红鳍东方鲀源哈维弧菌毒力基因检测及分型研究
|
摘要
为探明红鳍东方鲀源哈维弧菌所携带毒力基因的种类对致病性的影响,以从辽宁地区患病红鳍东方鲀中分离得到的24株哈维弧菌为材料,根据GenBank公布的基因序列,设计合成引物,采用聚合酶链式反应(Polymerase Chain Reaction,PCR)对7种毒力基因flaB、flaC、toxS、zot、tcpA、tdh和tlh进行检测。结果表明,24株哈维弧菌中均检测出鞭毛蛋白flaB基因和flaC基因,其余5种基因均未检测到。同时对24株哈维弧菌进行了BOX-PCR扩增,结果将其分为4种基因型,记为A1-A4型。
In this paper,we detected virulence genetypes of Vibrio harvey isolated from Takifugu rubripes for further studying its pathogenicity.24 strains of V.harveyi isolated from infected Takifugu rubripes in Liaoning region were used for research.The PCR-primers were designed and synthesized according to the published sequence in the GenBank data.Polymerase Chain Reaction(PCR)was used to detect 7 virulence genes,including flaB,flaC,toxS,zot,tcpA,tdh and tlh.The results showed that both flaB and flaC gene were detected in all 24 strains of V.harveyi,and other 5 virulence genes were not detected.Meanwhile,the results of BOX-PCR indentation showed that 24 strains of V.harveyi were divided into 4 genotypes,respectively named A1-A4.
In this paper,we detected virulence genetypes of Vibrio harvey isolated from Takifugu rubripes for further studying its pathogenicity.24 strains of V.harveyi isolated from infected Takifugu rubripes in Liaoning region were used for research.The PCR-primers were designed and synthesized according to the published sequence in the GenBank data.Polymerase Chain Reaction(PCR)was used to detect 7 virulence genes,including flaB,flaC,toxS,zot,tcpA,tdh and tlh.The results showed that both flaB and flaC gene were detected in all 24 strains of V.harveyi,and other 5 virulence genes were not detected.Meanwhile,the results of BOX-PCR indentation showed that 24 strains of V.harveyi were divided into 4 genotypes,respectively named A1-A4.
引文
[1]刘问,钱冬,杨国梁,等.南美白对虾虾苗淡化期间发光病病原研究[J].集美大学学报(自然科学版),2004(4):300-304.
[2]石亚素,童国忠,薛超波,等.舟山养殖大黄鱼烂尾病中哈氏弧菌的分离鉴定及药敏试验[J].中国卫生检验杂志,2005(3):267-269.
[3]张静,施慧,谢建军,等.网箱养殖鲈鱼内脏白点病病原的分离与鉴定[J].浙江海洋学院学报(自然科学版),2009(2):176-182.
[4]梅冰,周永灿,徐先栋,等.斜带石斑鱼烂尾病病原菌的分离与鉴定[J].热带海洋学报,2010(6):118-124.
[5]陈政强,姚志贤,林茂,等.半滑舌鳎皮肤溃疡病病原研究[J].水产学报,2012,36(5):764-771.
[6]范文辉,黄倢,王秀华,等.养殖大菱鲆溃疡症病原菌的分离鉴定及系统发育分析[J].微生物学报,2005(5):665-670.
[7]张凤萍,彭志兰,张健,等.鮸鱼弧菌病病原菌(哈维氏弧菌)的分离与鉴定[J].微生物学报,2010(3):304-309.
[8]陈月忠,黄万红,蔡清海,等.闽南地区鲍溃烂病的研究[J].福建水产,2008(4):31-37.
[9]王斌,于兰萍,胡亮,等.红鳍东方鲀皮肤溃烂病病原菌的分离与鉴定[J].中国水产科学,2008(2):352-358.
[10]庞玲玲.哈维氏弧菌毒力相关基因的筛查及快速检测技术研究[D].青岛:中国海洋大学,2007.
[11]徐芝亮.哈维氏弧菌遗传多样性分析及毒力相关基因的检测[D].湛江:广东海洋大学,2012.
[12]杨凤环,李正楠,姬惜珠,等.BOX-PCR技术在微生物多样性研究中的应用[J].微生物学通报,2008(8):1282-1286.
[13]SECHI L A,DUPRE I,DERIU A,et al.Distribution of Vibrio cholerae virulence genes among different Vibrio species isolated in Sardinia,Italy[J].J Appl Microbiol,2000,88(3):475-481.
[14]VERSALOVIC J,SCHNEIDER M,BRUIJN F,et al.Genomic fingerprint of bacteria using repetitive sequence-based polymerase chain reaction[J].Methods in Molecular&Cellular Biology,1994,5(1):25-40.
[15]SAEED MO.Association of Vibrio harveyi,with mortalities in cultured marine fish in Kuwait[J].Aquaculture,1995,136:21-29.
[16]LIU P C,LEE K C,KOU G H,et al.Isolation of Vibrio harveyi,from Diseased Kuruma Prawns Penaeusjaponicus[J].Current Microbiology,1996,33(2):129-132.
[17]LEE S E,KIM S Y,JEONG B C,et al.A Bacterial Flagellin,Vibrio vulnificus Fla B,H as a Strong Mucosal Adjuvant Activity To Induce Protective Immunity[J].Infection&Immunity,2006,74(1):694.
[18]MILLIKAN D S,RUBY E G.Vibriofischeri Flagellin A Is Essential for Normal Motility and for Symbiotic Competence during Initial Squid Light Organ Colonization[J].Journal of Bacteriology,2004,186(13):4315-4325.
[19]徐芝亮.哈维氏弧菌遗传多样性分析及毒力相关基因的检测[D].湛江:广东海洋大学,2012.
[20]刘迪,房文红,周红霞,等.虾源哈维氏弧菌的致病性与生物学特性比较分析[J].海洋渔业,2017,39(2):197-205.
[21]MILLER V L,MEKALANOS J J.Synthesis of cholera toxin is positively regulated at the transcriptional level by tox R[J].Proceedings of the National Academy of Sciences of the United States of America,1984,81(11):3471-3475.
[22]SHI L,MIYOSHI S,HIVRA M,et al.Detection of Genes Encoding Cholera Toxin(CT),Zonula Occludens Toxin(ZOT),Accessory Cholera Enterotoxin(ACE)and Heat-Stable Enterotoxin(ST)in Vibrio mimicus Clinical Strains[J].Microbiology&Immunology,1998,42(12):823-828.
[23]RAJA N,SHAMSVDIN M N,SOMARANY W,et al.Detection and molecular characterization of the zot gene in Vibrio cholerae and V.alginolyticus isolates[J].Southeast Asian Journal of Tropical Medicine&Public Health,2001,32(1):100.
[24]SECHI L A,DUPREI,DERIV A,et al.Distribution of Vibrio cholerae,virulence genes among different Vibrio,species isolated in Sardinia,Italy[J].Journal of Applied Microbiology,2000,88(3):475-481.
[25]刘佳妍,金莉莉,王秋雨.细菌基因组重复序列PCR技术及其应用[J].微生物学杂志,2006(3):90-93.
[26]刘洋,隋新华,陈文新.华北及西北地区岩黄芪根瘤菌的表型及遗传多样性[J].生态学报,2005(5):1088-1094.
[27]TACAO M,AIVES A,SAAVEDRA M J,et al.BOX-PCR is an adequate tool for typing Aeromonasspp[J].Antonie Van Leeuwenhoek,2005,88:173-179.
[28]PROUDY I,BOUGIED,COTON E,et al.Genotypic characterization of Enterobacter sakazakii isolates by PFGE,BOX-PCR and sequencing of the fli C gene[J].Journal of Applied Microbiology,2008,104(1):26-34.
[29]徐芝亮,吴灶和,简纪常,等.哈维氏弧菌的分子遗传多样性研究[J].水产学报,2012(9):1450-1456.
[30]林海云,车建美,刘波,等.基于BOX-PCR和REP-PCR技术青枯雷尔氏菌遗传多样性分析[J].农业生物技术学报,2011(6):1099-1109.
[31]LOUWS F J,FUIBRIGHT D W,STEPHENS C T,et al.Specific genomic fingerprints of phytopathogenic Xanthomonas and Pseudomonas pathovars and strains generated with repetitive sequences and PCR[J].Applied&Environmental Microbiology,1994,60(7):2286.
[32]张红侠,冯瑞华,李俊,等.黄土高原地区大豆根瘤菌的遗传多样性和系统发育[J].微生物学报,2010(11):1466-1473.
[2]石亚素,童国忠,薛超波,等.舟山养殖大黄鱼烂尾病中哈氏弧菌的分离鉴定及药敏试验[J].中国卫生检验杂志,2005(3):267-269.
[3]张静,施慧,谢建军,等.网箱养殖鲈鱼内脏白点病病原的分离与鉴定[J].浙江海洋学院学报(自然科学版),2009(2):176-182.
[4]梅冰,周永灿,徐先栋,等.斜带石斑鱼烂尾病病原菌的分离与鉴定[J].热带海洋学报,2010(6):118-124.
[5]陈政强,姚志贤,林茂,等.半滑舌鳎皮肤溃疡病病原研究[J].水产学报,2012,36(5):764-771.
[6]范文辉,黄倢,王秀华,等.养殖大菱鲆溃疡症病原菌的分离鉴定及系统发育分析[J].微生物学报,2005(5):665-670.
[7]张凤萍,彭志兰,张健,等.鮸鱼弧菌病病原菌(哈维氏弧菌)的分离与鉴定[J].微生物学报,2010(3):304-309.
[8]陈月忠,黄万红,蔡清海,等.闽南地区鲍溃烂病的研究[J].福建水产,2008(4):31-37.
[9]王斌,于兰萍,胡亮,等.红鳍东方鲀皮肤溃烂病病原菌的分离与鉴定[J].中国水产科学,2008(2):352-358.
[10]庞玲玲.哈维氏弧菌毒力相关基因的筛查及快速检测技术研究[D].青岛:中国海洋大学,2007.
[11]徐芝亮.哈维氏弧菌遗传多样性分析及毒力相关基因的检测[D].湛江:广东海洋大学,2012.
[12]杨凤环,李正楠,姬惜珠,等.BOX-PCR技术在微生物多样性研究中的应用[J].微生物学通报,2008(8):1282-1286.
[13]SECHI L A,DUPRE I,DERIU A,et al.Distribution of Vibrio cholerae virulence genes among different Vibrio species isolated in Sardinia,Italy[J].J Appl Microbiol,2000,88(3):475-481.
[14]VERSALOVIC J,SCHNEIDER M,BRUIJN F,et al.Genomic fingerprint of bacteria using repetitive sequence-based polymerase chain reaction[J].Methods in Molecular&Cellular Biology,1994,5(1):25-40.
[15]SAEED MO.Association of Vibrio harveyi,with mortalities in cultured marine fish in Kuwait[J].Aquaculture,1995,136:21-29.
[16]LIU P C,LEE K C,KOU G H,et al.Isolation of Vibrio harveyi,from Diseased Kuruma Prawns Penaeusjaponicus[J].Current Microbiology,1996,33(2):129-132.
[17]LEE S E,KIM S Y,JEONG B C,et al.A Bacterial Flagellin,Vibrio vulnificus Fla B,H as a Strong Mucosal Adjuvant Activity To Induce Protective Immunity[J].Infection&Immunity,2006,74(1):694.
[18]MILLIKAN D S,RUBY E G.Vibriofischeri Flagellin A Is Essential for Normal Motility and for Symbiotic Competence during Initial Squid Light Organ Colonization[J].Journal of Bacteriology,2004,186(13):4315-4325.
[19]徐芝亮.哈维氏弧菌遗传多样性分析及毒力相关基因的检测[D].湛江:广东海洋大学,2012.
[20]刘迪,房文红,周红霞,等.虾源哈维氏弧菌的致病性与生物学特性比较分析[J].海洋渔业,2017,39(2):197-205.
[21]MILLER V L,MEKALANOS J J.Synthesis of cholera toxin is positively regulated at the transcriptional level by tox R[J].Proceedings of the National Academy of Sciences of the United States of America,1984,81(11):3471-3475.
[22]SHI L,MIYOSHI S,HIVRA M,et al.Detection of Genes Encoding Cholera Toxin(CT),Zonula Occludens Toxin(ZOT),Accessory Cholera Enterotoxin(ACE)and Heat-Stable Enterotoxin(ST)in Vibrio mimicus Clinical Strains[J].Microbiology&Immunology,1998,42(12):823-828.
[23]RAJA N,SHAMSVDIN M N,SOMARANY W,et al.Detection and molecular characterization of the zot gene in Vibrio cholerae and V.alginolyticus isolates[J].Southeast Asian Journal of Tropical Medicine&Public Health,2001,32(1):100.
[24]SECHI L A,DUPREI,DERIV A,et al.Distribution of Vibrio cholerae,virulence genes among different Vibrio,species isolated in Sardinia,Italy[J].Journal of Applied Microbiology,2000,88(3):475-481.
[25]刘佳妍,金莉莉,王秋雨.细菌基因组重复序列PCR技术及其应用[J].微生物学杂志,2006(3):90-93.
[26]刘洋,隋新华,陈文新.华北及西北地区岩黄芪根瘤菌的表型及遗传多样性[J].生态学报,2005(5):1088-1094.
[27]TACAO M,AIVES A,SAAVEDRA M J,et al.BOX-PCR is an adequate tool for typing Aeromonasspp[J].Antonie Van Leeuwenhoek,2005,88:173-179.
[28]PROUDY I,BOUGIED,COTON E,et al.Genotypic characterization of Enterobacter sakazakii isolates by PFGE,BOX-PCR and sequencing of the fli C gene[J].Journal of Applied Microbiology,2008,104(1):26-34.
[29]徐芝亮,吴灶和,简纪常,等.哈维氏弧菌的分子遗传多样性研究[J].水产学报,2012(9):1450-1456.
[30]林海云,车建美,刘波,等.基于BOX-PCR和REP-PCR技术青枯雷尔氏菌遗传多样性分析[J].农业生物技术学报,2011(6):1099-1109.
[31]LOUWS F J,FUIBRIGHT D W,STEPHENS C T,et al.Specific genomic fingerprints of phytopathogenic Xanthomonas and Pseudomonas pathovars and strains generated with repetitive sequences and PCR[J].Applied&Environmental Microbiology,1994,60(7):2286.
[32]张红侠,冯瑞华,李俊,等.黄土高原地区大豆根瘤菌的遗传多样性和系统发育[J].微生物学报,2010(11):1466-1473.