副溶血弧菌主要毒力因子三重荧光定量PCR检测方法的建立与应用
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摘要
为建立快捷、准确、高效检测副溶血弧菌的方法,以副溶血弧菌种属特异性基因toxR和毒力因子tdh、trh基因序列,分别设计3对特异性引物及相应TaqMan探针,并在toxR、tdh和trh基因的3个探针的5'端分别标记FAM、HEX、CY5荧光报告基团,3'端标记BHQ1淬灭荧光基团,通过优化反应体系和反应参数,确定最佳反应体系,建立一种基于Taq Man探针定量检测副溶血弧菌的三重荧光定量PCR方法。结果:本试验所建立三重荧光定量PCR方法与其他菌株无交叉反应,其最低检出限达到了10 CFU/m L,重复性试验中每组变异系数均小于1%;检测人工染菌的虾肉和贝类样品时,最低检出限亦达到10 CFU/m L,且整个检测扩增时间大约为1 h。结果表明,本研究建立的三重荧光PCR检测方法,特异性强、敏感性高、重复性好且耗时短,是高通量检测致病性副溶血弧菌的有效手段。
To establish a rapid,accurate and efficient method for detection of Vibrio parahaemolyticus (VP),three pairs of specific primers and corresponding Taq Man probes were designed according to the species-specific gene toxR and the virulence genes tdh and trh of V. parahaemolyticus. The 5-ends of the probes for toxR,tdh and trh were individually labeled with FAM,HEX and CY5. The 3' -ends were all labeled with quencher BHQ1. The concentration and reaction parameters of each reaction were optimized to build a triple Real-time PCR based on the Taq Man probe method for the quantitative detection of V. parahaemolyticus. The results showed that the specificity of the assay had no cross-reaction with other pathogens,the minimum detection limit was 10 CFU/m L,and the variation coefficient of the repeated experiments was less than 1%. The triple Real-time PCR was used to detect shrimp and shellfish samples contaminated by V. parahaemolyticus,and the minimum detection reached 10 CFU/m L. The whole detection lasted approximately 1 h. A conclusion could be drawn that a new triple Realtime PCR method established in this study possessed strong specificity,high sensitivity,good repeatability and ideal convenience. It was an effective method for high-throughput detection of pathogenic V. parahaemolyticus.
To establish a rapid,accurate and efficient method for detection of Vibrio parahaemolyticus (VP),three pairs of specific primers and corresponding Taq Man probes were designed according to the species-specific gene toxR and the virulence genes tdh and trh of V. parahaemolyticus. The 5-ends of the probes for toxR,tdh and trh were individually labeled with FAM,HEX and CY5. The 3' -ends were all labeled with quencher BHQ1. The concentration and reaction parameters of each reaction were optimized to build a triple Real-time PCR based on the Taq Man probe method for the quantitative detection of V. parahaemolyticus. The results showed that the specificity of the assay had no cross-reaction with other pathogens,the minimum detection limit was 10 CFU/m L,and the variation coefficient of the repeated experiments was less than 1%. The triple Real-time PCR was used to detect shrimp and shellfish samples contaminated by V. parahaemolyticus,and the minimum detection reached 10 CFU/m L. The whole detection lasted approximately 1 h. A conclusion could be drawn that a new triple Realtime PCR method established in this study possessed strong specificity,high sensitivity,good repeatability and ideal convenience. It was an effective method for high-throughput detection of pathogenic V. parahaemolyticus.
引文
[1]Wang R,Zhong Y,Gu X,et al.The pathogenesis,detection,and prevention of Vibrio parahaemolyticus[J].Front Microbiol,2015,6(32):144.
[2]Cabello F C,Espejo R T,Hernandez M C,et al.Vibrio parahaemolyticus O3:K6 epidemic diarrhea,Chile,2005[J].Emerg Infect Dis,2007,13(4):655-656.
[3]Martinez-Urtaza J,Lozano-Leon A,De Paola A,et al.Characterization of pathogenic Vibrio parahaemolyticus isolates from clinical sources in Spain and comparison with Asian and North American pandemic isolates[J].J Clin Microbiol,2004,42(10):4672-4678.
[4]侯兵兵,陈昌国,李娜.TaqMan-探针荧光定量PCR鉴定副溶血弧菌方法的建立[J].现代检验医学杂志,2018(1):40-43.
[5]Park J Y,Jeon S,Kim J Y,et al.Multiplex real-time polymerase chain reaction assays for simultaneous detection of Vibrio cholerae,Vibrio parahaemolyticus,and Vibrio vulnificus[J].Osong Public Health Res Perspect,2013,4(3):133-139.
[6]高正琴,邢进,冯育芳,等.TaqMan MGB探针实时荧光定量PCR快速检测布鲁氏菌[J].中国人兽共患病学报,2011,27(11):995-1000.
[7]周朝东,黄哲甦.荧光定量PCR技术在药品检验领域中的应用[J].天津药学,2018(2):65-71.
[8]何再平,王权,陈永军,等.致病性副溶血弧菌三重PCR检测方法的建立和评价[J].中国动物传染病学报,2015,23(6):48-55.
[9]王立红,韩东升,韩崇旭,等.副溶血弧菌大流行株的感染及基因分型研究现状[J].临床检验杂志,2018(4):283-284.
[10]毕水莲,罗永文,关小莺.PCR衍生技术快速检测副溶血弧菌的研究进展[J].江西农业学报,2017,29(8):105-109.
[11]贾丹.2013-2016年沿海地区虾源副溶血弧菌的特性分析及致病性研究[D].上海:上海海洋大学,2017.
[12]Pascual J,Macián M C,Arahal D R,et al.Multilocus sequence analysis of the central clade of the genus Vibrio by using the 16SrRNA,rec A,pyr H,rpo D,gyr B,rct B and toxR genes[J].Int J Syst Evol Microbiol,2010,60(Pt 1):154-165.
[13]Venkateswaran K,Dohmoto N,Harayama S.Cloning and nucleotide sequence of the gyr B gene of Vibrio parahaemolyticus and its application in detection of this pathogen in shrimp[J].Appl Environ Microbiol,1998,64(2):681-687.
[14]Teh C S J,Chua K H,Thong K L.Simultaneous differential detection of human pathogenic and nonpathogenic Vibrio species using multiplex PCR based on gyr B and pntA genes[J].Appl Microbiol,2010,108(6):1940-1945.
[15]凌娇,王权,白雪瑞.副溶血弧菌和霍乱弧菌双重荧光定量PCR快速检测方法的建立与应用[J].畜牧与兽医,2018(4):93-99.
[16]Kim Y B,Okuda J,Matsumoto C,et al.Identification of Vibrio parahaemolyticus strains at the species level by PCR targeted to the toxR gene[J].Clin Microbiol,1999,37(4):1173-1177.
[17]Chen S,Ge B.Development of a toxR-based loop-mediated isothermal amplification assay for detecting Vibrio parahaemolyticus[J].BMC Microbiol,2010,10(1):1-9.
[18]Letchumanan V,Chan K G,Lee L H.Vibrio parahaemolyticus:a review on the pathogenesis,prevalence,and advance molecular identification techniques[J].Front Microbiol,2014,5(705):705.
[19]Nordstrom J L,Vickery M C,Blackstone G M,et al.Development of a multiplex real-time PCR assay with an internal amplification control for the detection of total and pathogenic Vibrio parahaemolyticus bacteria in oysters[J].Appl Environ Microbiol,2007,73(18):5840-5847.
[20]Bej A K,Patterson D P,Brasher C W,et al.Detection of total and hemolysin-producing Vibrio parahaemolyticus in shellfish using multiplex PCR amplification of tl,tdh and trh[J].J Microbiol Method,1999,36(3):215-225.
[21]张蔚,潘劲草,陈坤.副溶血性弧菌的分子流行病学研究进展[J].国外医学:流行病学传染病学分册,2004,31(3):182-184.
[22]Ronholm J,Petronella N,Leung C C,et al.Genomic features of environmental and clinical Vibrio parahaemolyticus isolates lacking recognized virulence factors are dissimilar[J].Appl Environ Microbiol,2015,82(4):1102-1113.
[23]金周浩,宋达锋,顾青.副溶血弧菌毒力基因的检测研究[J].中国食品学报,2008,8(3):143-146.
[24]蒋鲁岩.用复合PCR同步检测食品中霍乱弧菌、副溶血弧菌及创伤弧菌[J].检验检疫科学,2000(6):5-7.
[25]Collin B,Rehnstam-Holm A S.Occurrence and potential pathogenesis of Vibrio cholerae,Vibrio parahaemolyticus and Vibrio vulnificus on the South Coast of Sweden[J].FEMS Microbiol Ecol,2011,78(2):306-313.
[26]Cantet F,Hervioheath D,Caro A,et al.Quantification of Vibrio parahaemolyticus,Vibrio vulnificus and Vibrio cholerae in French Mediterranean coastal lagoons.[J].Res Microbiol,2013,164(8):867-874.
[27]Bauer A,Rrvik L M.A novel multiplex PCR for the identification of Vibrio parahaemolyticus,Vibrio cholerae and Vibrio vulnificus[J].Lett Appl Microbiol,2007,45(4):371-375.
[28]高兴,辛文文,高姗,等.11种(株)食源性细菌基因芯片检测方法的建立[J].生物技术通报,2013(12):123-128.
[29]高世光,李莉,冯华炜,等.TaqMan探针双重荧光PCR法检测副溶血性弧菌毒力基因[J].中国卫生检验杂志,2016(9):1244-1246.
[2]Cabello F C,Espejo R T,Hernandez M C,et al.Vibrio parahaemolyticus O3:K6 epidemic diarrhea,Chile,2005[J].Emerg Infect Dis,2007,13(4):655-656.
[3]Martinez-Urtaza J,Lozano-Leon A,De Paola A,et al.Characterization of pathogenic Vibrio parahaemolyticus isolates from clinical sources in Spain and comparison with Asian and North American pandemic isolates[J].J Clin Microbiol,2004,42(10):4672-4678.
[4]侯兵兵,陈昌国,李娜.TaqMan-探针荧光定量PCR鉴定副溶血弧菌方法的建立[J].现代检验医学杂志,2018(1):40-43.
[5]Park J Y,Jeon S,Kim J Y,et al.Multiplex real-time polymerase chain reaction assays for simultaneous detection of Vibrio cholerae,Vibrio parahaemolyticus,and Vibrio vulnificus[J].Osong Public Health Res Perspect,2013,4(3):133-139.
[6]高正琴,邢进,冯育芳,等.TaqMan MGB探针实时荧光定量PCR快速检测布鲁氏菌[J].中国人兽共患病学报,2011,27(11):995-1000.
[7]周朝东,黄哲甦.荧光定量PCR技术在药品检验领域中的应用[J].天津药学,2018(2):65-71.
[8]何再平,王权,陈永军,等.致病性副溶血弧菌三重PCR检测方法的建立和评价[J].中国动物传染病学报,2015,23(6):48-55.
[9]王立红,韩东升,韩崇旭,等.副溶血弧菌大流行株的感染及基因分型研究现状[J].临床检验杂志,2018(4):283-284.
[10]毕水莲,罗永文,关小莺.PCR衍生技术快速检测副溶血弧菌的研究进展[J].江西农业学报,2017,29(8):105-109.
[11]贾丹.2013-2016年沿海地区虾源副溶血弧菌的特性分析及致病性研究[D].上海:上海海洋大学,2017.
[12]Pascual J,Macián M C,Arahal D R,et al.Multilocus sequence analysis of the central clade of the genus Vibrio by using the 16SrRNA,rec A,pyr H,rpo D,gyr B,rct B and toxR genes[J].Int J Syst Evol Microbiol,2010,60(Pt 1):154-165.
[13]Venkateswaran K,Dohmoto N,Harayama S.Cloning and nucleotide sequence of the gyr B gene of Vibrio parahaemolyticus and its application in detection of this pathogen in shrimp[J].Appl Environ Microbiol,1998,64(2):681-687.
[14]Teh C S J,Chua K H,Thong K L.Simultaneous differential detection of human pathogenic and nonpathogenic Vibrio species using multiplex PCR based on gyr B and pntA genes[J].Appl Microbiol,2010,108(6):1940-1945.
[15]凌娇,王权,白雪瑞.副溶血弧菌和霍乱弧菌双重荧光定量PCR快速检测方法的建立与应用[J].畜牧与兽医,2018(4):93-99.
[16]Kim Y B,Okuda J,Matsumoto C,et al.Identification of Vibrio parahaemolyticus strains at the species level by PCR targeted to the toxR gene[J].Clin Microbiol,1999,37(4):1173-1177.
[17]Chen S,Ge B.Development of a toxR-based loop-mediated isothermal amplification assay for detecting Vibrio parahaemolyticus[J].BMC Microbiol,2010,10(1):1-9.
[18]Letchumanan V,Chan K G,Lee L H.Vibrio parahaemolyticus:a review on the pathogenesis,prevalence,and advance molecular identification techniques[J].Front Microbiol,2014,5(705):705.
[19]Nordstrom J L,Vickery M C,Blackstone G M,et al.Development of a multiplex real-time PCR assay with an internal amplification control for the detection of total and pathogenic Vibrio parahaemolyticus bacteria in oysters[J].Appl Environ Microbiol,2007,73(18):5840-5847.
[20]Bej A K,Patterson D P,Brasher C W,et al.Detection of total and hemolysin-producing Vibrio parahaemolyticus in shellfish using multiplex PCR amplification of tl,tdh and trh[J].J Microbiol Method,1999,36(3):215-225.
[21]张蔚,潘劲草,陈坤.副溶血性弧菌的分子流行病学研究进展[J].国外医学:流行病学传染病学分册,2004,31(3):182-184.
[22]Ronholm J,Petronella N,Leung C C,et al.Genomic features of environmental and clinical Vibrio parahaemolyticus isolates lacking recognized virulence factors are dissimilar[J].Appl Environ Microbiol,2015,82(4):1102-1113.
[23]金周浩,宋达锋,顾青.副溶血弧菌毒力基因的检测研究[J].中国食品学报,2008,8(3):143-146.
[24]蒋鲁岩.用复合PCR同步检测食品中霍乱弧菌、副溶血弧菌及创伤弧菌[J].检验检疫科学,2000(6):5-7.
[25]Collin B,Rehnstam-Holm A S.Occurrence and potential pathogenesis of Vibrio cholerae,Vibrio parahaemolyticus and Vibrio vulnificus on the South Coast of Sweden[J].FEMS Microbiol Ecol,2011,78(2):306-313.
[26]Cantet F,Hervioheath D,Caro A,et al.Quantification of Vibrio parahaemolyticus,Vibrio vulnificus and Vibrio cholerae in French Mediterranean coastal lagoons.[J].Res Microbiol,2013,164(8):867-874.
[27]Bauer A,Rrvik L M.A novel multiplex PCR for the identification of Vibrio parahaemolyticus,Vibrio cholerae and Vibrio vulnificus[J].Lett Appl Microbiol,2007,45(4):371-375.
[28]高兴,辛文文,高姗,等.11种(株)食源性细菌基因芯片检测方法的建立[J].生物技术通报,2013(12):123-128.
[29]高世光,李莉,冯华炜,等.TaqMan探针双重荧光PCR法检测副溶血性弧菌毒力基因[J].中国卫生检验杂志,2016(9):1244-1246.
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