食品过敏原牡蛎成分PCR检测方法的初步研究
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摘要
目的:建立基于PCR技术的过敏原牡蛎成分快速检测方法,结合溶解曲线及特异的Tm值与Ct值,用于不同产品的牡蛎成分检测。方法:根据NCBI上的牡蛎线粒体序列,通过DNA Star等软件设计引物,分别采用普通PCR和SYBR Green I实时荧光PCR的方法建立食品过敏原牡蛎成分的检测方法,对十种牡蛎阳性样品和二十余种阴性样品进行实验,并且对几种牡蛎相关食品进行检测。结果:研究建立的检测方法可以检测出含牡蛎成分0.1%的样品,其中荧光PCR的灵敏度达到0.01 ng/μL,特征峰的Tm温度为80.08℃,能够检测出牡蛎相关食品中的牡蛎成分。结论:该方法是一种操作安全方便、成本较低、特异、灵敏的实时荧光PCR方法,对于收集到的食品相关产品以及保健品中牡蛎粉的检出率为100%,该方法可广泛应用于食品中牡蛎成分的快速鉴定。
Objective: A PCR based method combined with melting curve analysis and site-specific Tm and Ct analysis was established to detect oyster as allergen in different types of food. Methods: According to the DNA sequences of oyster mitochondria from NCBI database,oyster-specific primers for both conventional PCR and SYBR Green I RT-qPCR were designed by DNA star software.10 samples were confirmed to contain oyster( positive) and 20 samples were negative in the oyster presentation.Some samples made of the oyster were used in our experiments as positive controls. Results: The minimum detectable proportion of oyster component in food samples was 0.1%( w/w),with a 0.01 ng/μL detection limit of qPCR and a melt curve peak at Tm 80.08 ℃ by this methods.Conclusion: Compared with the existing methods for oyster detection in food industry,the qPCR based protocol established here was step-wise,cost-effective with higher specificity and sensitivity.In this study,the detection efficiency of oyster components in all collected samples was 100%,which indicated this method would be widely applied for quick qualification of oyster component in food industry.
Objective: A PCR based method combined with melting curve analysis and site-specific Tm and Ct analysis was established to detect oyster as allergen in different types of food. Methods: According to the DNA sequences of oyster mitochondria from NCBI database,oyster-specific primers for both conventional PCR and SYBR Green I RT-qPCR were designed by DNA star software.10 samples were confirmed to contain oyster( positive) and 20 samples were negative in the oyster presentation.Some samples made of the oyster were used in our experiments as positive controls. Results: The minimum detectable proportion of oyster component in food samples was 0.1%( w/w),with a 0.01 ng/μL detection limit of qPCR and a melt curve peak at Tm 80.08 ℃ by this methods.Conclusion: Compared with the existing methods for oyster detection in food industry,the qPCR based protocol established here was step-wise,cost-effective with higher specificity and sensitivity.In this study,the detection efficiency of oyster components in all collected samples was 100%,which indicated this method would be widely applied for quick qualification of oyster component in food industry.
引文
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[8]刘志刚,邬玉兰,吴淑勤,等.虾夷扇贝过敏原tropomyosin的克隆表达、纯化及免疫学鉴定[J].水生生物学报,2010,34(5):979-983.
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[10]沈海旺,陈亨莉,曹敏杰,等.甲壳类动物4种过敏原的序列分析、抗原表位预测及三维结构建模[J].免疫学杂志,2012(7):613-619.
[11]Lopata A L,Lehrer S B. New insights into seafood allergy[J]. Current Opinion in Allergy and Clinical Immunology,2009,9(3):270-277.
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[13]Taylor S L.Molluscan shellfish allergy[J].Advances in Food and Nutrition Research,2008,54:139-177.
[14]董晓伟,姜国良,李立德,等.牡蛎综合利用的研究进展[J].海洋科学,2004,28(4):62-65.
[15]Shuang Li,Qi Li,Hong Yu,et al. Genetic variation and population structure of the Pacific oyster Crassostrea gigasin the northwestern Pacific inferred from mitochondrial COI sequences[J].Fisheries Science,2015,81:1071-1082.
[16]李咏梅,陈秀荔,赵永贞,等.钦州湾牡蛎线粒体16SrRNA和COI基因片段的序列变异分析[J].广东海洋大学学报,2009,29(3):11-18.
[2]刘欣,姚晗珺.世界主要贸易国对食品过敏原的法规和要求及对中国的借鉴[J].世界农业,2011(8):59-61.
[3]Sicherer S H,Sampson H A.Food allergy:Recent advance in pathophysiology and treatment[J].Annual Review of Medicine,2009,60:261-277.
[4]Caleb K,Venu G.Sex disparity in food allergy:evidence From the Pubmed database[J].Journal of Allergy,2009(2009):159845.
[5]王薇.食品过敏原标识管理,我国还需加把劲[N].中国食品报,2013-05-28.
[6]黄榕芳.软体类海产品主要过敏原的基因克隆及生物信息学分析[D].青岛:中国海洋大学,2014.
[7]伍慧妍,何颖,许晨霞,等.牡蛎过敏原Crag1的二级结构及其B细胞和T细胞表位分析[J].中国医药导报,2013,10(19):12-15.
[8]刘志刚,邬玉兰,吴淑勤,等.虾夷扇贝过敏原tropomyosin的克隆表达、纯化及免疫学鉴定[J].水生生物学报,2010,34(5):979-983.
[9]曹际娟,赵彤彤,刘冉,等.PCR检测食品中致敏原虾源性成分[J].食品安全质量检测学报,2012,3(4):274-278.
[10]沈海旺,陈亨莉,曹敏杰,等.甲壳类动物4种过敏原的序列分析、抗原表位预测及三维结构建模[J].免疫学杂志,2012(7):613-619.
[11]Lopata A L,Lehrer S B. New insights into seafood allergy[J]. Current Opinion in Allergy and Clinical Immunology,2009,9(3):270-277.
[12]Ayuso R.Update on the diagnosis and treatment of shellfish allergy[J].Current Allergy and Asthma Reports,2011,11(4):309-316.
[13]Taylor S L.Molluscan shellfish allergy[J].Advances in Food and Nutrition Research,2008,54:139-177.
[14]董晓伟,姜国良,李立德,等.牡蛎综合利用的研究进展[J].海洋科学,2004,28(4):62-65.
[15]Shuang Li,Qi Li,Hong Yu,et al. Genetic variation and population structure of the Pacific oyster Crassostrea gigasin the northwestern Pacific inferred from mitochondrial COI sequences[J].Fisheries Science,2015,81:1071-1082.
[16]李咏梅,陈秀荔,赵永贞,等.钦州湾牡蛎线粒体16SrRNA和COI基因片段的序列变异分析[J].广东海洋大学学报,2009,29(3):11-18.