泰国香米质量安全分子指纹检测技术研究
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摘要
本研究采用RAPD、多重荧光PCR-毛细管-SSCP、2DE、MALDI-TOF等现代分子生物学技术,开展泰国茉莉香米品种鉴定和纯度检测、香米新陈度检测、食源性致病菌高通量快速鉴定方法研究,并进行方法标准化研究,目的是加强我国进出口食品安全保障技术储备,为口岸大米的查验提供先进的科学手段,提高我国大米质量和安全的监管水平,保护消费者利益,促进大米进出口贸易的正常运行。
研究共分三个部分,第一部分是泰国茉莉香米的品种鉴定和纯度检测。采用RAPD指纹技术,通过对120条随机引物的筛选,收集泰国茉莉香米法定品种KDML105和RD15以及具有代表性的非茉莉香米共计32个水稻品种的指纹图谱数据,根据指纹数据进行品种聚类关系分析,表明RAPD指纹分类与传统分类基本一致。为了提高方法的可操作性,将RAPD方法部分转化为SCAR,建立了R2-449 RAPD和R5-1107SCAR构成的泰国茉莉香米品种鉴定和纯度检测方法。上述两个分子标记双隐性者为泰国茉莉香米。通过多次实验证实了该方法对泰国茉莉香米法定品种KDML105和RD15的分辨能力、稳定性和定量准确性。
第二部分为泰国香米新陈度鉴定方法研究。针对目前大米新陈度脂肪酸检测标准方法所存在的一些限制,采用2DGE技术,对6个不同年份的泰国香米样品进行蛋白质组分析,并对主要蛋白点进行肽指纹图谱分析,结合水稻蛋白质组数据库,对蛋白质进行鉴定。结果显示蛋白质指纹图谱呈现与香米年份一致的变化规律,碱性蛋白先降解,谷蛋白是变化最快的蛋白。2008年和2007年份样品差异明显,说明该方法具有较高的分辨率,另外由于蛋白质指纹信息量大,有利于对大米储藏的生物学过程进行分析,了解大米品质的变化规律,为粮食的生产、储存和流通过程中各个环节的质量控制提供科学指导。
第三部分为大米产品中食源性致病菌快速鉴定方法研究。针对传统的微生物鉴定方法耗时费力,以及常规PCR检测方法需要对特定微生物设计特定引物的情况,利用多重荧光PCR-CE-SSCP技术高通量、快速、自动化的特点,建立16srRNA和gyrB基因的多重荧光PCR体系,通过毛细管电泳对扩增片段的构象多态性进行分析,收集了25个标准菌株的指纹数据,采用RAPD分析方法对菌种进行聚类分析,结果显示SSCP聚类关系和传统分类基本一致。方法利用分子内标相对位置来检测信号峰,多次实验证实了SSCP信号峰具有很好的重现性。另外,由于近年来新出现阪崎杆菌日益受到关注,例如国际上多次报道了从婴幼儿米粉中检出该菌。因此研究针对42株从食品基质分离的阪崎杆菌进行了SSCP分析,为阪崎杆菌检测方法的进一步优化以及该菌的菌群特征提供了参考数据。
In this paper, modern molecular techniques including RAPD, multplex PCR-CE-SSCP, 2DE, MALDI-TOF were applied in methodology research for Thailand jasmine rice authentication, storage quality detection and foodborne pathogen identification. The further standardization of these methods would promote official supervision of imported Thailand jasmine rice, protection of consumer’s right and development of rice import and export trade.
The first part was jasmine rice identification and purity detection. Totally 32 rice cultivars including reference cultivar (KDML105 and RD15) and representative non-jasmine rice cultivars were studied. RAPD fingerprint analysis demonstrated that phylogenetic relationship of the 32 samples was generally consistent with practical kinship. In order to simplify the method for routine detection at CIQ lab, two RAPD markers (R2-449 and R5-1107) were selected for which RAPD and SCAR methods were established. Thailand jasmine rice was identified by its recessive trait of the two markers. A serious of experiments proved that the method possess high resolution, good reproducibility and accuracy.
The second part was about storage quality inspection. 2DGE was used to analyze 6 jasmine rice samples of different production year.The PMF of major peptide spots were analyzed and protein identiy were determined on the basis both of PMF and data in international rice proteomics database. The results showed that the chaning pattern of 2DE profiles were consistent with the crop year. Significant change between 2008 and 2007 corp year was observed, indicating high resolution of the method. Not only could the method be applied as a helpful complement of present standard for storage quality evaluation, but also reveal biological process during storage which is important informantion for rice production, processing and circulation.
The third part was fast identification method for foodborne pathogens. Comapred to traditional microbiological identification method and routine PCR method, multiplex PCR-CE-SSCP was characterized as faster, higher throughput and more automatic. Here 16srRNA and gyrB gene based multiplex PCR-CE-SSCP system were established and DNA fingerprint data of 25 referrence strains was collected. Phylogenetic analysis showed that the taxonomy tree was generally consistent with traditionl taxamony relationship. Signal peak was identified by relative position to inner markers instead of retaining time and good reproducibility was proved. Considering the fact that newly emergent foodborne pathogen, E.sakasaki, was frequently found in infant formula rice powder, we further analyzed 42 E.sakasaki isoltates separated from food, the data of which was believed to be helpful for optimization of present detection method and for further research of E.sakasaki population in food.
研究共分三个部分,第一部分是泰国茉莉香米的品种鉴定和纯度检测。采用RAPD指纹技术,通过对120条随机引物的筛选,收集泰国茉莉香米法定品种KDML105和RD15以及具有代表性的非茉莉香米共计32个水稻品种的指纹图谱数据,根据指纹数据进行品种聚类关系分析,表明RAPD指纹分类与传统分类基本一致。为了提高方法的可操作性,将RAPD方法部分转化为SCAR,建立了R2-449 RAPD和R5-1107SCAR构成的泰国茉莉香米品种鉴定和纯度检测方法。上述两个分子标记双隐性者为泰国茉莉香米。通过多次实验证实了该方法对泰国茉莉香米法定品种KDML105和RD15的分辨能力、稳定性和定量准确性。
第二部分为泰国香米新陈度鉴定方法研究。针对目前大米新陈度脂肪酸检测标准方法所存在的一些限制,采用2DGE技术,对6个不同年份的泰国香米样品进行蛋白质组分析,并对主要蛋白点进行肽指纹图谱分析,结合水稻蛋白质组数据库,对蛋白质进行鉴定。结果显示蛋白质指纹图谱呈现与香米年份一致的变化规律,碱性蛋白先降解,谷蛋白是变化最快的蛋白。2008年和2007年份样品差异明显,说明该方法具有较高的分辨率,另外由于蛋白质指纹信息量大,有利于对大米储藏的生物学过程进行分析,了解大米品质的变化规律,为粮食的生产、储存和流通过程中各个环节的质量控制提供科学指导。
第三部分为大米产品中食源性致病菌快速鉴定方法研究。针对传统的微生物鉴定方法耗时费力,以及常规PCR检测方法需要对特定微生物设计特定引物的情况,利用多重荧光PCR-CE-SSCP技术高通量、快速、自动化的特点,建立16srRNA和gyrB基因的多重荧光PCR体系,通过毛细管电泳对扩增片段的构象多态性进行分析,收集了25个标准菌株的指纹数据,采用RAPD分析方法对菌种进行聚类分析,结果显示SSCP聚类关系和传统分类基本一致。方法利用分子内标相对位置来检测信号峰,多次实验证实了SSCP信号峰具有很好的重现性。另外,由于近年来新出现阪崎杆菌日益受到关注,例如国际上多次报道了从婴幼儿米粉中检出该菌。因此研究针对42株从食品基质分离的阪崎杆菌进行了SSCP分析,为阪崎杆菌检测方法的进一步优化以及该菌的菌群特征提供了参考数据。
In this paper, modern molecular techniques including RAPD, multplex PCR-CE-SSCP, 2DE, MALDI-TOF were applied in methodology research for Thailand jasmine rice authentication, storage quality detection and foodborne pathogen identification. The further standardization of these methods would promote official supervision of imported Thailand jasmine rice, protection of consumer’s right and development of rice import and export trade.
The first part was jasmine rice identification and purity detection. Totally 32 rice cultivars including reference cultivar (KDML105 and RD15) and representative non-jasmine rice cultivars were studied. RAPD fingerprint analysis demonstrated that phylogenetic relationship of the 32 samples was generally consistent with practical kinship. In order to simplify the method for routine detection at CIQ lab, two RAPD markers (R2-449 and R5-1107) were selected for which RAPD and SCAR methods were established. Thailand jasmine rice was identified by its recessive trait of the two markers. A serious of experiments proved that the method possess high resolution, good reproducibility and accuracy.
The second part was about storage quality inspection. 2DGE was used to analyze 6 jasmine rice samples of different production year.The PMF of major peptide spots were analyzed and protein identiy were determined on the basis both of PMF and data in international rice proteomics database. The results showed that the chaning pattern of 2DE profiles were consistent with the crop year. Significant change between 2008 and 2007 corp year was observed, indicating high resolution of the method. Not only could the method be applied as a helpful complement of present standard for storage quality evaluation, but also reveal biological process during storage which is important informantion for rice production, processing and circulation.
The third part was fast identification method for foodborne pathogens. Comapred to traditional microbiological identification method and routine PCR method, multiplex PCR-CE-SSCP was characterized as faster, higher throughput and more automatic. Here 16srRNA and gyrB gene based multiplex PCR-CE-SSCP system were established and DNA fingerprint data of 25 referrence strains was collected. Phylogenetic analysis showed that the taxonomy tree was generally consistent with traditionl taxamony relationship. Signal peak was identified by relative position to inner markers instead of retaining time and good reproducibility was proved. Considering the fact that newly emergent foodborne pathogen, E.sakasaki, was frequently found in infant formula rice powder, we further analyzed 42 E.sakasaki isoltates separated from food, the data of which was believed to be helpful for optimization of present detection method and for further research of E.sakasaki population in food.
引文
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2.黄超武,胡事君;水稻香味性状的遗传分析;谷类作物品质性状遗传研究进展,江苏科技出版社,1990:87~92。
3.傅荣昭等;DNA分子标记技术及其在药用植物研究上的应用前景;生物工程进展, 1998, 18(4) :14– 17。
4.黄淑贞等;湖南香稻产地土壤特性与稻米品质的关系;湖南农业科学,1990(4):37-40。
5.杨晓蓉等;不同储藏条件下稻谷脂肪酸值变化和霉变相关性研究;谷物化学与品质分析,2006,35:49-52。
6.万忠民,徐宁;不同储藏温度下稻谷的品质劣变;粮食与食品工业,2004,11:49-51。
7.任顺成等;大米陈化过程中谷蛋白与大米质构特性的变化;中国粮油学报,2002,17:42-46。
8.罗正荣等;应用RAPD技术进行柿种类和品种鉴定;日本园艺学会杂志,1995,64:535-541。
9.任光俊等;水稻香味的遗传;四川农业大学学报,1994,12(3):392-395。
10.陆铮等;北京市售国产婴幼儿配方奶粉及婴幼儿食品带染阪崎肠杆菌调查;中国卫生检验杂志,2008,18:140-141。
11.任光俊等;水稻香味的遗传分析;西南农业学报,1999,12(2):24-27。
12. Cespedes A. etal., Application of polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) to identification of flatfish species. Journal of AOAC International 1999,82(4):903-907.
13. Armstrong J etal, Computer program distributed by the Australian National University. http://life.anu.edu.au/molecular/software/rapd.html 1994.
14. Asensio L.etal., PCR-SSCP: a simple method for the authentication of grouper(Epin ephelusguaza),wreck fish (Polyprion americanus),and Nile perch(Lates niloticus)fillets. Journal of Agricultural and Food Chemistry 2001, 49(4): 1720-1723.
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16. Buttery R.G. etal. Quantitative analysis of 2-acetyl-1-pyrroline in rice. J.Agric.Food Chem, 1986,34:112-114.
17. Colombo,F.etal., Identification of tuna species by computer-assisted and cluster analysis of PCR-SSCP electrophoretic patterns. Food control 2005,16(1):51-53.
18. Case R. J etal , Applied and environmental microbiology 2007,73(1):278–288.
19. Chung J.H etal Electrophoresis 2007,28: 2416–2423.
20. Dong Y. etal. Trisomic genetic analysis of aroma in three Japanese native rice varieries. Euphytica 2001,117:191-196.
21. Fernandez A.etal., Genetic differentiation between the clam species Ruditapes decussates(grooved carpet sheel)and Venerupis pullastra(pullet carpet shell)by PCR-SSCP analysis. Journal of the Science of Food and Agriculture2002, 82(8):881-885.
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2.黄超武,胡事君;水稻香味性状的遗传分析;谷类作物品质性状遗传研究进展,江苏科技出版社,1990:87~92。
3.傅荣昭等;DNA分子标记技术及其在药用植物研究上的应用前景;生物工程进展, 1998, 18(4) :14– 17。
4.黄淑贞等;湖南香稻产地土壤特性与稻米品质的关系;湖南农业科学,1990(4):37-40。
5.杨晓蓉等;不同储藏条件下稻谷脂肪酸值变化和霉变相关性研究;谷物化学与品质分析,2006,35:49-52。
6.万忠民,徐宁;不同储藏温度下稻谷的品质劣变;粮食与食品工业,2004,11:49-51。
7.任顺成等;大米陈化过程中谷蛋白与大米质构特性的变化;中国粮油学报,2002,17:42-46。
8.罗正荣等;应用RAPD技术进行柿种类和品种鉴定;日本园艺学会杂志,1995,64:535-541。
9.任光俊等;水稻香味的遗传;四川农业大学学报,1994,12(3):392-395。
10.陆铮等;北京市售国产婴幼儿配方奶粉及婴幼儿食品带染阪崎肠杆菌调查;中国卫生检验杂志,2008,18:140-141。
11.任光俊等;水稻香味的遗传分析;西南农业学报,1999,12(2):24-27。
12. Cespedes A. etal., Application of polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) to identification of flatfish species. Journal of AOAC International 1999,82(4):903-907.
13. Armstrong J etal, Computer program distributed by the Australian National University. http://life.anu.edu.au/molecular/software/rapd.html 1994.
14. Asensio L.etal., PCR-SSCP: a simple method for the authentication of grouper(Epin ephelusguaza),wreck fish (Polyprion americanus),and Nile perch(Lates niloticus)fillets. Journal of Agricultural and Food Chemistry 2001, 49(4): 1720-1723.
15. Binz T.etal., SSCP analysis of Mhc class IIB genes in the three spine stickleback; Journal of Fish Biology 2001,58(3):887-890.
16. Buttery R.G. etal. Quantitative analysis of 2-acetyl-1-pyrroline in rice. J.Agric.Food Chem, 1986,34:112-114.
17. Colombo,F.etal., Identification of tuna species by computer-assisted and cluster analysis of PCR-SSCP electrophoretic patterns. Food control 2005,16(1):51-53.
18. Case R. J etal , Applied and environmental microbiology 2007,73(1):278–288.
19. Chung J.H etal Electrophoresis 2007,28: 2416–2423.
20. Dong Y. etal. Trisomic genetic analysis of aroma in three Japanese native rice varieries. Euphytica 2001,117:191-196.
21. Fernandez A.etal., Genetic differentiation between the clam species Ruditapes decussates(grooved carpet sheel)and Venerupis pullastra(pullet carpet shell)by PCR-SSCP analysis. Journal of the Science of Food and Agriculture2002, 82(8):881-885.
22. Glaszmann J.C. Isozymes and classification of Asian rice varieties. Theor.Appl.Genet, 1987, 74: 21-30.
23. Grim B.C. etal, Screening for 2-Acetyl-1-qyrroline in the headspace of rice using spmepgc-MS. J.Agric.Food Chem,2001,49:245-249.
24. Hong H etal, Journal of Microbiological Methods, 2007,69: 52–64.
25. Ip etal, Journal of clinical microbiology,2006, 44(3): 970–975.
26. Jin Q S etal. Identification and potential use of a RAPD marker for aroma in rice. J. Genet.&Breed. 1996 ,50 :367～370.
27. Kostia S.etal., Applicability of SSCP analysis for MHC genotyping:gingerprinting of Ovar-DRB1 ex on 2 alleles from Finnish and Russian breeds, Animal Genetics 1998, 29(6):453-455.
28. Shirasawa K. etal., Dot-bolt-SNP analysis for practical plant breeding and cultivar identification in rice. Theor Appl Genet 2006, 113:147-155.
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