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PCR-RFLP技术在布氏菌鉴定与分型中的应用 被引量:1

Application of PCR-RFLP technique on identification and genotyping of Brucella spp
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摘要 目的利用PCR-RFLP技术扩增7株布氏菌临床分离株16S RNA基因片段,为该菌的分子诊断学、遗传学和流行病学研究提供实验依据。方法参考GenBank公布的布氏菌ATCC 25840标准株序列,设计针对该菌16S RNA保守区的特异性引物,从临床分离培养7株布氏菌,提取DNA并行16S RNA片段的PCR扩增。将PCR产物克隆至PGEM-T载体进行测序并选用合适的限制性内切酶进行RFLP分析,将分析结果用于临床株的同源性及变异位点的研究,并对感染患者的临床资料进行回顾性分析,研究不同布氏菌基因型与感染患者临床特征的相关性。结果利用PCR扩增出的目的片段与预期一致,大小约为1500bp。测序及同源性分析表明,7株临床分离株之间存在98.88%的同源性和11个变异位点。RFLP结果将7株菌分为4个基因型,临床资料回顾性分析患者不同基因型与临床表现之间无明显联系。结论利用PCR技术扩增布氏菌16SRNA基因片段可以实现对该菌的早期诊断,为布氏菌的分子遗传学和流行病学提供依据。 Objective To amplify the 16S RNA fragments of 7 clinically isolated strains of Brucella spp. by PCR-RFLP technique, so as to provide experimental basis for the studies on diagnostics, genetics and epidemiology of Brucella spp. Methods According to the gene sequence of ATCC 25840 standard strain in GenBank, special primers for the 16S RNA conservative area in the Brucella spp. were designed. DNA extraction and PCR amplification of the 16S RNA fragments were performed with the 7 isolated strains. PCR products were then sequenced and RFLP analysis was conducted with appropriate restricted enzymes to study the homology and the mutation sites in those strains. Meanwhile, the clinical data of infected patients were retrospectively analyzed to evaluate the relationship between the clinical features and genotypes of Brucella infection. Results The amplified target fragments were about 1500bp in length and consistent with what was expected. The sequencing and homology analysis showed a 98.88% homology and 11 mutation sites among the 7 isolated strains. Four genotypes were identified by RFLP. Retrospective analysis of the clinical data indicated that no obvious relationship existed between the genotypes and the clinical features. Conclusions Amplifying 16S RNA fragments by PCR technique is a feasible method to make an early diagnosis of Brucella infection. The 7 clinically isolated strains are different in genotypes and 16S RNA fragment is a highly conservative fragment in bacterial genome with some mutations. The research provides evidence for the genetics and epidemiology of brucellosis.
机构地区 解放军
出处 《解放军医学杂志》 CAS CSCD 北大核心 2010年第3期241-243,共3页 Medical Journal of Chinese People's Liberation Army
基金 军队"十一五"科技攻关课题(06G143)
关键词 布氏菌属 RNA 核糖体 16S 聚合酶链反应 多态性 限制性片段长度 Brucella RNA ribosomal 16S polymerase chain reaction polymorphism estriction fragment length
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  • 1Daffonchio D, Cherif A, Brusetti L, et al. Nature of polymorphisms in 16S-23S rRNA gene intergenic transcribed spacer fingerprinting of Bacillus and related genera[J]. Appl Environ Microbiol, 2003, 69(9): 5128 - 5137.
  • 2Del Vecchio V G, V Kapatral, R J Redkar, et al. The genome sequence of the facultative intracellular pathogen Brucella melitensis [J ]. Proc Natl Acad Sci, 2002, 99: 443 - 448.
  • 3Fox K F, A Fox, M Nagpal, et al. Identification of Brucella by Riboso mal-Spacer-Region PCR and Differentiation of Brucella canis from Other Brucella spp. Pathogenic for Humans by Carbohydrate Profiles [J]. J Clin Microbiol, 1998, 36 (11): 3217 - 3222.
  • 4Nagpal M L, K F Fox, A Fox. Utility of 16S - 23S rRNA spacer reion methodology:how similar are interspace regions within a genome and between strains for closely related organisms? [J ]. J Microbiol Methods, 1998, 33: 211 - 219.
  • 5Rijpens N P, G Jannes, M Van Asbroeck, et al. Direct detection of rucella spp. in raw milk by reverse hybridization with 16S- 23S rRNA spacer probes [ J ]. Appl Environ Microbiol, 1996, 62: 1683 - 1688.
  • 6Scheinert P, R Krausse, U Ullman, et al. Molecular differentiation of bacteria by PCR amplification of the 16S - 23S rRNA spacer region [J] .J Microbiol Methods, 1996, 26:103 - 117.
  • 7Rossetti O L, A I Arese, M L Boschiroli, et al. Cloning of Brucella abortus gene and characterization of expressed 26 - kilodalton periplasmic protein: potential use for diagnosis [ J ]. J Clin Microbiol, 1996, 34:165 - 169.
  • 8Vizcaino N, A Cloeckaert, M S Zygmunt, et al. Molecular characterization of a Brucella species large DNA fragment deleted in Brucella abortus strains:evidence for a locus involved in the synthesis of a polysaccharide[J ]. Infect Immun, 1999, 67: 2700 - 2712.
  • 9殷继明.布鲁氏菌属DNA的多态性[J].中国地方病防治,2002,17(6):341-346. 被引量:10

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  • 1Norrnile D. Emerging infectious diseases. Scientists puzzle over Ebola Reston virus in pigs[J]. Science, 2009, 323(5913):451.
  • 2Ksinzek TO, Erdman D, Goldsmith CS, et al. A novel coronavirus associated with severe acute respiratory syndrome[J]. N Engl J Med, 2003, 348 (20) : 1953-1988.
  • 3Drosten C, Gunther S, Preiser W, et al. Identification of a novel coronavirus in patients with severe acute respiratory syndrome[J]. N Engl J Med, 2003, 348(20):1967-1976.
  • 4Tran TH, Nguyen TL, Nguyen TD, et al. Avian influenza A (H5N1) in 10 patients in Vietnam[J]. N Engl J Med, 2004, 350(12):1179-1188.
  • 5Tang J, Wang C, Feng Y, et at. Streptococcal toxic shock syndrome caused by Streptococcus suis serotype 2[J]. PLoS Med, 2006, 3(5) :e151.
  • 6Cohen J. Swine flue outbreak. Flu researchers train sights on novel tricks of novel H1N1[J]. Science, 2009, 324(5929):870-871.
  • 7Canton R. Role of the microbiology laboratory in infectious disease surveillance, alert and response[J]. Clin Microbiol Infect, 2005, 11(1):3-8.
  • 8Satcher D. Emerging infections: getting ahead of the curve[J]. Emerg Infect Dis, 1995, 1(1):1-6.
  • 9Tang YW, Kilic A, Yang Q, et al. StaphPlex syatem for rapid and simultaneous identification of antibiotic resistance determinants and Panton-Valentine blood cultures[J]. J Clin Microbiol, 2007,45(7) :2105-2109.
  • 10Ilina EN, Borovskaya AD, Malakhova MM, et al. Direct bacterial profiling by MALDI-TOF mass spectrometry for identification of pathogenic Neisseria [J]. J Mol Diag, 2009, 11(1): 75-86.

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