基质辅助激光解析电离飞行时间质谱(MALDI-TOF MS)在鸭源鸡杆菌快速鉴定中的应用
|
摘要
按照MALDI-TOF MS要求进行56株鸡杆菌分离株的样品制备,点靶并获取样品质谱图,运用Biotyper分析软件对所获取的质谱图鉴定分析;同时对这些菌株进行荧光定量PCR检测、gyrB基因的PCR扩增和序列分析,进行辅助分析鉴定。结果显示,56株鸡杆菌分离株经MALDI-TOF MS和gyrB基因序列分析均鉴定为鸭源鸡杆菌,阳性率为100.0%;荧光定量PCR鉴定出55株鸭源鸡杆菌,阳性率为98.2%(55/56)。结果表明,MALDI-TOF MS可用于鸭源鸡杆菌分离株的快速鉴定,操作简便,准确率高。
Matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDITOF MS)was applied for rapid identification of 56 Gallibacterium strains.The bacteria sample were prepared by method of MALDI-TOF MS,then added to the target polished steel plate and the mass spectra were acquired.The spectra were analyzed by the Biotyper analysis software.At the same time,these strains were identified by real-time fluorescence quantitative PCR and gyrBgene sequence analysis,for auxiliary analysis and identification.All 56 strains were identified as Gallibacterium anatis rapidly by MALDI-TOF MS,showing 100.0% positive rate,which is consistent with the identification of gyrB gene sequence analysis.55 strains of Gallibacterium anatis were identified by real time fluorescence quantitative PCR with a 98.2%(55/56)detection rate.MALDITOF MS can be used to identify Gallibacterium anatis,as a fast,simple,accurate diagnostic tools in veterinary clinic.
Matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDITOF MS)was applied for rapid identification of 56 Gallibacterium strains.The bacteria sample were prepared by method of MALDI-TOF MS,then added to the target polished steel plate and the mass spectra were acquired.The spectra were analyzed by the Biotyper analysis software.At the same time,these strains were identified by real-time fluorescence quantitative PCR and gyrBgene sequence analysis,for auxiliary analysis and identification.All 56 strains were identified as Gallibacterium anatis rapidly by MALDI-TOF MS,showing 100.0% positive rate,which is consistent with the identification of gyrB gene sequence analysis.55 strains of Gallibacterium anatis were identified by real time fluorescence quantitative PCR with a 98.2%(55/56)detection rate.MALDITOF MS can be used to identify Gallibacterium anatis,as a fast,simple,accurate diagnostic tools in veterinary clinic.
引文
[1]BISGAARD M,KORCZAK B M,BUSSE H J,et al.Classification of the taxon 2and taxon 3complex of bisgaard within Gallibacterium and description of Gallibacterium melopsittaci sp.nov.,Gallibacterium trehalosifermentans sp.nov.and Gallibacterium salpingitidis sp.nov.[J].Int J Syst Evol Microbiol,2009,59(4):735-744.
[2]JORDAN F T,WILLIAMS N J,WATTRET A,et al.Observations on salpingitis,peritonitis and salpingoperitonitis in a layer breeder flock[J].Vet Rec,2005,157(19):573-577.
[3]PERSSON G,BOJESEN A M,Bacterial determinants of importance in the virulence of Gallibacterium anatis in poultry[J].Vet Rec,2015,46(1):57.
[4]PAUDEL S,ALISPAHIC M,LIEBHART D,et al.Assessing pathogenicity of Gallibacterium anatis in a natural infection model:the respiratory and reproductive tracts of chickens are targets for bacterial colonization[J].Avian Pathol,2013,42(6):527-535.
[5]BISGAARD M.Ecology and significance of pasteurellaceae in animals[J].Zbl Bakt-int J Med M,1993,279(1):7-26.
[6]AUBIN G G,HALOUN A,TREILHAUD M,et al.Gallibacterium anatis bacterium in a human[J].J Clin Microbiol,2013,51(11):3897-3899.
[7]BOJESEN A M,VAZQUEZ M E,ROBLES F,et al.Specific identification of Gallibacterium by a PCR using primers targeting the 16SrRNA and 23SrRNAgenes[J].Vet Microbiol,2007,123(1/3):262-268.
[8]HUANG-FU H P,ZHAO J,YANG X,et al.Development and preliminary application of a quantitative PCR assay for detecting gtxA-containing Gallibacteriumspecies in chickens[J].Avian Dis,2012,56(2):315-320.
[9]WANG C,ROBLES F,RAMIREZ S,et al.Culture-independent identification and quantification of Gallibacterium anatis(G.anatis)by real-time quantitative PCR[J].Avian Pathol,2016,45(5):538-544.
[10]HUANG-FU H P,WANG C Q,CHEN L,et al.Molecular identification of Gallibacteriumfrom hen and analysis of it’s 16SrRNA Gene[J].Agri Sci Tech,2009,10(1):43-46,55.
[11]BADER O.MALDI-TOF MS-based species identification and typing approaches in medical mycology[J].Proteomics,2013,13(5):788-799.
[12]VAN VEEN S Q,CLAAS E C,KUIJPER E J.Highthroughput identification of bacteria and yeast bymatrix-assisted laser desorption/ionization time of flight mass spectrometry in conventional medical microbiology laboratories[J].J Clin Microbiol,2010,48(3):900-907.
[13]周千渝,张延国,黄成才,等.食源性致病菌MALDI-TOF MS检测方法的建立与应用[J].食品工业科技,2015,36(18):59-64.
[14]战晓微,傅俊范,郑秋月,等.沙门菌MALDI-TOF MS检测方法的建立[J].现代食品科技,2011,27(5):595-597.
[15]鲍春梅,崔恩博,陈鹏,等.MALDI-TOF MS鉴定宋内志贺菌的初步应用研究[J].传染病信息,2012,25(1):10-13.
[16]王耀,曹际娟,赵昕,等.单增李斯特菌MALDI-TOF MS鉴定与分型研究[J].食品科学,2012,33(3):194-198.
[17]DALLAGASSA C B,HUERGO L F,STETS M I,et al.Matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis of Escherichia coli categories[J].Genet Mol Res,2014,13(1):716-722.
[18]YAMAMOTO S,HARAYAMA S.PCR amplification and direct sequencing of gyrB genes with universal primers and their application to the detection and taxonomic analysis of Pseudomonas putida strains[J].Appl Environ Microb,1995,61(3):1104.
[19]董青,史静柯,王海花,等.郑州地区7株鸭源鸡杆菌的MALDI-TOF MS鉴定及其生物学特性分析[J].河南农业科学,2017,46(10):143-147.
[20]王海花,苗丽,王亚宾,等.MALDI-TOF MS对猪源肠球菌的快速检测[J].中国兽医学报,2018,38(2):276-280.
[21]皇甫和平,杨霞,赵军,等.豫陕两省14株鸡杆菌的gyrB、16SrRNA和rpoB基因序列比较分析[J].畜牧兽医学报,2012,43(7):1103-1110.
[2]JORDAN F T,WILLIAMS N J,WATTRET A,et al.Observations on salpingitis,peritonitis and salpingoperitonitis in a layer breeder flock[J].Vet Rec,2005,157(19):573-577.
[3]PERSSON G,BOJESEN A M,Bacterial determinants of importance in the virulence of Gallibacterium anatis in poultry[J].Vet Rec,2015,46(1):57.
[4]PAUDEL S,ALISPAHIC M,LIEBHART D,et al.Assessing pathogenicity of Gallibacterium anatis in a natural infection model:the respiratory and reproductive tracts of chickens are targets for bacterial colonization[J].Avian Pathol,2013,42(6):527-535.
[5]BISGAARD M.Ecology and significance of pasteurellaceae in animals[J].Zbl Bakt-int J Med M,1993,279(1):7-26.
[6]AUBIN G G,HALOUN A,TREILHAUD M,et al.Gallibacterium anatis bacterium in a human[J].J Clin Microbiol,2013,51(11):3897-3899.
[7]BOJESEN A M,VAZQUEZ M E,ROBLES F,et al.Specific identification of Gallibacterium by a PCR using primers targeting the 16SrRNA and 23SrRNAgenes[J].Vet Microbiol,2007,123(1/3):262-268.
[8]HUANG-FU H P,ZHAO J,YANG X,et al.Development and preliminary application of a quantitative PCR assay for detecting gtxA-containing Gallibacteriumspecies in chickens[J].Avian Dis,2012,56(2):315-320.
[9]WANG C,ROBLES F,RAMIREZ S,et al.Culture-independent identification and quantification of Gallibacterium anatis(G.anatis)by real-time quantitative PCR[J].Avian Pathol,2016,45(5):538-544.
[10]HUANG-FU H P,WANG C Q,CHEN L,et al.Molecular identification of Gallibacteriumfrom hen and analysis of it’s 16SrRNA Gene[J].Agri Sci Tech,2009,10(1):43-46,55.
[11]BADER O.MALDI-TOF MS-based species identification and typing approaches in medical mycology[J].Proteomics,2013,13(5):788-799.
[12]VAN VEEN S Q,CLAAS E C,KUIJPER E J.Highthroughput identification of bacteria and yeast bymatrix-assisted laser desorption/ionization time of flight mass spectrometry in conventional medical microbiology laboratories[J].J Clin Microbiol,2010,48(3):900-907.
[13]周千渝,张延国,黄成才,等.食源性致病菌MALDI-TOF MS检测方法的建立与应用[J].食品工业科技,2015,36(18):59-64.
[14]战晓微,傅俊范,郑秋月,等.沙门菌MALDI-TOF MS检测方法的建立[J].现代食品科技,2011,27(5):595-597.
[15]鲍春梅,崔恩博,陈鹏,等.MALDI-TOF MS鉴定宋内志贺菌的初步应用研究[J].传染病信息,2012,25(1):10-13.
[16]王耀,曹际娟,赵昕,等.单增李斯特菌MALDI-TOF MS鉴定与分型研究[J].食品科学,2012,33(3):194-198.
[17]DALLAGASSA C B,HUERGO L F,STETS M I,et al.Matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis of Escherichia coli categories[J].Genet Mol Res,2014,13(1):716-722.
[18]YAMAMOTO S,HARAYAMA S.PCR amplification and direct sequencing of gyrB genes with universal primers and their application to the detection and taxonomic analysis of Pseudomonas putida strains[J].Appl Environ Microb,1995,61(3):1104.
[19]董青,史静柯,王海花,等.郑州地区7株鸭源鸡杆菌的MALDI-TOF MS鉴定及其生物学特性分析[J].河南农业科学,2017,46(10):143-147.
[20]王海花,苗丽,王亚宾,等.MALDI-TOF MS对猪源肠球菌的快速检测[J].中国兽医学报,2018,38(2):276-280.
[21]皇甫和平,杨霞,赵军,等.豫陕两省14株鸡杆菌的gyrB、16SrRNA和rpoB基因序列比较分析[J].畜牧兽医学报,2012,43(7):1103-1110.