉴定及16S rRNA序列分析

从云南某规模化养猪场病猪肺脏分离到1株革兰氏阴性小杆菌,经细菌生化鉴定、PCR鉴定和16S rRNA序列比对鉴定为副猪嗜血杆菌。抗生素药物敏感试验结果表明,分离菌株对四环素、红霉素、氯霉素、头孢噻吩高敏;对庆大霉素、氧氟沙星、诺氟沙星中敏;对磺胺甲唑耐药。16S rRNA分析结果表明,该分离株与GenBank中的Hps参考株AB078973(基因登录号)同源性为100%,将分离菌株鉴定为副猪嗜血杆菌。16S rRNA遗传进化关系表明,分离株与副猪嗜血杆菌3株血清5型参考株AB078972、AB078973、AB078974的16S rRNA序列位于一个分支上,遗传进化关系最近,它们之间的核苷酸同源性在99.0%~99.4%之间,初步鉴定为血清5型副猪嗜血杆菌,致病性试验结果表明,分离菌株对小白鼠有强致病性,命名为YN-1株。     

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.     

新疆某牛场大肠杆菌的分离鉴定及16S rRNA序列分析

为了调查新疆某规模化奶牛场犊牛腹泻的原因并确定病原,无菌采集15份腹泻犊牛粪样进行病原的分离培养,采用形态学观察、生化试验、血清学和致病性分析等方法鉴定分离菌株,利用大肠杆菌16S rRNA通用引物进行基因序列扩增并测序,使用DNAStar软件将分离菌株测序结果与GenBank中的其他菌株序列进行同源性比对,采用Mega 6.0软件依据16S rRNA序列构建分离株系统进化树并进行分析,同时对分离菌株的黏附性基因进行鉴定。结果从15份样品中分离获得2株分离菌株;试验显示,分离株均发酵葡萄糖,MR试验、吲哚和甲基红试验呈阳性,硫化氢、氧化酶、DNA酶、VP试验呈阴性。2株分离菌株血清型均为O78,且均具有致病性。药敏试验显示,分离菌株均对环丙沙星、卡那霉素、氟哌酸、庆大霉素、磺胺甲唑、头孢哌酮、壮观霉素、多黏菌素B、呋喃妥因敏感。分离株16S rRNA基因序列与大肠杆菌菌株NF738(GenBank登录号:MT649856)同源性为≥99.7%,且处于同一大分支。经PCR鉴定,分离菌株具有K88黏附性基因。本研究结果证实了引起新疆某规模化奶牛场犊牛腹泻的病原为致病性产肠毒素型大肠杆菌。     

高寒地区菊芋青贮物料耐低温乳酸菌的筛选与鉴定

为筛选可提升高寒地区菊芋(Helianthus tuberosus)青贮发酵品质的耐低温乳酸菌,本研究以‘青芋2号’菊芋青贮物料为材料,利用平板培养法分离乳酸菌株,分析菌株生理生化特性,并通过16S rRNA测序进行菌株种属鉴定。结果表明,菊芋青贮物料中共分离得到34株乳酸菌株,经低温及生理生化筛选获得13株耐低温乳酸菌株,包括11株同型发酵乳酸菌和2株异型发酵乳酸菌,其耐酸性和耐盐性均较强;经产酸和生长能力对比,GN02菌株生长最快(OD=2.63),GN10菌株具有更强的产酸能力(pH=3.59),XN25菌株兼具较强的生长和产酸能力;16S rRNA测序鉴定出9株植物乳杆菌(Lactiplantibacillus plantarum)、1株戊糖乳杆菌(Lactiplantibacillus pentosus)、1株棒状乳杆菌(Lactiplantibacillus coryniformis)、1株短乳杆菌(Lactiplantibacillus brevis)及1株无法鉴定。本研究筛选出适合高寒地区菊芋青贮发酵的耐低温植物乳杆菌、戊糖乳杆菌、短乳杆菌各1株。     

猪链球菌的分离、16S rRNA鉴定及药敏试验

从湖南某猪场病猪组织中分离出6株细菌,通过培养形态、菌落特征、细菌染色特性、镜检、16S rRNA基因扩增及序列BLAST分析等系统鉴定,确定为猪链球菌,6株分离菌的16S rRNA基因序列与收录菌株同源性为99%以上。药物敏感性试验结果表明,分离细菌对头孢喹肟、先锋霉素、青霉素药物高度敏感。     

酸奶中乳酸杆菌分离鉴定及其与女性生殖道感染有关的生理生化特性研究

对国内外市售酸奶中添加的乳酸杆菌益生菌进行分离鉴定,对其产H2O2量及与宫颈癌HeLa细胞黏附能力进行分析,并初步判定酸奶中的乳酸杆菌是否对女性生殖道健康具有潜在的保健作用.实验共分离鉴定出10株乳酸杆菌,其中,嗜酸性乳杆菌5株、干酪乳杆菌1株、瑞士乳杆菌1株、鼠李糖乳杆菌1株、植物乳杆菌1株和德式乳杆菌1株.只有瑞士乳杆菌产H2O2量较高且与HeLa细胞的黏附作用较强,其余乳酸杆菌产H2O2的能力偏低.大部分国内外市售酸奶产品中添加的益生菌不具备抑制女性生殖道感染有关的生理生化特性,只有瑞士乳杆菌可能对女性生殖系统的健康具有保护作用.     

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。     

乳酸杆菌属对抗生素的耐药性

以分离自不同地区的发酵乳制品的44株乳杆菌为研究对象,通过对其进行16S rRNA的基因序列分析,鉴定为干酪乳杆菌、植物乳杆菌、发酵乳杆菌、瑞士乳杆菌和保加利亚乳杆菌.同时,研究了这5种乳杆菌的抗生素耐药性.结果显示,乳杆菌对不同的抗生素表现出不同的耐药性.     

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.     

嗜热链球菌对瑞士乳杆菌发酵乳后酸化的影响

瑞士乳杆菌是潜在的功能发酵乳发酵剂,为改善瑞士乳杆菌的发酵特性,使用嗜热链球菌配合瑞士乳杆菌用于发酵乳制备。结果表明：嗜热链球菌与瑞士乳杆菌混合发酵将凝乳时间缩短了3.5 h,黏度提高了1.8 倍,改善了乳清析出和质构;嗜热链球菌的使用有效减弱了发酵乳的后酸化,贮藏20 d发酵乳的pH值仍大于4.2。为研究这一现象的原因,测定发酵乳中的活菌数、乳糖消耗量、谷氨酸脱羧酶和β-半乳糖苷酶活性,结果表明,混合发酵乳贮藏期间具有较低的谷氨酸脱羧酶和β-半乳糖苷酶活性,乳糖利用量较低,这是后酸化减弱的主要原因。与嗜     

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.     

Rapid identification and differentiation of pathogenic clostridia in gas gangrene by polymerase chain reaction based on the 16S-23S rDNA spacer region

In cattle, sheep, and other ruminants, clostridial myonecrosis (gas gangrene) is mostly caused by Clostridium chauvoei, C septicum, C novyi and C sordellii. A polymerase chain reaction (PCR) system using common primers designed from multiple alignment of the 16S rRNA and 23S rRNA genes of Clostridium species was developed to identify pathogenic clostridia. The PCR was performed with total DNA from 26 strains which included seven different Clostridia species. These bacteria were differentiated at species level by the different PCR product patterns. To characterise the 16S-23S rDNA spacer regions of these clostridia further, most PCR products of these bacteria were sequenced. The smallest PCR products of each bacterium represented the fundamental 16S-23S rDNA spacer region; larger PCR products of each bacterium were caused by insertion sequences, i.e. tRNA gene sequences. The authors' observations indicate that the PCR patterns of the 16S-23S rDNA spacer regions have the potential to be used as an identification marker of pathogenic clostridia in gas gangrene.     

Determination of intraspecies variations of the V2 region of the 16S rRNA gene of Streptococcus equi subsp. zooepidemicus

The 16S rRNA gene of 39 S. equi subsp. zooepidemicus strains and two S. equi subsp. equi strains was amplified by polymerase chain reaction and subsequently digested with the restriction enzyme Hinc II. A restriction profile with two fragments with sizes of 1250 bp and 200 bp could be observed for both S. equi subsp. equi strains and for 30 of the 39 S. equi subsp. zooepidemicus strains indicating a sequence variation within the V2 region of the 16S rRNA gene of the remaining nine S. equi subsp. zooepidemicus isolates. A segment of the 16S rRNA gene including the hypervariable V2 region of 11 S. equi subsp. zooepidemicus and two S. equi subsp. equi could be amplified by PCR and sequenced. The sequence of the V2 region of eight S. equi subsp. zooepidemicus strains appeared to be identical or almost identical to the sequence of the two S. equi subsp. equi strains. The sequence of the remaining three S equi subsp. zooepidemicus strains differed significantly from the sequence of S. equi subsp. equi. These differences allowed a division of S. equi subsp. zooepidemicus strains into two 16S rRNA types and might possibly have consequences for the taxonomic position of these phenotypically indistinguishable strains of one subspecies. A molecular typing could additionally be performed by amplification of the gene encoding the 16S-23S rRNA spacer region. A single amplicon of the spacer gene of 1100 bp could be observed for one S. equi subsp. zooepidemicus, an amplicon of 950 bp for two S. equi subsp. equi strains and 10 S. equi subsp. zooepidemicus strains, a amplicon of 780 bp for 27 S. equi subsp. zooepidemicus strains and a single amplicon of 600 bp for one S. equi subsp. zooepidemicus strain. The variations of the V2 region of the 16S rRNA gene and the size variations of the 16S-23S rRNA spacer gene were not related to each other. Both variations could be used for molecular typing of this species, possibly useful in epidemiological aspects.     

利用16S rRNA全长序列鉴定植物乳杆菌

从标明含有嗜酸乳杆菌的活菌胶囊中分离到一株乳杆菌Lal菌株，利用16S rRNA全序列分析法和传统分类法对Lal菌株进行分类鉴定表明：Lal菌株属于植物乳杆菌种新株系，该菌株现在已经被中国科学院北京微生物所菌种保藏中心收录保存，编号为ASl．2986。     

Molecular characterization of Streptococcus suis strains by 16S–23S intergenic spacer polymerase chain reaction and restriction fragment length polymorphism analysis

We developed a new molecular method of typing Streptococcus suis based on polymerase chain reaction (PCR) amplification of a large fragment of rRNA genes, including a part of the 16S and 23S genes and the 16S-23S intergenic spacer region (ISR), followed by restriction fragment length polymorphism (RFLP) analysis with RsaI or MboII endonuclease. The 16S-23S ISRs of 5 S. suis isolates were sequenced and compared. Size and sequence polymorphisms were observed between the S735 reference strain and the 4 wild-type strains. The genetic relationships between 138 independent S. suis strains belonging to various serotypes, isolated from swine or human cases, were determined. The discriminatory power of the method was > 0.95, the threshold value for interpreting typing results with confidence (0.954 with RsaI and 0.984 with RsaI plus MboII). The in vitro reproducibility was 100%. The strains isolated from humans were less genetically diverse than the strains isolated from pigs. For the first time, 2 molecular patterns (R6, M9) were significantly associated with S. suis serotype 2 strains. This genetic tool could be valuable in distinguishing individual isolates of S. suis during epidemiologic investigations.