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根据布鲁菌属特异性基因BCSP31和布鲁菌种间特异性标志IS711插入序列,设计合成了3对引物,以牛种布鲁菌544A、104M和羊种布鲁菌16M基因组DNA为模板,通过优化反应条件,建立了可同时检测布鲁菌属、牛种布鲁菌和羊种布鲁菌的多重PCR方法。牛种布鲁菌可扩增出301和114 bp 2条带,羊种布鲁菌可扩增出301和253 bp 2条带,该方法对牛种布鲁菌544A和羊种布鲁菌16M混合DNA模板的最小检出量为100 pg,对大肠杆菌O157∶H7、小肠结肠炎耶尔森菌等15种参照菌的核酸扩增结果均为阴性。应用该方法对吉林省某牛场的106份粪便进行检测,虎红平板凝集试验作对照,结果PCR检测9份为阳性,且全为牛种布鲁菌阳性,对应的虎红平板凝集试验也为阳性。结果表明,建立的多重PCR方法具有良好的敏感性和特异性,为布鲁菌病的鉴别诊断提供了一种分子检测工具。 相似文献
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《中国预防兽医学报》2016,(1)
正布鲁菌(Brucella)为革兰氏阴性胞内寄生菌,根据宿主偏爱性分为10个种~[1],除经典的羊种、牛种、猪种、犬种、绵羊附睾种与沙林鼠种外,还包括新发现的从海洋哺乳动物分离到的鳍种和鲸种布鲁菌,以及从红狐狸和土壤中分离到的田鼠型布鲁菌和从乳房移植物中分离到的湖浪布鲁菌~[2]。布鲁菌可以感染包括人在内的多种哺乳动物引起布鲁菌病,对畜牧业生产、人类健康和公共卫生安全危害严重。在感染细胞或机体内增殖能力和持续生存时间是 相似文献
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布鲁菌种属鉴定多重PCR方法的建立及初步应用 总被引：1，自引：0，他引：1
用BCSP31作为布鲁菌属特异性基因,以IS711基因拷贝数差异作为布鲁菌种间特异性标志,建立了布鲁菌种属特异性的多重PCR鉴定方法.用建立的多重PCR方法对11株(牛种A19,A544,A387;羊种M111,M28,M16;犬种RM6/66;绵羊种63/290;猪种S2,rS2,S1330)不同种来源的布鲁菌菌体和基因组进行鉴定,结果牛种菌能扩增大小分别为494,223,178 bp 3条带,羊种菌能扩增出大小分别为733,223,178 bp 3条带,犬种菌能扩增出大小分别为223,178 bp 2条带,绵羊种菌能扩增出大小分别为976,223,178 bp 3条带,猪种菌能扩增出大小分别为285,223,178 bp 3条带.均与预期一致;而作为对照的大肠杆菌、胸膜肺炎放线杆菌、多杀性巴氏杆菌、流产沙门菌和都柏林沙门菌,均未扩增出任何务带.结果表明,本研究所建立的方法具有良好的特异性,能够区分不同种源的布鲁菌,可用于布鲁菌种属的快速鉴定和流行病学调查. 相似文献
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鉴别牛羊布鲁菌实时荧光定量PCR方法的建立 总被引：1，自引：0，他引：1
[目的]建立准确、快速检测布鲁菌、羊种布鲁菌、牛种布鲁菌的方法。[方法]以BCSP31作为布鲁菌菌属特异性基因,以IS711插入元件作为布鲁菌菌种特异性标志,设计引物和Taqman探针;分别构建标准阳性质粒模板,将其10倍倍比稀释作多重实时荧光定量PCR标准曲线,评价多重实时荧光定量PCR反应体系的敏感性、特异性、重复性。[结果]建立了鉴别牛种、羊种布鲁菌的实时荧光定量PCR方法,并用该方法对临床样品进行检测。[结论]所建立的多重实时荧光定量PCR诊断方法能够快速、准确地鉴别牛种和羊种布鲁菌。 相似文献
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株非典型羊源布鲁菌的基因分型研究 《畜牧与饲料科学》2019,40(12):108-112
[目的]探讨采用基因分型方法对非典型布鲁菌鉴定的实用性,为非典型菌株的鉴别提供参考。[方法]采用常规鉴定方法和VITEK 2.0全自动细菌鉴定分析系统对菌株进行初步鉴定,利用AMOS-PCR进行种型鉴定,应用多位点可变数目串联重复序列分析方法(MLVA)确定菌株的基因型。[结果]常规鉴定结果显示试验菌株为疑似布鲁菌;VITEK 2.0全自动细菌鉴定系统显示3株试验菌株均为布鲁菌;AMOS-PCR扩增表明试验菌株均为羊种菌,MLVA聚类分析表明试验菌株与羊种2型布鲁菌紧密地聚为一类,属东地中海基因型,并在Panel 2B中发现了新的基因型(4-4-3-7-5),命名为CN2B-45。[结论]MLVA基因分型方法对非典型布鲁菌具有极高的分辨力,是非典型布鲁菌分型鉴别的最佳策略。 相似文献
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布鲁菌病是一种典型的人兽共患细菌病,胞内寄生是其感染宿主、并难以治疗的主要原因。目前有关布鲁菌胞内寄生的机制尚未清晰。在本试验中,通过克隆布鲁菌DnaK基因到表达载体pQE-80L,分离纯化His-DnaK融合蛋白,探讨DnaK蛋白调节宿主细胞凋亡的功能。试验表明,第一,His-DnaK处理的巨噬细胞Caspase3剪切被抑制;第二,His-DnaK处理的巨噬细胞TUNEL染色显著降低;第三,布鲁菌DnaK蛋白能抑制宿主细胞凋亡。这将为进一步阐明布鲁菌胞内寄生的分子机制奠定基础。 相似文献
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分子生物学技术在布鲁菌种型鉴定上的应用 总被引：1，自引：0，他引：1
由于布鲁菌种型较多、宿主广泛、传播途径多样,因此有必要对布鲁菌进行准确的种型鉴别,以利于该病的快速诊断、流行病学分析,并采取科学有效的防控措施。分子生物学技术的不断发展为布鲁菌种型鉴定提供了愈来愈丰富的手段,并逐渐成为布鲁菌遗传溯源研究的重要工具。MLVA和real-time PCR技术因具有重复性好、分辨率高等优势,成为布鲁菌种型鉴定研究关注的热点。多重PCR、RFLP、RAPD和REP等技术也在布鲁菌种型鉴定中得到广泛应用。为了解分子生物学技术在布鲁菌种型鉴定上的应用现状,对相关研究进行了综述。 相似文献
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布氏杆菌为世界性具重要公共卫生意义的人兽共患疫病原，分6个种。建立种间及种株间安全敏感、经济有效的快速鉴别诊断方法对布病防制及分子流行病学研究具有重要意义。布氏杆菌IS711和omp2基因具有种属特异性，可用于布氏杆菌的PCR分子诊断。其中IS711为转座因子，在不同种布菌种存在插入位置的多态性，外膜蛋白OMP2编码基因则存在反向重复序列及种株间的多态性。为此，分别采用复式-PCR、PCR和限制性酶切片段长多态性（RFLP）分析，对分属于B．mclitcnsis、B．suis和B．abortus的不同种布氏杆菌的不同种株，M5、M16、S2、S6和S19进行分子鉴别诊断。结果显示，根据IS711基因特定PCR扩增片段长多态性，可进行布氏杆菌种间的快速鉴别；而omp2编码基因PCR扩增片段PsrⅠ、KpnⅠ、NcoⅠ和Eco47 Ⅲ等4种限制酶片段长多态性，则可成为布氏杆菌菌株间特异的分子鉴别诊断标记，甚至疫苗株M5和野毒株M16之间的分子诊断标记。 相似文献
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A E Jensen N F Cheville C O Thoen A P MacMillan W G Miller 《Journal of veterinary diagnostic investigation》1999,11(2):152-157
Genomic DNA from reference strains and biovars of the genus Brucella was analyzed using pulsed-field gel electrophoresis (PFGE). Fingerprints were compared to estimate genetic relatedness among the strains and to obtain information on evolutionary relationships. Electrophoresis of DNA digested with the restriction endonuclease XbaI produced fragment profiles for the reference type strains that distinguished these strains to the level of species. Included in this study were strains isolated from marine mammals. The PFGE profiles from these strains were compared with those obtained from the reference strains and biovars. Isolates from dolphins had similar profiles that were distinct from profiles of Brucella isolates from seals and porpoises. Distance matrix analyses were used to produce a dendrogram. Biovars of B. abortus were clustered together in the dendrogram; similar clusters were shown for biovars of B. melitensis and for biovars of B. suis. Brucella ovis, B. canis, and B. neotomae differed from each other and from B. abortus, B. melitensis, and B. suis. The relationship between B. abortus strain RB51 and other Brucella biovars was compared because this strain has replaced B. abortus strain 19 for use as a live vaccine in cattle and possibly in bison and elk. These results support the current taxonomy of Brucella species and the designation of an additional genomic group(s) of Brucella. The PFGE analysis in conjunction with distance matrix analysis was a useful tool for calculating genetic relatedness among the Brucella species. 相似文献
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From the discovery of the Malta fever's agent to the discovery of a marine mammal reservoir, brucellosis has continuously been a re-emerging zoonosis 总被引：14，自引：0，他引：14
Godfroid J Cloeckaert A Liautard JP Kohler S Fretin D Walravens K Garin-Bastuji B Letesson JJ 《Veterinary research》2005,36(3):313-326
Brucellosis is not a sustainable disease in humans. The source of human infection always resides in domestic or wild animal reservoirs. The routes of infection are multiple: food-borne, occupational or recreational, linked to travel and even to bioterrorism. New Brucella strains or species may emerge and existing Brucella species adapt to changing social, cultural, travel and agricultural environment. Brucella melitensis is the most important zoonotic agent, followed by Brucella abortus and Brucella suis. This correlates with the fact that worldwide, the control of bovine brucellosis (due to B. abortus) has been achieved to a greater extent than the control of sheep and goat brucellosis (due to B. melitensis), these latter species being the most important domestic animals in many developing countries. The long duration and high cost of treatment of human brucellosis reduces the efficacy of the therapy. There is no human vaccine for brucellosis and the occurrence of brucellosis is directly linked to the status of animal brucellosis in a region. In this context, the Word Health Organization has defined the development of a human vaccine, besides the implementation of control and eradication programs in animals, as a high priority. The pathogenicity for humans of B. suis biovars 1, 3 and 4 is well established, whereas B. suis biovar 2 seems to be less pathogenic. Indeed, although hunters and pig farmers have repeatably experienced infectious contact with B. suis biovar 2 (found in wild boar and outdoor-rearing pigs in Europe), isolation of B. suis biovar 2 from human samples have only been seldom reported. Marine mammal brucellosis, due to two new proposed Brucella species i.e. B. cetaceae and B. pinnipediae, represents a new zoonotic threat but the pathogenicity for humans of the different Brucella species found in cetaceans and pinnipeds still has to be clearly established. 相似文献
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De Hertogh B Lahlimi L Lambert C Letesson JJ Depiereux E 《Veterinary microbiology》2008,127(3-4):369-378
The genome sequences of three Brucella biovars and of some species close to Brucella sp. have become available, leading to new relationship analysis. Moreover, the automatic genome annotation of the pathogenic bacteria Brucella melitensis has been manually corrected by a consortium of experts, leading to 899 modifications of start sites predictions among the 3198 open reading frames (ORFs) examined. This new annotation, coupled with the results of automatic annotation tools of the complete genome sequences of the B. melitensis genome (including BLASTs to 9 genomes close to Brucella), provides numerous data sets related to predicted functions, biochemical properties and phylogenic comparisons. To made these results available, alphaPAGe, a functional auto-updatable database of the corrected sequence genome of B. melitensis, has been built, using the entity-relationship (ER) approach and a multi-purpose database structure. A friendly graphical user interface has been designed, and users can carry out different kinds of information by three levels of queries: (1) the basic search use the classical keywords or sequence identifiers; (2) the original advanced search engine allows to combine (by using logical operators) numerous criteria: (a) keywords (textual comparison) related to the pCDS's function, family domains and cellular localization; (b) physico-chemical characteristics (numerical comparison) such as isoelectric point or molecular weight and structural criteria such as the nucleic length or the number of transmembrane helix (TMH); (c) similarity scores with Escherichia coli and 10 species phylogenetically close to B. melitensis; (3) complex queries can be performed by using a SQL field, which allows all queries respecting the database's structure. The database is publicly available through a Web server at the following url: http://www.fundp.ac.be/urbm/bioinfo/aPAGe. 相似文献
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羊种布鲁氏菌分子生物学研究进展 总被引：1，自引：0，他引：1
综述了近年来羊种布鲁氏菌的研究概况,着重介绍了羊种布鲁氏菌基因组、致病因子、致病机理以及分子生物学检测方法等方面的研究进展,并对未来的研究发展趋势提出见解,为最终使该菌所引发的相关疾病得到有效控制提供参考. 相似文献
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A O?ate H Folch 《Zentralblatt für Veterin?rmedizin. Reihe B. Journal of veterinary medicine. Series B》1989,36(5):397-400
The proteins constituents of Brucella abortus, Brucella melitensis and Brucella ovis were analyzed SDS-PAGE. From the comparison appears that the three species of Brucella studied shows a different electrophoretic pattern specially at the level of small peptides. On the contrary when two strains of B. abortus are analyzed no differences can be noticed. 相似文献
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R Smith J C Kapatsa S J Sherwood T A Ficht J W Templeton L G Adams 《American journal of veterinary research》1990,51(4):518-523
The reactivity of bovine lymphocytes to 4 species of Brucella was tested in thymidine-uptake assays, using long-term cultured lymphocytes and freshly obtained blood mononuclear cells. Lymphocytes were taken from cows that had been challenge exposed with a virulent strain of B abortus at midgestation. The cows were classified retrospectively as being naturally resistant or susceptible to brucellosis. Lymphocytes taken from these cows had 3 patterns of reactivity with species of Brucella: pattern 1 was defined by reactivity with 4 species (B abortus, B canis, B suis, and B melitensis); pattern 2 was defined by reactivity with all these species, except B melitensis; pattern 3 was defined by reactivity with B abortus and B canis, but not with B suis or B melitensis. There was a statistically significant correlation between susceptibility to brucellosis and expression of lymphocyte cross-reactivity with B suis (P less than 0.01) and with B melitensis (P less than 0.001). 相似文献
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An evaluation of the polymerase chain reaction (PCR) for detection of Brucella melitensis DNA in bovine and ovine semen was performed. Since semen contains different components that inhibit PCR amplification, a protocol was used to purify Brucella-DNA from bovine and ovine semen samples prior to conducting amplification of the targeted DNA. When separated fractions of naturally Brucella contaminated semen were analyzed by the PCR, most of B. melitensis DNA were present in the seminal fluid and non-sperm fractions.The PCR examination results for detection of B. melitensis DNA in different semen fractions were compared with the results for traditional cultural methods of Brucella from semen. The PCR was more sensitive than the traditional cultural methods since it detected Brucella-DNA in 12 (10%) out of 120 semen samples while direct culture detected only 7 (5.8%) in the same semen samples. The limit of detection by PCR was 100 CFU/ml of semen. In addition, the results of PCR were available in one day, whereas isolation and identification of Brucella organisms required days or even weeks. The PCR may be used as a supplementary test for detection of B. melitensis in semen. 相似文献
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The galE gene of Streptomyces lividans was used to probe a cosmid library harbouring Brucella melitensis 16M DNA and the nucleotide sequence of a 2.5 kb ClaI fragment which hybridised was determined. An open reading frame encoding a predicted polypeptide with significant homology to UDP-galactose-4-epimerases of Brucella arbortus strain 2308 and other bacterial species was identified. DNA sequences flanking the B. melitensis galE gene shared no identity with other gal genes and, as for B. abortus, were located adjacent to a mazG homologue. A plasmid which encoded the B. melitensis galE open reading frame complemented a galE mutation in Salmonella typhimurium LB5010, as shown by the restoration of smooth lipopolysaccharide (LPS) biosynthesis, sensitivity to phage P22 infection and restoration of UDP-galactose-4-epimerase activity. The galE gene on the B. melitensis 16M chromosome was disrupted by insertional inactivation and these mutants lacked UDP-galactose-4-epimerase activity but no discernible differences in LPS structure between parent and the mutants were observed. One B. melitensis 16M galE mutant, Bm92, was assessed for virulence in CD-1 and BALB/c mice and displayed similar kinetics of invasion and persistence in tissues compared with the parent bacterial strain. CD-1 mice immunised with B. melitensis 16M galE were protected against B. melitensis 16M challenge. 相似文献