<4D F736F F D D323738B2DCBCCABEEA5F6E65775F504352BCECB2E2CAB3C6B7D6D0D6C2C3F4D4ADCFBAD4B4D0D4B3C9B7D62DD0A3B6D4B7B5BBD833A1AD>

Size: px

Start display at page:

Download "<4D F736F F D D323738B2DCBCCABEEA5F6E65775F504352BCECB2E2CAB3C6B7D6D0D6C2C3F4D4ADCFBAD4B4D0D4B3C9B7D62DD0A3B6D4B7B5BBD833A1AD>"

扁爱新觉罗
7 years ago
Views:

1 3 4 Vol. 3 No Journal of Food Safety and Quality Aug., 2012 PCR 曹际娟 *, 赵彤彤, 刘冉, 孙铭英, 刘洋 (, ) 摘要 : 目的 PCR 方法 16S rrna, PCR, 16S rrna 结果 PCR,,,, min, 0.05% 结论, 关键词 : PCR; ; ; Detection of shrimp-derived components of allergen in food by PCR CAO Ji-Juan *, ZHAO Tong-Tong, LIU Ran, SUN Ming-Ying, LIU Yang (Liaoning Entry-Exit Inspection and Quarantine Bureau, Dalian , China) ABSTRACT: Objective To develop a method for the detection of shrimp-derived components of allergen in food by PCR. Methods The primers for PCR were designed based on 16S rrna genes through comparing a large number of nucleic acid sequences published by National Center for Biotechnology Information (NCBI) among different shrimp species. Results Totally 56 kinds of samples including shrimps, crabs, fish, shellfish and octopus were subjected to the detection by PCR. The results indicated that PCR could successfully identify the shrimp-derived components. In order to explore the effect of food processing on detection sensitivity, fish power containing shrimp powder were treated by heating at 133 C for 30 min. The detection sensitivity of shrimp-derived components in fish powder reached up to 0.05%. Conclusion The new established method is highly specific, sensitive and accurate, and can be applied for the detection of allergen shrimp-derived components in foods. KEY WORDS: PCR; allergen; shrimp-derived components; detection,,,, [1-3],, [4-5] 1981 Hoffman [6] Antigen II, 38 kda, Jeoung [7] (Pen a I), 36 kda 1990 基金项目 : (2007GYJ036) * 通讯作者 :,,, cjj0909@163.com

2 4, : PCR 275 Morgan [8], 13, 16~166 kda Pen a I, [9], [10-12],, PCR (NCBI),, 16S rrna,, PCR 1 料与方法 1.1 样品和 DNA 提取 14, 5, 鱖鯷 24, 12,, 56 PCR Universal Genomic DNA Extraction Kit Ta- KaRa, Ver.3.0) DNA, min,, (10%, 5%, 0.5%, 0.1%, 0.05%, 0.01%, 0%), DNA PCR, 1.2 引物设计和合成 (NCBI),, 16S rrna,, DNAMAN 8.0, 16S rrna, : Shrimp1 F 5 -GATTAAGTTACTTTAGGGATAACAGCG-3 Shrimp1 R 5 -TAAAGGTCGAACAGACCTTCTCACT-3, 135 bp Shrimp2 F 5 -AAGTCTAGC TGCCCACTG-3 Shrimp2 R 5 -GTCAACCATCAT CAAGCC-3, 119 bp ( )( PAGE ), PCR 扩增和电泳检测 PCR 25 µl, PCR : 10 PCR 2 µl, 10 µmol/l 1 µl, dntp(10 mmol/l) 2 µl, Taq DNA (5 U/µL) 0.2 µl, DNA( ng DNA) 1 µl, dh 2 O 25 µl PCR : 94 3 min; 94 1 min, 66 1 min, 72 1 min, 35 ; 72 7 min ; 4, PCR 2% 2 实验结果 2.1 虾源性成分特异性基因 DNA 片段的扩增 56 DNA, Shrimp1 PCR,, bp DNA,, Shrimp1 PCR Shrimp2 PCR,, 16 DNA,,,, 2 Shrimp2 PCR

3 276 3 PCR, Blast 2.2 检测方法的灵敏度 M: 100 bp DNA Ladder Marker; 1: ; 2: ; 3: ; 4: ; 5: ; 6: ; 7: ; 8: ; 9: ; 10: M: 100 bp DNA Ladder Marker; 1: Prawn; 2: Pandalus borealis; 3: Dried peeled shrimp; 4: Acetes chinensis Hansen; 5: Oratosquilla oratorical; 6: Orithyia sinica; 7: Salmon; 8: Oysters; 9: Octopus; 10: Scallops 1 Shrimp1 PCR Fig. 1 PCR amplification of shrimp-derived components using Shrimp 1 primers, Shrimp1, DNA,, Tartaglia M [13], min 10% 5% 0.5% 0.1% 0.05%, 0.01% 0% min, DNA PCR, 6, PCR 0.05% 2.3 不同食品样品的检测情况 M: 100 bp DNA Ladder Marker; 1: ; 2: ; 3: ; 4: ; 5:; 6: ; 7: ; 8: ; 9: ; 10: M: 100 bp DNA Ladder Marker; 1: Prawn; 2: Sea crabs; 3: Eriocheir sinensis; 4: Chinoecetes bairdi; 5: Salmon; 6: Redfish; 7: Microstomuskitt; 8: Oysters; 9: Octopus; 10: Scallops 2 Shrimp2 PCR Fig. 2 PCR amplification of shrimp-derived components using Shrimp 2 primers Shrimp1 Shrimp2 PCR 1, PCR,, Shrimp1 Shrimp2 PCR,,,, [1-2],, Shrimp1, Shrimp2, PCR,, Shrimp1 PCR,,,, DNA, 0.05%, Shrimp1 PCR,, ( ) 3 讨论, [14-15] lge,,,,,,,, DNA PCR DNA,, PCR

4 4, : PCR 277 表 1 两对引物检测虾源性成分的试验结果 Table 1 Detection results of shrimp-derived components using two pairs of primers Shrimp1 Shrimp2 ( ) ( ) Shrimp1 Shrimp2 1 Prawn Dired peeled shrimp + 2 Crangon septemspinosa Acetes chinensis Hansen + 3 Macrobrachium nipponense Oratosquilla oratoria + 4 Alpheus japonicus Orithyia sinica + 5 Pandalus borealis + 16 Charybdis japonica + 6 Metapenaeus ensis Portunus trituberculatus + 7 Sea shrimp Eriocheir Sinensis 8 Brachium nipponense Chinoecetes bairdi 9 Lobster Octopus 10 Procambarus clarkii Whitebait 11 Penaeus japonicus Short necked clam : +, DNA, PCR, PCR,,, NCBI,, 16S rrna NCBI, Tax BLAST, 100%, DNA,,, 0.05% PCR,, 参考文献 [1] Dean DM, Hugh AS, Ronald AS. Food Allergy: Adverse reactions to foods and food additives(second Edition) [M]. USA Blackwell science inc, [2] Schafer T, Bohler E, Ruhdorfer S, et al. Epidemiology of food allergy/food intolerance in adults: associations with other manifestations of atopy [J]. Allergy, 2001, 56(12): [3] Daul CB, Morgan JE, Lehrer SB. The natural history of shrimp hypersensitivity [J]. J Allergy Clin Immunol, 1990, 86: [4],. l [J]., 2000, 2(2): 106. [5],. l [J]., 2003, l6(8): 877. [6] Hoffman DR, Day Jr, Miller JS. The major heat stable allergen of shrimp [J]. Ann Allergy, 1981, 47: [7] Jeoung BJ, Reese G, Hauck P, et a1. Quantification of the major brown shrimp allergen Pen a 1(tropomyosin)by a monoclonal antibody-based sandwich ELISA [J]. J Allergy Clin Immunol, 1997, 100(2): [8] Morgan JE, Daul CB, Lehrer SB. The relationships among shrimp-specific IgG subclass antibodies and immediate adverse reactions to shrimp challenge [J]. J Allergy Clin Immunol, 1990, 86: 387. [9] Reese G, Ayuso R, Carle T, et a1. IgE-Binding Epitopes of Shrimp Tropomyosin, the Major Allergen Pen a 1 [J]. Int Arch Allergy Immunol, 1999, l18: [10],,,. [J]., 2007, 28(4): [11],,,.

5 278 3 [J]., 2005, 30(2): [12] Naqpal S, Rajappa L, Metcalfe DD, et al. Isolation and characterization of heat stable allergens from shrimp (Penaeus inclicus) [J]. J Allergy Clin Immunol, 1989, 83: [13] Tartaglia M, Saulle E, Pestalozza S, et al. Detection of bovine mitochondrial DNA in ruminant feeds: a molecular approach to test for the presence of bovine-derived materials [J]. J Food Prot, 1998, 61(5): [14] Byun MW, Kim JH. Effects of Gamma Radiation on the Conformational and Antigenic Properties of a Heat-Stable Major Allergen in Brown Shrimp [J]. J Food Prot, 2000, (7): [15],,,. Western blotting [J]., 2004, 24(9): 747. ( 责任编辑 : 张宏梁 ) 作者简介曹际娟,,, cjj0909@163.com ˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆ,,,,, (), 2013,, jfoodsq@126.com

<4D6963726F736F667420576F7264202D203239352D323939D5C5BAA3D3A25F6E65775FCAB3C6B7D6D0A6C22DC8E9C7F2B5B0B0D7B9FDC3F4D4ADBCECB2E2B7BDB7A8B5C4B1C8BDCF2DA1AD>

<4D6963726F736F667420576F7264202D203239352D323939D5C5BAA3D3A25F6E65775FCAB3C6B7D6D0A6C22DC8E9C7F2B5B0B0D7B9FDC3F4D4ADBCECB2E2B7BDB7A8B5C4B1C8BDCF2DA1AD> 3 4 Vol. 3 No. 4 2012 8 Journal of Food Safety and Quality Aug., 2012 β- 张海英 1, 刘旸 1, 吴冬雪 1, 梁靖 1, 张霞 1, 2* (1., 300461; 2., 300457) 摘要 : 目的 β- ELISA 方法 β-, ELISA 结果 ELISA β-, 结论 ELISA 关键