山羊奇异变形杆菌分离鉴定及其16S-23S rRNA ISR序列RFLP分析

2012年初,山东菏泽某羊场的羊群发病,从发病羊器官分离到2株病原细菌。对病原细菌进行鉴定,并对其与已知同种异源菌株相似性差异进行分析。从患病山羊内脏器官分离细菌,经形态特征、培养特性、生化试验、血清学试验及致病性试验进行鉴定;再通过设计通用引物扩增16S-23SrRNA ISR(intergenic spacer region)序列,将PCR产物经HinfⅠ单酶切获得3条可视条带,同时对扩增条带中的主带测序并进行系统发育分析。结果表明,分离菌株为奇异变形杆菌;分离菌株同本实验室保存的兔源与鸡源奇异变形杆菌PCR-RFLP结果一致;分离菌株PCR产物同GenBank收录的HI4320株奇异变形杆菌及本实验室保存的兔源与鸡源奇异变形杆菌进行序列比较,分离羊源菌株与兔源菌株相似性为94.8%、与鸡源菌株相似性为96.0%～98.2%,与人源HI4320株相似性为96.9%。研究证实发病羊致病病原为奇异变形杆菌,其与鸡源、兔源和人源奇异变形杆菌的亲缘关系较近。     

奇异变形杆菌分离株23S rRNA序列分析

通过PCR反应扩增7株鸡奇异变形杆菌和1株兔奇异变形杆菌的23SrRNA基因片段(1045bp),经克隆测序,用DNA Star分析软件将所获得的序列与GenBank中收录的奇异变形杆菌、普通变形杆菌以及其他相近属的23S rRNA基因序列进行同源性比较,并由此构建奇异变形杆菌系统进化发生树。结果表示,本实验室保存的7株鸡奇异变形杆菌核苷酸序列与GenBank中收录的奇异变形杆菌核苷酸序列同源性为99.4%～99.8%,与兔奇异变形杆菌核苷酸序列同源性为98.8%～99.3%,与普通变形杆菌的核苷酸序列同源性为95.4%～96.2%,而与其他相近属同源性只有92.9%～93.4%。结果表明,23S rRNA基因序列分析可以作为鉴定奇异变形杆菌的一种快速、简便的方法。     

水貂奇异变形杆菌的分离鉴定及16S rRNA基因序列分析

从辽宁某貂场发病水貂中分离到1株致病菌,命名为PMSD株,通过形态学观察和生化试验等常规鉴定发现符合奇异变形杆菌特性,进一步经VITEK 2Compact 30全自动细菌鉴定及药敏分析系统鉴定该株细菌为奇异变形杆菌。药敏试验结果显示PMSD株对氨基糖苷类药物、喹诺酮类药物等敏感,而对β-内酰胺类药物和磺胺类药物等不敏感。以细菌16SrRNA基因为模板应用通用引物进行PCR扩增,得到PMSD株的16SrRNA基因序列,长约1 504bp,提交到GenBank中,登录号:KM229530。将该序列与GenBank中序列进行BLAST比对,结果发现与其匹配度最高的均是奇异变形杆菌各株系的16SrRNA序列,均高达99%以上。选取其中前20株作为参考序列,运用生物学软件构建系统发育树并进行同源性比对,结果表明,分离菌PMSD株与20个代表菌株的同源性为98.9%~99.7%,其中与BB2000株、HI4320株、B1株和FCC141株同源性最高,为99.7%。本研究为预防和控制奇异变形杆菌引起的水貂疾病奠定了一定的基础。     

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.     

鸡奇异变形杆菌的分离鉴定和16S rRNA基因序列测定与系统进化分析

从山东泰安一鸡场发病雏鸡眼中分离到1株致病菌(编号为QY),通过细菌形态学等常规鉴定符合奇异变形杆菌(Proteus mirabilis)特性。用奇异变形杆菌阳性血清诊断结果呈阳性,人工感染证明该菌株是造成该鸡场雏鸡大批发病死亡的致病菌。药敏试验结果显示对头孢类、恩诺沙星等高度敏感,而对青霉素和复合磺胺等不敏感。以细菌16SrRNA基因通用引物进行PCR扩增,得到QY的16SrRNA基因序列,长约1 453bp(GenBank,登录号为GU477712)。将该序列与GenBank中序列进行Blast比对,发现与其匹配度最高的均是奇异变形杆菌各株系的16SrRNA序列,均高达98%以上。运用DNAStar软件与其中10株奇异变形杆菌分离株构建系统进化树,结果表明,分离株(QY菌株)与10个代表菌株的同源性均为98.9%～99.9%,其中与AB272366同源性最高为99.9%。从分子水平证明该菌是奇异变形杆菌并分析了其遗传进化规律,为鸡奇异变形杆菌的鉴定及其引起的疾病的诊断与治疗提供了参考。     

山羊奇异变形杆菌的分离鉴定及16SrDNA和ZapA基因的PCR-RFLP分析

从发病山羊分离到5株病原菌,对分离株进行游散行为分析,生化试验、动物试验、药敏试验及16SrDNA和ZapA基因克隆测序,选用限制性内切酶对分离株16SrDNA和ZapA基因进行PCR-RFLP分析。结果表明分离株出现游散生长现象,对丁胺卡那霉素和复达欣敏感,小鼠LD5。为2．5000×10 ^7CFU／mL-4．4453×10^7CFU／mL;分离株16SrDNA与奇异变形杆菌的同源率为99．5％～99．8％,ZapA基N与奇异变形杆菌的同源率为99．5％;PCR—RFLP分析发现,分离株酶切片段数目和大小与标准株相同。结果表明分离株是奇异变形杆菌,多态性较为单一。     

熟肉制品中奇异变形杆菌的分离鉴定及药物敏感性分析

熟肉制品的特点是可直接食用,而其在食用前又很容易受到致病性微生物污染,所以保证其食用安全性尤其重要。为了解熟肉制品中微生物携带情况,对从沈阳市某熟食店购买的猪头肉和鸡肝中分离到的2株微生物进行了培养鉴定,从形态特征、培养特性、生化试验、16S rRNA基因序列的角度进行分析,并对药物敏感性进行了调查。结果显示,2株分离菌均为短杆状革兰氏阴性菌;均发酵葡萄糖、产生硫化氢,甲基红试验和三糖铁试验阳性;与GenBank中公布的奇异变形杆菌的同源性均在98%以上,综合分析后确定分离的2株菌均为奇异变形杆菌(分别命名为SZ2101菌株和SJ2124菌株);药敏试验结果显示,2株分离菌对β-内酰胺类、喹诺酮类、氨基糖苷类以及大环内酯类的阿奇霉素等药物不同程度敏感;对磺胺类及大环内酯类的红霉素不敏感。结果表明,从熟肉制品中分离的2株菌均为奇异变形杆菌,研究结果为肉品污染防控和食品安全防护提供了重要的参考资料。     

一株致病性奇异变形杆菌的分离与鉴定

从江苏某猪场病猪中分离到一株病原菌,通过纯化、生化鉴定以及16SrDNA序列分析技术鉴定,结果表明,该分离株是奇异变形杆菌。奇异变形杆菌病已成为危害我国养猪业的一种新的细菌性传染病,并且还可引起人类的原发性或继发性感染,造成人类食物中毒,其作为人畜共患传染病病原,应引起高度重视。     

3株鸽源奇异变形杆菌的分离与鉴定

对从江苏省某鸽场病死鸽脏器中分离的细菌进行分离鉴定。通过细菌培养、革兰染色镜检及生化特性测定等对分离株进行初步鉴定;扩增分离株的16S rDNA片段并测序,Blast搜索比对确定细菌的种属;通过动物致病性试验和药敏试验来检测分离株的致病力及耐药性。结果:3株细菌的16S rDNA序列测定和Blast分析表明与奇异变形杆菌参考株的同源性达99.4%～99.8%,确定为奇异变形杆菌;致病性试验显示奇异变形杆菌对鸽子和小鼠均有致病力;药敏试验表明奇异变形杆菌对头孢菌素类抗生素和氟苯尼考敏感,对青霉素类和磺胺类抗生素等有耐药性。结果表明,从鸽子脏器中所分离的细菌为奇异变形杆菌,具有较强的致病力和多重耐药性。     

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.     

Identification of Bartonella strains isolated from wild and domestic ruminants by a single-step PCR analysis of the 16S-23S intergenic spacer region

Of the 20 species or subspecies of Bartonella currently known, 7 cause various diseases in humans with many being zoonotic. However, some Bartonella species appear only to cause asymptomatic bacteraemia in their hosts. In ruminants, three Bartonella species (B. bovis, B. capreoli and B. schoenbuchensis) have recently been described. However, limited or no information has yet been published concerning their mode of transmission and their possible pathogenicity for domestic cattle. The phylogenetic relationship of these species with other bacteria of the Bartonella genus has only been recently investigated. It is therefore necessary to develop appropriate tools that will easily allow identification of these ruminant strains for epidemiological and clinical studies. A single-step PCR assay, based on the amplification of a fragment of the 16S-23S rRNA intergenic spacer (ITS), was evaluated for identification of Bartonella isolated from domestic cattle and from free-ranging or captive cervids. For each Bartonella species tested, the PCR assay led to a product that was unique either for its length or its sequence. All ruminant isolates tested could be easily differentiated among themselves and from the other Bartonella species. Furthermore, sequence analysis of the PCR products revealed a close relationship between all ruminant Bartonella strains. Therefore, ITS PCR testing appears to be a convenient tool for a quick diagnosis of ruminant Bartonella species.     

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.     

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.     

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 Arcanobacterium abortisuis by phenotypic properties and by sequencing the 16S-23S rDNA intergenic spacer region

The present study was designed to characterize phenotypically and genotypically nine Arcanobacterium abortisuis strains collected from specimen of pigs in a period of nine years. All nine A. abortisuis strains and A. abortisuis reference strain DSM 19515 displayed a synergistic hemolytic reaction with Staphylococcus aureus β-hemolysin, Rhodococcus equi, and Arcanobacterium haemolyticum indicator strains and showed the typical biochemical properties of this species. The species identity could be confirmed by identification and sequencing of the 16S-23S rDNA intergenic spacer region (ISR), which appeared to be a useful target for genotypic characterization of this bacterial species. The A. abortisuis strains of the present study were isolated from specimen of pigs together with various other bacterial species indicating that the pathogenic importance of this newly described species remains to be elucidated.     

Identification of porcine intestinal spirochetes by PCR-restriction fragment length polymorphism analysis of ribosomal DNA encoding 23S rRNA

The Brachyspira (formerly Serpulina) species rrl gene encoding 23S ribosomal RNA (rRNA) was used as a target for amplification of a 517bp DNA fragment by polymerase chain reaction (PCR). The primers for PCR amplification had sequences that were conserved among Brachyspira 23S rRNA gene and were designed from nucleotide sequences of Brachyspira hyodysenteriae, Serpulina intermedia, Brachyspira innocens and Brachyspira pilosicoli available from the GenBank database. Digestion of PCR-generated products from reference and field isolates of swine intestinal spirochetes with restriction enzymes Taq I and Alu I revealed five restriction fragment length polymorphism (RFLP) patterns. Each RFLP pattern corresponded to previously established genetic groups including B. hyodysenteriae (I), S. intermedia/B. innocens (II), Brachyspira murdochii (III), B. pilosicoli (IV) and B. alvinipulli (V). The 23S rRNA PCR/RFLP provided a relatively simple genotypic method for identification of porcine pathogenic B. hyodysenteriae and B. pilosicoli.