人工喷泉中一株军团菌的系统遗传学鉴定
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摘要
目的检测喷泉水中的1株军团菌并进行分子生物学鉴定和系统发育分析。方法采集潍坊市一处人工喷泉水水样,用细菌基因组试剂盒提取水样中的总细菌基因组DNA,使用针对军团菌属16S rRNA基因片段的特异性引物PCR扩增军团菌属16S rRNA基因片段并测序,与从GenBank获得的另外41株军团菌的16S rRNA的基因序列进行对比分析,采用PAUP*4.0b10软件对16S rRNA基因片段序列数据矩阵进行最大简约法分析并绘制基因进化树。结果 PCR扩增喷泉水提取物中军团菌属特异性16S rRNA基因片段,片段碱基数654 bp。测序分析该菌株(命名为WF-B)的16S rRNA基因序列与Legionella beliardensis 16S rRNA基因序列的遗传距离较近,相似度达99.8%,在构建的最大简约进化树中该菌与L.beliardensis以自检值97%聚类为一枝。结论该喷泉水中检出的军团菌WF-B与L.beliardensis的亲缘关系较近,相关部门应加强监管力度,降低其感染风险。
Objective To detect the molecular biological identification and phylogenetic analysis of an isolated strain of the water from the fountain in Weifang. Methods Water sample is collected from an artificial fountain in Weifang and extracted total bacterial genomic DNA using bacterial genome kit from the sample. Legionella 16S rRNA gene were obtained with Legionella 16S rRNA gene specific primers through PCR, and then sequenced which compared with other 41 strains of Legionella 16S rRNA gene sequences obtained from the GenBank. The 16S rRNA gene fragment sequences data matix was analysis using PAUP* 4.0b10 software to get the Maximum parsimony phylogenetic trees. Results We found the positive result with Legionella 16S rRNA gene specific primers through PCR. The genetic distance between sequence of the positive strain(named WF-B) and L. beliardensis is closer, the similarity is 99.8%, while in the Maximum parsimony phylogenic tree, WF-B was a sister to L. beliardensis with high bootstrap probability(BP=97%). Conclusion The WF-B detected in the fountain water of Weifang is closely related to L. beliardensis. Relevant departments should strengthen supervision and reduce the risk of infection.
Objective To detect the molecular biological identification and phylogenetic analysis of an isolated strain of the water from the fountain in Weifang. Methods Water sample is collected from an artificial fountain in Weifang and extracted total bacterial genomic DNA using bacterial genome kit from the sample. Legionella 16S rRNA gene were obtained with Legionella 16S rRNA gene specific primers through PCR, and then sequenced which compared with other 41 strains of Legionella 16S rRNA gene sequences obtained from the GenBank. The 16S rRNA gene fragment sequences data matix was analysis using PAUP* 4.0b10 software to get the Maximum parsimony phylogenetic trees. Results We found the positive result with Legionella 16S rRNA gene specific primers through PCR. The genetic distance between sequence of the positive strain(named WF-B) and L. beliardensis is closer, the similarity is 99.8%, while in the Maximum parsimony phylogenic tree, WF-B was a sister to L. beliardensis with high bootstrap probability(BP=97%). Conclusion The WF-B detected in the fountain water of Weifang is closely related to L. beliardensis. Relevant departments should strengthen supervision and reduce the risk of infection.
引文
[1] Brenner DJ,Steigerwalt AG,McDADE JE.Classification of the Legionnaires' disease bacterium:Legionella pneumophila,genus novum,species nova,of the family Legionellaceae,familia nova[J].Ann Inter Med,1979,90(4):656-8.
[2] Muder RR,Victor LY.Infection due to Legionella species other than L.pneumophila[J].Clin Infect Dis,2002,35(8):990-8.
[3] Verykiou S,Goodhead C,Parry G,et al.Legionella feeleii:an unusual organism associated with cutaneous infection in an immunocompromised patient[J].Clin Exp Dermatol,2018,43(3):300-2.
[4] Presti FL,Riffard S,Meugnier H,et al.Legionella gresilensis sp.nov.and Legionella beliardensis sp.nov.,isolated from water in France[J].Int J Syst Evol Micrbio,2001,51(6):1949-57.
[5] Wallis L,Robinson P.Soil as a source of Legionella pneumophila serogroup 1 (Lp1)[J].Au st NZL J Public Health,2005,29(6):518-20.
[6] Nieminen T,Pakarinen J,Tsitko I,et al.16S rRNA targeted sandwich hybridization method for direct quantification of mycobacteria in soils[J].J Microbiol Methods,2006,67(1):44-55.
[7] Miyamoto H,Yamamoto H,Arima K,et al.Development of a new seminested PCR method for detection of Legionella species and its application to surveillance of Legionellae in hospital cooling tower water[J].Appl Environ Microbiol,1997,63(7):2489-94.
[8] Thompson JD,Gibson TJ,Plewniak F,et al.The CLUSTAL_X windows interface:flexible strategies for multiple sequence alignment aided by quality analysis tools[J].Nucleic Acids Res,1997,25(24):4876-82.
[9] Swafford D.PAUP*.Phylogenetic analysis using parsimony (* and other methods)[J].Version.2002(4):b10.
[10] Kumar S,Stecher G,Tamura K.MEGA7:molecular evolutionary genetics analysis version 7.0 for bigger datasets[J].Mol Biol Evol,2016,33(7):1870-4.
[11] Felsenstein J.Confidence limits on phylogenies:an approach using the bootstrap[J].Evolution,1985,39(4):783-91.
[12] 吕恒,张锦海,陈凤娟,等.军团菌检测方法概述[J].东南国防医药,2014,16(2):177-80.
[13] 赵利伟,胡朝晖,陈文聪,等.PCR-酶切技术快速鉴定军团菌[J].微生物学通报,2013,40(8):1514-20.
[14] 刘航.EMA-qPCR检测公共场所气溶胶中存活的嗜肺军团菌[D].北京:中国疾病预防控制中心,2014.
[15] 杨斌斌,李恒,管艳,等.潍坊地区人工喷泉中一株军团菌的分子生物学鉴定[J].中国病原生物学杂志,2017,12(5):412-5.
[16] 于纪棉,李如松,倪健波,等.嗜肺军团菌微滴式数字PCR检测方法的建立[J].中国卫生检验杂志,2017,27(9):1233-9
[17] Presti FL,Riffard S,Vandenesch F,et al.Identification of Legionella species by random amplified polymorphic DNA profiles[J].J clin Microbiol,1998,36(11):3193-7.
[18] Yang G,Benson R,Pelish T,et al.Dual detection of Legionella pneumophila and Legionella species by real-time PCR targeting the 23S-5S rRNA gene spacer region[J].Clin Microbiol Infect,2010,16(3):255-61.
[19] Zhan XY,Hu CH,Zhu QY.Targeting single-nucleotide polymorphisms in the 16S rRNA gene to detect and differentiate Legionella pneumophila and non-Legionella pneumophila species[J].Arch Microbiol,2016,198(6):591-4.
[20] Janczarek M,Palusinska-Szysz M.PCR method for the rapid detection and discrimination of Legionella spp.based on the amplification of pcs,pmtA,and 16S rRNA genes[J].J Appl Genetics,2016,57(2):251-61.
[21] Avni T,Bieber A,Green H,et al.Diagnostic acuracy of PCR alone and compared to urinary antigen testing for detection of Legionella spp.:a systematic review[J].J Clin Microbiol,2016,54(2):401-11.
[22] Ditommaso S,Ricciardi E,Giacomuzzi M,et al.Legionella in water samples:How can you interpret the results obtained by quantitative PCR?[J].Mol Cell Probes,2015,29(1):7-12.
[23] Campocasso A,Boughalmi M,Fournous G,et al.Legionella tunisiensis sp.nov.and Legionella massiliensis sp.nov.,isolated from environmental water samples[J].Int J Syst Evol Micrbio,2012,62(12):3003-6.
[24] Palmer A,Painter J,Hassler H,et al.Legionella clemsonensis sp.nov.:a green fluorescing Legionella strain from a patient with pneumonia[J].Microbiol Immunol,2016,60(10):694-701.
[25] Ahmadrajabi R,Shakibaie MR,Iranmanesh Z,et al.Prevalence of mip virulence gene and PCR-base sequence typing of Legionella pneumophila from cooling water systems of two cities in Iran[J].Virulence,2016,7(5):602-9.
[26] Collins S,Jorgensen F,Willis C,et al.Real-time PCR to supplement gold-standard culture-based detection of Legionella in environmental samples[J].J Appl Microbiol,2015,119(4):1158-69.
[27] 刘文强,贾玉萍,赵宏坤.16S rRNA在细菌分类鉴定研究中的应用[J].动物医学进展,2006,27(11):15-8.
[28] 王卓,吴琼,杨慎江,等.长白山地区蜱类和鼠类中查菲埃立克体感染调查及16S rRNA序列分析[J].中国病原生物学杂志,2017,12(10):971-4,978.
[29] 滑留帅,王璟,徐照学,等.16S rRNA基因高通量测序分析牛粪发酵细菌多样性[J].农业工程学报,2016,32(1):311-5.
[2] Muder RR,Victor LY.Infection due to Legionella species other than L.pneumophila[J].Clin Infect Dis,2002,35(8):990-8.
[3] Verykiou S,Goodhead C,Parry G,et al.Legionella feeleii:an unusual organism associated with cutaneous infection in an immunocompromised patient[J].Clin Exp Dermatol,2018,43(3):300-2.
[4] Presti FL,Riffard S,Meugnier H,et al.Legionella gresilensis sp.nov.and Legionella beliardensis sp.nov.,isolated from water in France[J].Int J Syst Evol Micrbio,2001,51(6):1949-57.
[5] Wallis L,Robinson P.Soil as a source of Legionella pneumophila serogroup 1 (Lp1)[J].Au st NZL J Public Health,2005,29(6):518-20.
[6] Nieminen T,Pakarinen J,Tsitko I,et al.16S rRNA targeted sandwich hybridization method for direct quantification of mycobacteria in soils[J].J Microbiol Methods,2006,67(1):44-55.
[7] Miyamoto H,Yamamoto H,Arima K,et al.Development of a new seminested PCR method for detection of Legionella species and its application to surveillance of Legionellae in hospital cooling tower water[J].Appl Environ Microbiol,1997,63(7):2489-94.
[8] Thompson JD,Gibson TJ,Plewniak F,et al.The CLUSTAL_X windows interface:flexible strategies for multiple sequence alignment aided by quality analysis tools[J].Nucleic Acids Res,1997,25(24):4876-82.
[9] Swafford D.PAUP*.Phylogenetic analysis using parsimony (* and other methods)[J].Version.2002(4):b10.
[10] Kumar S,Stecher G,Tamura K.MEGA7:molecular evolutionary genetics analysis version 7.0 for bigger datasets[J].Mol Biol Evol,2016,33(7):1870-4.
[11] Felsenstein J.Confidence limits on phylogenies:an approach using the bootstrap[J].Evolution,1985,39(4):783-91.
[12] 吕恒,张锦海,陈凤娟,等.军团菌检测方法概述[J].东南国防医药,2014,16(2):177-80.
[13] 赵利伟,胡朝晖,陈文聪,等.PCR-酶切技术快速鉴定军团菌[J].微生物学通报,2013,40(8):1514-20.
[14] 刘航.EMA-qPCR检测公共场所气溶胶中存活的嗜肺军团菌[D].北京:中国疾病预防控制中心,2014.
[15] 杨斌斌,李恒,管艳,等.潍坊地区人工喷泉中一株军团菌的分子生物学鉴定[J].中国病原生物学杂志,2017,12(5):412-5.
[16] 于纪棉,李如松,倪健波,等.嗜肺军团菌微滴式数字PCR检测方法的建立[J].中国卫生检验杂志,2017,27(9):1233-9
[17] Presti FL,Riffard S,Vandenesch F,et al.Identification of Legionella species by random amplified polymorphic DNA profiles[J].J clin Microbiol,1998,36(11):3193-7.
[18] Yang G,Benson R,Pelish T,et al.Dual detection of Legionella pneumophila and Legionella species by real-time PCR targeting the 23S-5S rRNA gene spacer region[J].Clin Microbiol Infect,2010,16(3):255-61.
[19] Zhan XY,Hu CH,Zhu QY.Targeting single-nucleotide polymorphisms in the 16S rRNA gene to detect and differentiate Legionella pneumophila and non-Legionella pneumophila species[J].Arch Microbiol,2016,198(6):591-4.
[20] Janczarek M,Palusinska-Szysz M.PCR method for the rapid detection and discrimination of Legionella spp.based on the amplification of pcs,pmtA,and 16S rRNA genes[J].J Appl Genetics,2016,57(2):251-61.
[21] Avni T,Bieber A,Green H,et al.Diagnostic acuracy of PCR alone and compared to urinary antigen testing for detection of Legionella spp.:a systematic review[J].J Clin Microbiol,2016,54(2):401-11.
[22] Ditommaso S,Ricciardi E,Giacomuzzi M,et al.Legionella in water samples:How can you interpret the results obtained by quantitative PCR?[J].Mol Cell Probes,2015,29(1):7-12.
[23] Campocasso A,Boughalmi M,Fournous G,et al.Legionella tunisiensis sp.nov.and Legionella massiliensis sp.nov.,isolated from environmental water samples[J].Int J Syst Evol Micrbio,2012,62(12):3003-6.
[24] Palmer A,Painter J,Hassler H,et al.Legionella clemsonensis sp.nov.:a green fluorescing Legionella strain from a patient with pneumonia[J].Microbiol Immunol,2016,60(10):694-701.
[25] Ahmadrajabi R,Shakibaie MR,Iranmanesh Z,et al.Prevalence of mip virulence gene and PCR-base sequence typing of Legionella pneumophila from cooling water systems of two cities in Iran[J].Virulence,2016,7(5):602-9.
[26] Collins S,Jorgensen F,Willis C,et al.Real-time PCR to supplement gold-standard culture-based detection of Legionella in environmental samples[J].J Appl Microbiol,2015,119(4):1158-69.
[27] 刘文强,贾玉萍,赵宏坤.16S rRNA在细菌分类鉴定研究中的应用[J].动物医学进展,2006,27(11):15-8.
[28] 王卓,吴琼,杨慎江,等.长白山地区蜱类和鼠类中查菲埃立克体感染调查及16S rRNA序列分析[J].中国病原生物学杂志,2017,12(10):971-4,978.
[29] 滑留帅,王璟,徐照学,等.16S rRNA基因高通量测序分析牛粪发酵细菌多样性[J].农业工程学报,2016,32(1):311-5.
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