热加工食品和水产品中致病菌检测技术体系建立
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摘要
热加工食品和水产品中多种致病菌是威胁人类食品安全和身体健康的重要因素。目前国内外对热加工食品和水产品中致病菌的检测方法还多停留在检测效率低、目标单一的水平上。本研究采用DNA聚合酶链反应(PCR)结合变性高效液相色谱(denaturing highperformance liquid chromatography,DHPLC)高通量核酸检测方法,对热加工食品和水产品中多种致病菌的高通量检测、快速分种、毒素编码基因筛选、致病菌突变株检测等高通量检测技术进行了系统研究,搭建相应技术平台,获得了以下试验结果:
1、在国内外首次应用多重PCR结合DHPLC在非变性条件下分离DNA片段的原理,通过优化多重PCR和DHPLC分析条件,建立了针对热加工食品中常见的蜡样芽胞杆菌、肉毒梭状芽胞杆菌、产气荚膜梭状芽胞杆菌等3种芽胞菌及其毒素编码基因的DHPLC高通量检测技术;在国内外首次建立了PCR-DHPLC非变性条件检测热加工食品中致病菌的特异性基因检测平台。弥补了芽胞菌检测因普通培养鉴定周期长、试剂质量和生化反应不稳定而导致的误检、漏检等问题。
2、在国内外首先进行了通用引物PCR-DHPLC非变性条件下,乳酸杆菌属检测方法的研究,解决了热加工食品中常见的乳酸杆菌属PCR-DHPLC法高通量检测技术难题,建立了乳酸杆菌属的PCR-DHPLC快速检测技术。为在食品安全和工业生产检测中对乳酸杆菌属的快速检测提供了科学、高效、快捷的检测手段。
3、解决了乳酸杆菌属的各个菌种间凭借DNA序列碱基差异而达到分类鉴别的高通量分种鉴定技术难题。采用DHPLC部分变性条件下分型鉴定方法,在国内外首先建立了乳酸杆菌属9个分类菌种的高通量分种鉴定方法,真正达到了一次增菌、一次检测,即可同时鉴别乳酸杆菌属9个分类菌种的分种鉴定的科学、高效、快捷的鉴定目的。部分变性条件下DHPLC检测乳酸杆菌9个菌种分型鉴定方法具有创新性。
4、本研究应用多重PCR结合DHPLC,解决了水产品中多种常见致病菌高通量快速检测技术难题,建立了从多种目标菌一次复合增菌、一次PCR-DHPLC完成检测的快速检测技术。以水产品中常见致病菌,霍乱弧菌、副溶血性弧菌、创伤弧菌、溶藻弧菌、拟态弧菌、肠出血性大肠埃希氏菌0157:H7、沙门氏菌、单核细胞增生李斯特氏菌、金黄色葡萄球菌等9种致病菌为研究对象,进行非变性条件下的九重PCR-DHPLC检测技术研究。在国内外首次建立了多重PCR-DHPLC非变性条件下,检测水产品中9种致病菌的高通量检测方法,建立了PCR-DHPLC检测水产品中致病菌的特异性基因检测平台。并进行了霍乱弧菌突变株的筛选检测,在进行水样检测时,分离并鉴定出霍乱弧菌突变株。
本研究成果实现了在一次操作过程中同时检测水产品中9种致病菌,使得致病菌的检测时间、检测数量和检测水平发生了很大的飞跃,从而达到对水产品中上述9种致病菌快速、准确、高灵敏检测的目的。
综上所述,本课题在国内外首次建立了热加工食品和水产品中多种致病菌的DHPLC精准、高通量检测技术,细菌快速分种鉴定技术,毒素编码基因筛选技术。并使其检测技术标准化,在研究成果基础上,制定了中华人民共和国出入境检验检疫行业标准;而且研究成果已经转化为检测试剂盒,申报了国家发明专利,具有重要的研究意义和应用价值。
The kinds of pathogenic bacterium in heat-processed food and aquatic products are the fatal factors to threat the food safety and physical health of human being.Currently,the domestic and foreign inspection method to the heat-processed food and aquatic products mostly be in the lever of low efficiency and single target.This research has materialized the system study on the high throughput technology like the high throughput inspection,toxin code gene filter,quick classifying,pathogenic bacterium mutant cluster inspection,and identification diagnosis of kinds of pathogenic bacterium of heat-processed food and aquatic products through PCR combing high throughput nuclear acid inspection method of denaturing high performance liquid chromatography,DHPLC and get the following test results by building the corresponding technology platform.
1.The high throughput technology of three kinds of foodborne pathogenic bacterium like the common bacillus cereus,Clostridium bobulinm and cl.perfringens and its toxin code gene which aim at the heat-processed food has been developed with the application of PCR combining with the principal of separating DNA fragment under non-denaturing condition by denaturing high performance liquid chromatography,DHPLC and through the optimization of multi-PCR and DHPLC analysis conditions.As well as the specific gene inspection platform to detect the pathogenic bacterium of heat-processed food through PCR-DHPLC inspection has been established.The researched and developed multi PCR-DHPLC to inspect the three kinds of sporogenes under non-denaturing condition is creative.
2.The high throughput inspection technical difficult of common Lactobacillus PCR-DHPLC in heat-processed food has been solved and the Lactobacillus quick inspection technology has been developed.With the application of PCR-DHPLC non denaturing analysis condition,based on the inspection level of genus,the food lactobillus inspection method has been developed,which remedies the problems of error and leak inspection caused by long period,unstable reagent quality and biochemical reaction of common culture identifying,and provides a scientific,high efficient and quick inspection medium to the lactobillus quick inspection during the food safety and industry production inspection.
3.The high throughput strain-classifying technical difficult to reach classification identification between lactobillus strain relying on the difference of general primer and DNA nucleotide base, the high throughput strain-classifying identifying method of the nine classified strain of lactobillus has been developed with the application of high throughput type-classifying identifying method under DHPLC partial denaturing condition and aiming at the nine classified strains of lactobillus,the scientific,high efficient and quick identifying purpose of the type-classifying of nine classified strains of lactobillus can be identified at one time by one enrichment,one inspection has been really reached.The type-classifying identification technology of nine strains of lactobillus by DHPLC inspection under partial denaturing condition is creative.
4.The technical difficult of high throughput quick inspection to the common pathogenic bacterium of aquatic products has been solved and the high throughput quick inspection technology of completing the inspection from kinds of target bacterium,one time compound enrichment and one time PCR-DHPLC has been created.Taking the nine microorganisms like the common pathogenic bacterium,comma bacillus,V.parahemolyticus,V.vulnificus,V. alginnolyficus,V.mimicus,enterohemorrhagic escherichia coli 0157:H7,salmonella,Listeria monocytogenes and staphyloccocus aureus rosenbach in aquatic products as research target, this reach has developed the PCR-DHPLC quick inspection under non-denaturing condition and the specific gene inspection platform to detect the pathogenic bacterium of heat-processed food through PCR-DHPLC inspection,and carried out the filter inspection to of comma bacillus mutant cluster.Nine kinds of pathogenic bacterium of aquatic products are inspected together at one operation process has made the inspection time,quantity and level greatly leaped.Therefore to reach the quick,accurate and high sensitive inspection purpose to the abovementioned nine kinds of microorganism.The researched and developed multi PCR-DHPLC to inspect the nine kinds of common pathogenic bacterium of aquatic products under non-denaturing condition is creative.
Above all,this research has firstly developed the DHPLC accurate and high throughput inspection technology of kinds of pathogenic bacterium of heat-processed food and aquatic products,quick type-classifying identifying technology of pathogenic bacterium,toxin code gene filter technology as well as filled in the vacancy of this research.Meanwhile,it has standardized the inspection technology,and formulated the sector standard of Entry-exit inspection and quarantine of P.R.C based on the research basis;and the research achievement has been transformed and declared the state invention patent with important research meaning and application value.
1、在国内外首次应用多重PCR结合DHPLC在非变性条件下分离DNA片段的原理,通过优化多重PCR和DHPLC分析条件,建立了针对热加工食品中常见的蜡样芽胞杆菌、肉毒梭状芽胞杆菌、产气荚膜梭状芽胞杆菌等3种芽胞菌及其毒素编码基因的DHPLC高通量检测技术;在国内外首次建立了PCR-DHPLC非变性条件检测热加工食品中致病菌的特异性基因检测平台。弥补了芽胞菌检测因普通培养鉴定周期长、试剂质量和生化反应不稳定而导致的误检、漏检等问题。
2、在国内外首先进行了通用引物PCR-DHPLC非变性条件下,乳酸杆菌属检测方法的研究,解决了热加工食品中常见的乳酸杆菌属PCR-DHPLC法高通量检测技术难题,建立了乳酸杆菌属的PCR-DHPLC快速检测技术。为在食品安全和工业生产检测中对乳酸杆菌属的快速检测提供了科学、高效、快捷的检测手段。
3、解决了乳酸杆菌属的各个菌种间凭借DNA序列碱基差异而达到分类鉴别的高通量分种鉴定技术难题。采用DHPLC部分变性条件下分型鉴定方法,在国内外首先建立了乳酸杆菌属9个分类菌种的高通量分种鉴定方法,真正达到了一次增菌、一次检测,即可同时鉴别乳酸杆菌属9个分类菌种的分种鉴定的科学、高效、快捷的鉴定目的。部分变性条件下DHPLC检测乳酸杆菌9个菌种分型鉴定方法具有创新性。
4、本研究应用多重PCR结合DHPLC,解决了水产品中多种常见致病菌高通量快速检测技术难题,建立了从多种目标菌一次复合增菌、一次PCR-DHPLC完成检测的快速检测技术。以水产品中常见致病菌,霍乱弧菌、副溶血性弧菌、创伤弧菌、溶藻弧菌、拟态弧菌、肠出血性大肠埃希氏菌0157:H7、沙门氏菌、单核细胞增生李斯特氏菌、金黄色葡萄球菌等9种致病菌为研究对象,进行非变性条件下的九重PCR-DHPLC检测技术研究。在国内外首次建立了多重PCR-DHPLC非变性条件下,检测水产品中9种致病菌的高通量检测方法,建立了PCR-DHPLC检测水产品中致病菌的特异性基因检测平台。并进行了霍乱弧菌突变株的筛选检测,在进行水样检测时,分离并鉴定出霍乱弧菌突变株。
本研究成果实现了在一次操作过程中同时检测水产品中9种致病菌,使得致病菌的检测时间、检测数量和检测水平发生了很大的飞跃,从而达到对水产品中上述9种致病菌快速、准确、高灵敏检测的目的。
综上所述,本课题在国内外首次建立了热加工食品和水产品中多种致病菌的DHPLC精准、高通量检测技术,细菌快速分种鉴定技术,毒素编码基因筛选技术。并使其检测技术标准化,在研究成果基础上,制定了中华人民共和国出入境检验检疫行业标准;而且研究成果已经转化为检测试剂盒,申报了国家发明专利,具有重要的研究意义和应用价值。
The kinds of pathogenic bacterium in heat-processed food and aquatic products are the fatal factors to threat the food safety and physical health of human being.Currently,the domestic and foreign inspection method to the heat-processed food and aquatic products mostly be in the lever of low efficiency and single target.This research has materialized the system study on the high throughput technology like the high throughput inspection,toxin code gene filter,quick classifying,pathogenic bacterium mutant cluster inspection,and identification diagnosis of kinds of pathogenic bacterium of heat-processed food and aquatic products through PCR combing high throughput nuclear acid inspection method of denaturing high performance liquid chromatography,DHPLC and get the following test results by building the corresponding technology platform.
1.The high throughput technology of three kinds of foodborne pathogenic bacterium like the common bacillus cereus,Clostridium bobulinm and cl.perfringens and its toxin code gene which aim at the heat-processed food has been developed with the application of PCR combining with the principal of separating DNA fragment under non-denaturing condition by denaturing high performance liquid chromatography,DHPLC and through the optimization of multi-PCR and DHPLC analysis conditions.As well as the specific gene inspection platform to detect the pathogenic bacterium of heat-processed food through PCR-DHPLC inspection has been established.The researched and developed multi PCR-DHPLC to inspect the three kinds of sporogenes under non-denaturing condition is creative.
2.The high throughput inspection technical difficult of common Lactobacillus PCR-DHPLC in heat-processed food has been solved and the Lactobacillus quick inspection technology has been developed.With the application of PCR-DHPLC non denaturing analysis condition,based on the inspection level of genus,the food lactobillus inspection method has been developed,which remedies the problems of error and leak inspection caused by long period,unstable reagent quality and biochemical reaction of common culture identifying,and provides a scientific,high efficient and quick inspection medium to the lactobillus quick inspection during the food safety and industry production inspection.
3.The high throughput strain-classifying technical difficult to reach classification identification between lactobillus strain relying on the difference of general primer and DNA nucleotide base, the high throughput strain-classifying identifying method of the nine classified strain of lactobillus has been developed with the application of high throughput type-classifying identifying method under DHPLC partial denaturing condition and aiming at the nine classified strains of lactobillus,the scientific,high efficient and quick identifying purpose of the type-classifying of nine classified strains of lactobillus can be identified at one time by one enrichment,one inspection has been really reached.The type-classifying identification technology of nine strains of lactobillus by DHPLC inspection under partial denaturing condition is creative.
4.The technical difficult of high throughput quick inspection to the common pathogenic bacterium of aquatic products has been solved and the high throughput quick inspection technology of completing the inspection from kinds of target bacterium,one time compound enrichment and one time PCR-DHPLC has been created.Taking the nine microorganisms like the common pathogenic bacterium,comma bacillus,V.parahemolyticus,V.vulnificus,V. alginnolyficus,V.mimicus,enterohemorrhagic escherichia coli 0157:H7,salmonella,Listeria monocytogenes and staphyloccocus aureus rosenbach in aquatic products as research target, this reach has developed the PCR-DHPLC quick inspection under non-denaturing condition and the specific gene inspection platform to detect the pathogenic bacterium of heat-processed food through PCR-DHPLC inspection,and carried out the filter inspection to of comma bacillus mutant cluster.Nine kinds of pathogenic bacterium of aquatic products are inspected together at one operation process has made the inspection time,quantity and level greatly leaped.Therefore to reach the quick,accurate and high sensitive inspection purpose to the abovementioned nine kinds of microorganism.The researched and developed multi PCR-DHPLC to inspect the nine kinds of common pathogenic bacterium of aquatic products under non-denaturing condition is creative.
Above all,this research has firstly developed the DHPLC accurate and high throughput inspection technology of kinds of pathogenic bacterium of heat-processed food and aquatic products,quick type-classifying identifying technology of pathogenic bacterium,toxin code gene filter technology as well as filled in the vacancy of this research.Meanwhile,it has standardized the inspection technology,and formulated the sector standard of Entry-exit inspection and quarantine of P.R.C based on the research basis;and the research achievement has been transformed and declared the state invention patent with important research meaning and application value.
引文
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