高通量基因芯片在压载水中检测致病菌的应用
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摘要
在口岸压载水检测中发现有多种食源性致病菌的存在,给公共卫生和口岸生态环境带来极大风险。目前对食源性致病菌的检测主要是通过细菌的培养及生化鉴定、荧光PCR等方法,但是不能同时检测多种致病菌。本研究针对一种能检测12种食源致病菌的芯片进行了应用研究,用参比菌株和实验室自分离的菌株检测该芯片的特异性,结果表明该芯片的特异性良好,在所检测的菌株之间无交叉反应。在DNA水平上可以检测到110~7 000 copy的DNA分子,在菌裂解液的水平上可以检测到104~105copy数目的菌体,样品添加实验证明1 cfu/m L的菌浓度就可以通过增菌后芯片检测到。该芯片可鉴别金黄色葡萄球菌、阪崎肠杆菌、肠出血性大肠杆菌O157∶H7、霍乱弧菌、志贺氏痢疾杆菌、空肠弯曲杆菌、单增李斯特菌、副溶血性弧菌、弯曲肠杆菌、沙门氏菌、志贺氏菌、蜡样芽孢杆菌和产气荚膜杆菌等。研究证明该芯片的灵敏度比PCR水平略低,但是其具备高通量、快速、准确,简单易操作等优点,可以应用于口岸压载水中携带食源致病菌的快速检测。
The potential risk of foodborne pathogens in the ballast water to act as a major vector for public health and port ecological environment has long been recognised. At present,the foodborne pathogens is detected by culture and biochemical identification,Real-time PCR and other methods,which can not detect multiple pathogenic bacteria. This study focused on the application of a high-throughput DNA microarray chip p that can detect 12 kinds of foodborne pathogens. By the chip,every control strain of pathogen can be specifically detected without inferring with other organism. In the level of DNA molecule,the chip can detect the minimal 110 copies for the most sensitive Shigella,and the maximal 7000 copies for Staphylococcus aureus. Pathogens with copies of 104-105,can be detected directly by the thermal lysis bacteria. ballast water supplied with five kinds of pathogens of 1 cfu/ml can be detected in 2 hours followed with several hours of culture. the method for the simultaneous foodborne pathogens besides Staphylococcus aureus,Salmonella spp,and Shigella spp, Listeria monocytogenes, Escherichia coli O157: H7, Vibrio parahaemolyticus,Vibrio cholera,Bacillus cereus Clostridium perfringens,Campylobacter,Cronobacter sakazakii,Shigella Castellani could be detected. The minimum limit of the chip is higher than the PCR methods,while the chip of this study was high-throughput,rapid and efficient in the detection,which could be a useful method for the simultaneous detection of foodborne pathogens in the ballast water
The potential risk of foodborne pathogens in the ballast water to act as a major vector for public health and port ecological environment has long been recognised. At present,the foodborne pathogens is detected by culture and biochemical identification,Real-time PCR and other methods,which can not detect multiple pathogenic bacteria. This study focused on the application of a high-throughput DNA microarray chip p that can detect 12 kinds of foodborne pathogens. By the chip,every control strain of pathogen can be specifically detected without inferring with other organism. In the level of DNA molecule,the chip can detect the minimal 110 copies for the most sensitive Shigella,and the maximal 7000 copies for Staphylococcus aureus. Pathogens with copies of 104-105,can be detected directly by the thermal lysis bacteria. ballast water supplied with five kinds of pathogens of 1 cfu/ml can be detected in 2 hours followed with several hours of culture. the method for the simultaneous foodborne pathogens besides Staphylococcus aureus,Salmonella spp,and Shigella spp, Listeria monocytogenes, Escherichia coli O157: H7, Vibrio parahaemolyticus,Vibrio cholera,Bacillus cereus Clostridium perfringens,Campylobacter,Cronobacter sakazakii,Shigella Castellani could be detected. The minimum limit of the chip is higher than the PCR methods,while the chip of this study was high-throughput,rapid and efficient in the detection,which could be a useful method for the simultaneous detection of foodborne pathogens in the ballast water
引文
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[9]畅修军,刘桂华,孔祥云,等.2002-2007年吉林省食品中食源性致病菌监测结果分析[J].中国国境卫生检疫杂志,2008,18(7):1400-1402.CHANG X J,LIU G H,KONG X Y,et al.Monitoring results of foodborne pathogens in food in Jilin Province in2002-2007[J].Chinese Frontier Health Quarantine,2008,18(7):1400-1402.
[10]龚霄,陈盼.革兰氏阳性食源性致病菌[J].肉类研究,2008(5):53-60.GONG X,CHEN P.G+Food-borne Pathogens Bacteria[J].MEAT RESEARCH,2008(5):53-60.
[11]赵新,王永,兰青阔,等.四种食源性致病菌多重PCR检测技术的研究及应用[J].天津农业科学,2009,15(6):6-8.ZHAO X,WANG Y,LAN Q K,et al.Research and application of multiplex PCR for four foodborne pathogens[J].Tianjin Agricultural Sciences,2009,15(6):6-8.
[12]郝玉莲,孙皆宜,李艾,等.正交优化多重PCR反应体系检测3种食源性致病菌的研究[J].安徽农业科学,2010,38(2):602-605.HAO Y L,SUN J Y,LI A,et al.Application of Orthogonal Design to Optimize Multiplex PCR Reaction System for Detection of Three Food-borne Bacteria Pathogens[J].Journal of Anhui Agri.Sci.,2010,38(2):602-605.
[13]KAWASAKI S,HORIKOSHI N,OKADA Y,et al.Multiplex PCR for simultaneous detection of Salmonella spp.,Listeria monocytogenes,and Escherichia coli O157:H7 in meat samples[J].Journal of Food Protect,2005,68(3):551-556.
[2]贾俊涛,赵丽青,李伟才,等.入境船舶压载水中细菌调查的多维尺度分析[J].中国国境卫生检疫杂志,2009(1):31-34.JIA J T,ZHAO L Q,LI W C,et al.Multidimensional scaling analysis of bacteria in ballast water from entry ships[J].Chinese Frontier Health Quarantine,2009(1):31-34.
[3]李云峰,吴刚,薛芳,宋锋林,蔡鹏.16S r DNA序列分析法在国际船舶压载舱沉积物病原微生物检测中的应用[J].中国国境卫生检疫杂志,2009,(6):475-477.LI Y F,WU G,XUE F,et al.Application of 16S r DNA Sequence Analysis Method in Pathogens Detection from Ballast Sediments of International Vessels[J].Chinese Frontier Health Quarantine,2009,(6):475-477.
[4]李春丽,刘兵,周君,等.宁波港压载水的细菌多样性研究[J].生态科学,2012,(06):636-644+670.LI C L,LIU B,ZHOU J,et al.Study on genetic diversity of bacteria in ballast water from Ningbo port[J].Ecological Science,2012,(06):636-644+670.
[5]RUIZ G M,RAWLINGS T K,DOBBS F C,et al.Global spread of microorganisms by ships[J].Nature,2000,408:49-50.
[6]MCCARTHY S A,KHAMBATY F M.International dissemination of epidemic Vibrio cholera by cargo shipballast and other nonpotable waters[J].Appl Environ Microbiol,1994,60(7):2597-601.
[7]张红波.我国食品安全现状分析及其对策[J].中国安全科学学报,2004,14(1):15-17.ZHANG H B.Analysis on Current Situation of Food Safety in China and Its Countermeasures[J].China Safety Science Journal,2004,14(1):15-17.
[8]张芳,马国柱,潘立,等.陕西省2002-2006年食源性致病菌污染状况[J].中国公共卫生,2008,24(2):222-224.ZHANG F,MA G Z,PAN L,et al.Surveillance and analysis for food-borne pathogens in retailed foods in Shaanxi province,2002-2006[J].Chin J Public Health,2008,24(2):222-224.
[9]畅修军,刘桂华,孔祥云,等.2002-2007年吉林省食品中食源性致病菌监测结果分析[J].中国国境卫生检疫杂志,2008,18(7):1400-1402.CHANG X J,LIU G H,KONG X Y,et al.Monitoring results of foodborne pathogens in food in Jilin Province in2002-2007[J].Chinese Frontier Health Quarantine,2008,18(7):1400-1402.
[10]龚霄,陈盼.革兰氏阳性食源性致病菌[J].肉类研究,2008(5):53-60.GONG X,CHEN P.G+Food-borne Pathogens Bacteria[J].MEAT RESEARCH,2008(5):53-60.
[11]赵新,王永,兰青阔,等.四种食源性致病菌多重PCR检测技术的研究及应用[J].天津农业科学,2009,15(6):6-8.ZHAO X,WANG Y,LAN Q K,et al.Research and application of multiplex PCR for four foodborne pathogens[J].Tianjin Agricultural Sciences,2009,15(6):6-8.
[12]郝玉莲,孙皆宜,李艾,等.正交优化多重PCR反应体系检测3种食源性致病菌的研究[J].安徽农业科学,2010,38(2):602-605.HAO Y L,SUN J Y,LI A,et al.Application of Orthogonal Design to Optimize Multiplex PCR Reaction System for Detection of Three Food-borne Bacteria Pathogens[J].Journal of Anhui Agri.Sci.,2010,38(2):602-605.
[13]KAWASAKI S,HORIKOSHI N,OKADA Y,et al.Multiplex PCR for simultaneous detection of Salmonella spp.,Listeria monocytogenes,and Escherichia coli O157:H7 in meat samples[J].Journal of Food Protect,2005,68(3):551-556.