食品安全已成为一个重要的公共卫生问题,快速、准确地监测和检测食源性致病菌是控制和预防人类食源性疾病的最有效方法之一。由于食品基质的复杂性、细菌的多样性及不同生长和复制特性,给食源性致病菌检测带来了重大挑战。传统微生物检...食品安全已成为一个重要的公共卫生问题,快速、准确地监测和检测食源性致病菌是控制和预防人类食源性疾病的最有效方法之一。由于食品基质的复杂性、细菌的多样性及不同生长和复制特性,给食源性致病菌检测带来了重大挑战。传统微生物检测方法耗时费力,不足以满足不可培养活菌细胞和现场快速食品检测的要求。因此,近年来针对食源性致病菌开发了各种免疫检测技术,比传统方法更加灵敏、简单和高效,具有广阔的应用前景。该文结合食源性致病菌亚致死损伤、活的不可培养(viable but non-culturable,VBNC)和休眠3种代谢状态的生物学特征及抗体的类型和特点,综述了当前用于食源性致病菌常见的免疫技术的检测原理、优缺点和应用,并对现有方法的局限性和未来发展方向进行讨论,以期为食源性致病菌免疫检测技术的开发和利用提供参考。展开更多
Objective Development of monoclonal antibody based sandwich enzyme linked immunosorbant assay （sELISA） for rapid detection of Salmonella enterica serovar typhi （S. typhi） from food and water samples and optimizati...Objective Development of monoclonal antibody based sandwich enzyme linked immunosorbant assay （sELISA） for rapid detection of Salmonella enterica serovar typhi （S. typhi） from food and water samples and optimization of enrichment procedures for use with the developed sELISA to increase the detection sensitivity of the assay. Methods Spleen cells from BALB/c mice immunized with flagellin （H=d） antigen of S. typhi were fused with Sp2/0 myeloma cells. The hybridoma cell line specific to H=d antigen was established, characterized and ascites raised against one of these clones. The hyperimmune serum to flagellin antigen was raised in New Zealand White rabbits. An sELISA was developed using polyclonal antibody as capture and monoclonal antibody as detection antibody. To design the efficient culture strategies for use with the sELISA, different pre-enrichment and enrichment broths were evaluated. The media included buffered peptone water （BPW） and brain heart infusion broth for pre-enrichment and selenite F broth and Rappaport-Vassiliadis broth as enrichment broths. The developed sELISA with preceding enrichment step in BPW （Enrichment-ELISA） was evaluated in various food samples artificially inoculated with S. typhi bacteria. Various food （30） and water （35） samples collected from field were also tested by Enrichment-ELISA and culture method. Results Out of four specific clones to H=d antigen, one clone （# 2/56, IgG2a isotype） was used in sELISA. The sELISA had the detection limit of 10^4-10^5 cfu of S. typhi. Of the various broths used with sELISA, BPW was found to yield maximum ELISA values. Enrichment-ELISA, when tested in artificially inoculated food samples, generally, could detect 10^2 S. typhi cfu/mL within 10 h from various food rinses （meat, vegetable） and milk samples. After overnight enrichment in BPW, as less as 2 bacteria per 10 mL of milk, meat rinse, and chicken rinse could be detected. Only one of the field samples （water） gave false positive result by Enrichment-ELISA. Conclusion In comparison to culture, the Enrichment-ELISA is a rapid, sensitive, and specific method for detection of S. typhi from food or water samples. This method may be used as rapid screening procedure for environmental monitoring during outbreak situation.展开更多
Background:Colistin(polymyxin E)is a kind of peptide antibiotic which has been approved in animal production for the purposes of disease prevention,treatment,and growth promotion.However,the wide use of colistin in an...Background:Colistin(polymyxin E)is a kind of peptide antibiotic which has been approved in animal production for the purposes of disease prevention,treatment,and growth promotion.However,the wide use of colistin in animal feed may accelerate the spread of colistin-resistance gene MCR-1 from animal production to human beings,and its residue in animal-origin food may also pose serious health hazards to humans.Thus,it is necessary to develop corresponding analytical methods to monitor the addition of colistin in animal feed and the colistin residue in animal-origin food.Results:A one-step enzyme-linked immunosorbent assay(ELISA)and a lateral flow immunochromatographic assay(LFIA)for colistin were developed based on a newly developed monoclonal antibody.The ELISA showed a 50%inhibition value(IC50)of 9.7 ng/m L with assay time less than 60 min,while the LFIA had a strip reader-based detection limit of 0.87 ng/m L in phosphate buffer with assay time less than 15 min.For reducing the non-specific adsorption of colistin onto sample vial,the components of sample extraction solution were optimized and proved to greatly improve the assay accuracy.The spiked recovery experiment showed that the recoveries of colistin from feed,milk and meat samples were in the range of 77.83%to 113.38%with coefficient of variations less than 13%by ELISA analysis and less than 18%by LFIA analysis,respectively.Furthermore,actual sample analysis indicated that the two immunoassays can produce results consistent with instrumental analysis.Conclusions:The developed assays can be used for rapid qualitative or quantitative detection of colistin in animal feed and food.展开更多
文摘食品安全已成为一个重要的公共卫生问题,快速、准确地监测和检测食源性致病菌是控制和预防人类食源性疾病的最有效方法之一。由于食品基质的复杂性、细菌的多样性及不同生长和复制特性,给食源性致病菌检测带来了重大挑战。传统微生物检测方法耗时费力,不足以满足不可培养活菌细胞和现场快速食品检测的要求。因此,近年来针对食源性致病菌开发了各种免疫检测技术,比传统方法更加灵敏、简单和高效,具有广阔的应用前景。该文结合食源性致病菌亚致死损伤、活的不可培养(viable but non-culturable,VBNC)和休眠3种代谢状态的生物学特征及抗体的类型和特点,综述了当前用于食源性致病菌常见的免疫技术的检测原理、优缺点和应用,并对现有方法的局限性和未来发展方向进行讨论,以期为食源性致病菌免疫检测技术的开发和利用提供参考。
文摘Objective Development of monoclonal antibody based sandwich enzyme linked immunosorbant assay （sELISA） for rapid detection of Salmonella enterica serovar typhi （S. typhi） from food and water samples and optimization of enrichment procedures for use with the developed sELISA to increase the detection sensitivity of the assay. Methods Spleen cells from BALB/c mice immunized with flagellin （H=d） antigen of S. typhi were fused with Sp2/0 myeloma cells. The hybridoma cell line specific to H=d antigen was established, characterized and ascites raised against one of these clones. The hyperimmune serum to flagellin antigen was raised in New Zealand White rabbits. An sELISA was developed using polyclonal antibody as capture and monoclonal antibody as detection antibody. To design the efficient culture strategies for use with the sELISA, different pre-enrichment and enrichment broths were evaluated. The media included buffered peptone water （BPW） and brain heart infusion broth for pre-enrichment and selenite F broth and Rappaport-Vassiliadis broth as enrichment broths. The developed sELISA with preceding enrichment step in BPW （Enrichment-ELISA） was evaluated in various food samples artificially inoculated with S. typhi bacteria. Various food （30） and water （35） samples collected from field were also tested by Enrichment-ELISA and culture method. Results Out of four specific clones to H=d antigen, one clone （# 2/56, IgG2a isotype） was used in sELISA. The sELISA had the detection limit of 10^4-10^5 cfu of S. typhi. Of the various broths used with sELISA, BPW was found to yield maximum ELISA values. Enrichment-ELISA, when tested in artificially inoculated food samples, generally, could detect 10^2 S. typhi cfu/mL within 10 h from various food rinses （meat, vegetable） and milk samples. After overnight enrichment in BPW, as less as 2 bacteria per 10 mL of milk, meat rinse, and chicken rinse could be detected. Only one of the field samples （water） gave false positive result by Enrichment-ELISA. Conclusion In comparison to culture, the Enrichment-ELISA is a rapid, sensitive, and specific method for detection of S. typhi from food or water samples. This method may be used as rapid screening procedure for environmental monitoring during outbreak situation.
基金financially supported by Beijing Advanced Innovation Center for Food Nutrition and Human HealthBasic Research Program of Science and Technology(2014FY111000).
文摘Background:Colistin(polymyxin E)is a kind of peptide antibiotic which has been approved in animal production for the purposes of disease prevention,treatment,and growth promotion.However,the wide use of colistin in animal feed may accelerate the spread of colistin-resistance gene MCR-1 from animal production to human beings,and its residue in animal-origin food may also pose serious health hazards to humans.Thus,it is necessary to develop corresponding analytical methods to monitor the addition of colistin in animal feed and the colistin residue in animal-origin food.Results:A one-step enzyme-linked immunosorbent assay(ELISA)and a lateral flow immunochromatographic assay(LFIA)for colistin were developed based on a newly developed monoclonal antibody.The ELISA showed a 50%inhibition value(IC50)of 9.7 ng/m L with assay time less than 60 min,while the LFIA had a strip reader-based detection limit of 0.87 ng/m L in phosphate buffer with assay time less than 15 min.For reducing the non-specific adsorption of colistin onto sample vial,the components of sample extraction solution were optimized and proved to greatly improve the assay accuracy.The spiked recovery experiment showed that the recoveries of colistin from feed,milk and meat samples were in the range of 77.83%to 113.38%with coefficient of variations less than 13%by ELISA analysis and less than 18%by LFIA analysis,respectively.Furthermore,actual sample analysis indicated that the two immunoassays can produce results consistent with instrumental analysis.Conclusions:The developed assays can be used for rapid qualitative or quantitative detection of colistin in animal feed and food.
