Preliminary Study on PCR Detection Method for Food Allergen Oyster
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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。Abstract: 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.
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Keywords:
- food allergen /
- oysters /
- PCR /
- qualitative detection
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