近海副溶血弧菌和甲肝病毒检测方法建立及沉积物细菌多样性分析
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摘要
海洋环境中存在着大量的病原微生物，其中既有导致海洋生物疾病的病原微生物、人类传染病病原微生物，亦有导致人与海洋生物共患的病原微生物。这些病原微生物的存在和散播，对海水养殖业构成了很大威胁，同时又可通过食物链进入人体，导致人传染病爆发流行。因此，开展海洋病原微生物的监测，对于评估海产食品安全，进行疾病的早期预警预报，减少相关的经济损失，具有重要的应用推广价值。
近年来，随着我国沿海城乡人口的日益增长，城乡生活污水的排海量亦逐年增加，致使生活污水中大量的人类病原微生物随之进入海洋环境，分布于海水、生物体以及沉积物当中。它们不仅对海洋生物可造成巨大危害，进而影响海洋生态系统的稳定性，而且还可通过与海水、沉积物等的接触，或海产品的食用而危害人体健康。1988年上海因食用毛蚶引发甲型肝炎大流行，即是海水中甲肝病毒(Hepatitis A virus，HAV)通过贝类进入食物链导致人疾病爆发的典型实例。排污入海口处沉积物中细菌的存在，对人体健康存在着一定的潜在威胁，亦有必要对排污入海口处沉积物中细菌进行检测。另外，目前病害已经成为制约海水养殖业健康发展的瓶颈，凡纳滨对虾是世界养殖虾类产量最高的三大种类之一，近年来频频受到红体病的侵袭，对其进行病原微生物的分离鉴定及相应检测方法的建立是十分必要的。
在我国，对海洋病原微生物的检测一直沿用传统的检测手段，缺少系统、快速的检测方法，因而难以开展常规监测。因此，建立系统、特异、简便、快速、准确的海洋病原微生物检测方法是十分必要的。本研究选定凡纳滨对虾红体病病原菌—副溶血弧菌(Vibrio parahaemolyticus)、海水中以及贝类体内的甲肝病毒、排污入海口处沉积物中细菌作为研究代表，从实际应用出发，初步系统地建立起高效、灵敏的检测方法。并在我国首次将聚合酶链式反应-变性梯度凝胶电泳(Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis，PCR-DGGE)的方法应用于海洋沉积物中病原微生物多样性的分析。
本研究通过优势菌分离、回归感染实验、生理生化特性以及16S rRNA基因分析，将从患红体病的凡纳滨对虾体内分离到的病原菌确定为副溶血弧菌。以副溶血弧菌为抗原，免疫新西兰兔获得高免血清，建立了检测副溶血弧菌的三种免疫学检测方法，分别为间接酶联免疫吸附试验法(Indirect Enzyme-linked Immunosorbent Assay，iELISA)、斑点ELISA(Dot-ELISA)方法、间接荧光抗体(Indirect Fluorescent Antibody，IFA)检测方法。三种免疫学检测方法均具有较高的稳定性和特异性；灵敏性从高到低排列依次为：IFA、iELISA、Dot-ELISA；检测所用时间均很快速，分别为2h、9h、7h；所需仪器设备方面，Dot-ELISA方法所需仪器设备最简单，最适于现场调查应用。另外，根据副溶血弧菌耐热直接溶血毒素(TDH)的保守阅读框pR72H基因序列设计引物，采用热裂解法提取副溶血弧菌的DNA，建立了PCR方法。所建立的PCR方法灵敏度较高，检出限能够达到90～100CFU；特异性较强，能够在2h内完成检测。
采用吸附-洗脱的原理，建立了从海水中浓缩甲肝病毒的方法；采用氯仿去除脂蛋白、聚乙二醇浓缩的原理，建立了从贝类体内浓缩甲肝病毒的方法；在此基础之上，建立了检测甲肝病毒的“RT-PCR扩增—聚丙烯酰胺凝胶电泳—银染色方法”。将该方法应用到辽东湾重点
There are many pathogen microorganism in ocean, they can infect halobios and human. Marine pathogen microorganism pose a significant threat to seawater breed aquatics industry and human health, consequently marine pathogen microorganism monitoring have a important application spread potential for evaluating seafood safety and forecasting disease and lessening correlative economy loss.
Recently, with the coastal city population increasing, pathogen with the life sewage pouring sea increased, Then, pathogen distributed in sea water, sediment and organisms, so they influence marine ecosystem stability by hurt halobios, and threaten human body health by contacting seawater or sediment and taking seafood. In 1988, hepatitis A crazed on account of eated shellfish. Pathogenic microorganism in sediment nearby sewage inlet can harm human body. Otherwise, disease have already become a bottle-neck of restricting marine breed aquatics industry development, Litopenaeus vannamei is one of the three important kinds of breeding shrimp in the world, recently often affected by red body disease. So it is indispensable to monitoring pathogenic microorganism.
In china, methods to detecting marine pathogenic microorganism are still traditional not yet systemic and rapid, so as to routine monitoring is difficult. Therefore, it is indispensable to get methods which is systemic, special, simple; rapid and exact. In this paper, methods to detecting Vibrio parahaemolyticus which was the pathogen of the red body disease of Litopenaeus vannamei, hepatitis A viruses in seawater and shellfish, pathogenic microorganism in sediment nearby sewage inlet was studied and applied, and PCR-DGGE was applied for analysis of pathogenic microorganism diversity in sediment for the first time.
In this study, pathogen of the red body disease of Litopenaeus vannamei was Vibrio parahaemolyticus by dominant bacteria separating, regressing infection assy, biochemical identification and 16S rRNA homology analysis. Three kinds of methods to detecting Vibrio parahaemolyticus were finished by use of Vibrio parahaemolyticus antigen and rabbit anti- Vibrio parahaemolyticus IgG, namely iELISA, Dot-ELISA, IFA. Three kinds methods were stabile and special; they had different sensitivity, IFA was highest and iELISA was higher and Dot-ELISA was lowests; they were all rapid, finished time were 2h, 9h and 7h respectively; they need different apparatus, Dot-ELISA is fittest for locally survey due to not requiring more apparatus. Otherwise, PCR for detecting Vibrio parahaemolyticus was developed on the basis of the primer designed by
近年来，随着我国沿海城乡人口的日益增长，城乡生活污水的排海量亦逐年增加，致使生活污水中大量的人类病原微生物随之进入海洋环境，分布于海水、生物体以及沉积物当中。它们不仅对海洋生物可造成巨大危害，进而影响海洋生态系统的稳定性，而且还可通过与海水、沉积物等的接触，或海产品的食用而危害人体健康。1988年上海因食用毛蚶引发甲型肝炎大流行，即是海水中甲肝病毒(Hepatitis A virus，HAV)通过贝类进入食物链导致人疾病爆发的典型实例。排污入海口处沉积物中细菌的存在，对人体健康存在着一定的潜在威胁，亦有必要对排污入海口处沉积物中细菌进行检测。另外，目前病害已经成为制约海水养殖业健康发展的瓶颈，凡纳滨对虾是世界养殖虾类产量最高的三大种类之一，近年来频频受到红体病的侵袭，对其进行病原微生物的分离鉴定及相应检测方法的建立是十分必要的。
在我国，对海洋病原微生物的检测一直沿用传统的检测手段，缺少系统、快速的检测方法，因而难以开展常规监测。因此，建立系统、特异、简便、快速、准确的海洋病原微生物检测方法是十分必要的。本研究选定凡纳滨对虾红体病病原菌—副溶血弧菌(Vibrio parahaemolyticus)、海水中以及贝类体内的甲肝病毒、排污入海口处沉积物中细菌作为研究代表，从实际应用出发，初步系统地建立起高效、灵敏的检测方法。并在我国首次将聚合酶链式反应-变性梯度凝胶电泳(Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis，PCR-DGGE)的方法应用于海洋沉积物中病原微生物多样性的分析。
本研究通过优势菌分离、回归感染实验、生理生化特性以及16S rRNA基因分析，将从患红体病的凡纳滨对虾体内分离到的病原菌确定为副溶血弧菌。以副溶血弧菌为抗原，免疫新西兰兔获得高免血清，建立了检测副溶血弧菌的三种免疫学检测方法，分别为间接酶联免疫吸附试验法(Indirect Enzyme-linked Immunosorbent Assay，iELISA)、斑点ELISA(Dot-ELISA)方法、间接荧光抗体(Indirect Fluorescent Antibody，IFA)检测方法。三种免疫学检测方法均具有较高的稳定性和特异性；灵敏性从高到低排列依次为：IFA、iELISA、Dot-ELISA；检测所用时间均很快速，分别为2h、9h、7h；所需仪器设备方面，Dot-ELISA方法所需仪器设备最简单，最适于现场调查应用。另外，根据副溶血弧菌耐热直接溶血毒素(TDH)的保守阅读框pR72H基因序列设计引物，采用热裂解法提取副溶血弧菌的DNA，建立了PCR方法。所建立的PCR方法灵敏度较高，检出限能够达到90～100CFU；特异性较强，能够在2h内完成检测。
采用吸附-洗脱的原理，建立了从海水中浓缩甲肝病毒的方法；采用氯仿去除脂蛋白、聚乙二醇浓缩的原理，建立了从贝类体内浓缩甲肝病毒的方法；在此基础之上，建立了检测甲肝病毒的“RT-PCR扩增—聚丙烯酰胺凝胶电泳—银染色方法”。将该方法应用到辽东湾重点
There are many pathogen microorganism in ocean, they can infect halobios and human. Marine pathogen microorganism pose a significant threat to seawater breed aquatics industry and human health, consequently marine pathogen microorganism monitoring have a important application spread potential for evaluating seafood safety and forecasting disease and lessening correlative economy loss.
Recently, with the coastal city population increasing, pathogen with the life sewage pouring sea increased, Then, pathogen distributed in sea water, sediment and organisms, so they influence marine ecosystem stability by hurt halobios, and threaten human body health by contacting seawater or sediment and taking seafood. In 1988, hepatitis A crazed on account of eated shellfish. Pathogenic microorganism in sediment nearby sewage inlet can harm human body. Otherwise, disease have already become a bottle-neck of restricting marine breed aquatics industry development, Litopenaeus vannamei is one of the three important kinds of breeding shrimp in the world, recently often affected by red body disease. So it is indispensable to monitoring pathogenic microorganism.
In china, methods to detecting marine pathogenic microorganism are still traditional not yet systemic and rapid, so as to routine monitoring is difficult. Therefore, it is indispensable to get methods which is systemic, special, simple; rapid and exact. In this paper, methods to detecting Vibrio parahaemolyticus which was the pathogen of the red body disease of Litopenaeus vannamei, hepatitis A viruses in seawater and shellfish, pathogenic microorganism in sediment nearby sewage inlet was studied and applied, and PCR-DGGE was applied for analysis of pathogenic microorganism diversity in sediment for the first time.
In this study, pathogen of the red body disease of Litopenaeus vannamei was Vibrio parahaemolyticus by dominant bacteria separating, regressing infection assy, biochemical identification and 16S rRNA homology analysis. Three kinds of methods to detecting Vibrio parahaemolyticus were finished by use of Vibrio parahaemolyticus antigen and rabbit anti- Vibrio parahaemolyticus IgG, namely iELISA, Dot-ELISA, IFA. Three kinds methods were stabile and special; they had different sensitivity, IFA was highest and iELISA was higher and Dot-ELISA was lowests; they were all rapid, finished time were 2h, 9h and 7h respectively; they need different apparatus, Dot-ELISA is fittest for locally survey due to not requiring more apparatus. Otherwise, PCR for detecting Vibrio parahaemolyticus was developed on the basis of the primer designed by
引文
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