基于16S rRNA和rpoB基因的分子系统发育分析在铜绿假单胞菌鉴定中的应用
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摘要
为对比16S rRNA和rpo B基因分子系统发育分析与传统表型分类法对铜绿假单胞菌的鉴定,评估16S rRNA和rpo B基因序列分析在铜绿假单胞菌鉴定中的应用,用表型分类方法对临床自动微生物鉴定系统鉴定为铜绿假单胞菌的23株分离株进行再鉴定,PCR扩增23株分离株16S rRNA和rpo B基因片段,并测序进行系统发育分析。结果表明,表型再鉴定结果与自动微生物鉴定系统鉴定结果一致。基于两个基因的系统发育分析均显示分离株p22与不动杆菌属序列聚为一枝,其余22株分离株与铜绿假单胞菌序列聚为一枝。因此p22应鉴定为不动杆菌,16S rRNA和rpo B基因序列分析均能准确鉴定铜绿假单胞菌并能较好建立假单胞菌属内种间关系。
16S rRNA and rpo B gene sequence analysis to identify of Pseudomonas aeruginosa was compared with conventional phenotypic methods to evaluate the application of 16 S rRNA and rpo B sequence analysis to identify P.aeruginosa; 23 isolated strains that were re-characterized with phenotypic classification method that was identified as P.aeruginosa by clinical automatic microbial identification system.16 S rRNA and rpo B gene fragments were amplified with PCR and sequenced for molecular phylogenic analysis.The results showed that the re-characterized results were consistent with those characterized with automatic microbial identification system.Using Bayesian phylogenetic analyses based on 16 S rRNA and rpo B genes,22 isolates are characterized as P.aeruginosa,whereas one isolate,p22,was classified as Acinetobacter sp.p22 should be identified as Acinetobacter sp.16 S rRNA and rpo B genes can accurate identify P.aeruginosa and could fairly establish the interspecific relation within the genus of Pseudomonas.
16S rRNA and rpo B gene sequence analysis to identify of Pseudomonas aeruginosa was compared with conventional phenotypic methods to evaluate the application of 16 S rRNA and rpo B sequence analysis to identify P.aeruginosa; 23 isolated strains that were re-characterized with phenotypic classification method that was identified as P.aeruginosa by clinical automatic microbial identification system.16 S rRNA and rpo B gene fragments were amplified with PCR and sequenced for molecular phylogenic analysis.The results showed that the re-characterized results were consistent with those characterized with automatic microbial identification system.Using Bayesian phylogenetic analyses based on 16 S rRNA and rpo B genes,22 isolates are characterized as P.aeruginosa,whereas one isolate,p22,was classified as Acinetobacter sp.p22 should be identified as Acinetobacter sp.16 S rRNA and rpo B genes can accurate identify P.aeruginosa and could fairly establish the interspecific relation within the genus of Pseudomonas.
引文
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[13]Stackebrandt E,Goebel BM.Taxonomic note:a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology[J].Int J Syst Bacteriol,1994,44(4):846-849.
[14]Marianelli C,Ciuchini F,Tarantino M,et al.Molecular characterization of the rpo B gene in Brucella species:new potential molecular markers for genotyping[J].Microbes Infect,2006,8(3):860-865.
[15]Mollet C,Drancourt M,Raoult D.rpo B sequence analysis as a novel basis for bacterial identification[J].Mol Microbiol,1997,26(5):1005-1011.
[16]Adékambi T,Drancourt M,Raoult D.The rpo B gene as a tool for clinical microbiologists[J].Trends Microbiol,2009,17(1):37-45.
[17]Pei AY,Oberdorf WE,Nossa CW,et al.Diversity of 16S rRNA genes within individual prokaryotic genomes[J].Appl Environ Microbiol,2010,76(12):3886-3897.
[18]Kim BJ,Kim CJ,Chun J,et al.Phylogenetic analysis of the genera Streptomyces and Kitasatospora based on partial RNA polymerase beta-subunit gene(rpo B)sequences[J].Int J Syst Evol Microbiol,2004,54(Pt 2):593-598.
[19]zen AI,Ussery DW.Defining the Pseudomonas genus:where do we draw the line with Azotobacter[J].Microb Ecol,2012,63(2):239-248.
[2]Farmer JJ,Davis BR,Hickman-Brenner FW,et al.Biochemical identification of new species and biogroups of Enterobacteriaceae isolated from clinical specimens[J].J Clin Microbiol,1985,21(1):46-76.
[3]Capurro A,Artursson K,Waller KP,et al.Comparison of a commercialized phenotyping system,antimicrobial susceptibility testing,and tuf gene sequence-based genotyping for specieslevel identification of coagulase-negative staphylococci isolated from cases of bovine mastitis[J].Vet Microbiol,2009,134(3-4):327-333.
[4]Baghal Asghari F,Nikaeen M,Mirhendi H.Rapid monitoring of Pseudomonas aeruginosa in hospital water systems:a key priority in prevention of nosocomial infection[J].FEMS MicrobiolLett,2013,343(1):77-81.
[5]Harris KA,Hartley JC.Development of broad-range 16S r DNA PCR for use in the routine diagnostic clinical microbiology service[J].J Med Microbiol,2003,52(Pt 8):685-691.
[6]Ait Tayeb L,Ageron E,Grimont F,et al.Molecular phylogeny of the genus Pseudomonas based on rpo B sequences and application for the identification of isolates[J].Res Microbiol,2005,156(5-6):763-773.
[7]Mollet C,Drancourt M,Raoult D.rpo B sequence analysis as a novel basis for bacterial identification[J].Mol Microbiol,1997,26(5):1005-1011.
[8]Seok Y,Shin H,Lee Y,et al.First report of bloodstream infection caused by Pseudomonas fulva[J].J Clin Microbiol,2010,48(7):2656-2657.
[9]Lane DJ.16S and 23S rRNA sequencing.In:Stackebrandt E,Goodfellow M(eds)Nucleic acids techniques in bacterial systematics[M].Chichester:John Wiley&Sons,1991:115-175.
[10]Diancourt L,Passet V,Verhoef J,et al.Multilocus sequence typing of Klebsiellapneumoniae nosocomial isolates[J].J ClinMicrobiol,2005,43(8):4178-4182.
[11]吴会桃,蔡芷荷,吴清平,等.细菌鉴定系统的应用研究进展[J].中国卫生检验杂志,2010,20(9):2381-2384.
[12]Harris KA,Hartley JC.Development of broad-range 16S r DNA PCR for use in the routine diagnostic clinical microbiology service[J].J Med Microbiol,2003,52(Pt 8):685-691.
[13]Stackebrandt E,Goebel BM.Taxonomic note:a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology[J].Int J Syst Bacteriol,1994,44(4):846-849.
[14]Marianelli C,Ciuchini F,Tarantino M,et al.Molecular characterization of the rpo B gene in Brucella species:new potential molecular markers for genotyping[J].Microbes Infect,2006,8(3):860-865.
[15]Mollet C,Drancourt M,Raoult D.rpo B sequence analysis as a novel basis for bacterial identification[J].Mol Microbiol,1997,26(5):1005-1011.
[16]Adékambi T,Drancourt M,Raoult D.The rpo B gene as a tool for clinical microbiologists[J].Trends Microbiol,2009,17(1):37-45.
[17]Pei AY,Oberdorf WE,Nossa CW,et al.Diversity of 16S rRNA genes within individual prokaryotic genomes[J].Appl Environ Microbiol,2010,76(12):3886-3897.
[18]Kim BJ,Kim CJ,Chun J,et al.Phylogenetic analysis of the genera Streptomyces and Kitasatospora based on partial RNA polymerase beta-subunit gene(rpo B)sequences[J].Int J Syst Evol Microbiol,2004,54(Pt 2):593-598.
[19]zen AI,Ussery DW.Defining the Pseudomonas genus:where do we draw the line with Azotobacter[J].Microb Ecol,2012,63(2):239-248.