基于改进的预扩增实时荧光定量聚合酶链式反应法检验虹鳟源性成分

Detection of Oncorhynchus mykiss source components based on improved pre-amplification real-time quantitative polymerase chain reaction method

中文关键词:  虹鳟源性成分  真实性检验  qPCR  预扩增 qPCR  

英文关键词:Oncorhynchus mykiss source  authenticity identification  quantitative real-time polymerase chain reaction  pre-amplified quantitative real-time polymerase chain reaction

中文摘要:

      目的 建立改进的预扩增实时荧光定量聚合酶链式反应法(quantitative real-time polymerase chain reaction, qPCR)法检验虹鳟源性成分的方法。方法 基于对现行标准中虹鳟qPCR方法进行验证和改进提高, 建立改进的预扩增qPCR方法, 并对两种qPCR方法进行比较。结果 经验证, 在采用标准中qPCR方法对虹鳟的近种鱼种银鲑、大西洋鲑等鱼种进行特异性验证时有交叉反应, 基于设置预扩增反应步骤的改进qPCR方法特异性良好, 对于银鲑、大西洋鲑等近种鱼种未出现交叉扩增。qPCR和预扩增qPCR方法的扩增效率分别为90.25%、104.91%, 最低检出限分别为2.99×10–3 ng/μL (0.83~8.87 pg/μL, 95%置信区间)和1.50×10–4 ng/μL (0.09~0.27 pg/μL, 95%置信区间)。结论 所改进的虹鳟源性成分预扩增qPCR方法具有高特异性、高灵敏度, 适用于食品样品中虹鳟源性成分的真实性和掺假检验。

英文摘要:

      Objective To establish a pre-amplified quantitative real-time polymerase chain reaction (qPCR) method for the detection of Oncorhynchus mykiss source components. Methods The study developed an enhanced pre-amplified qPCR method, which was based on the validation and improvement of the existing standard qPCR method and compared the performance of both methods. Results Specificity testing of the standard qPCR method revealed cross-reactivity with closely related species, such as Coho salmon and Atlantic salmon. The established improved pre-amplified qPCR method, which incorporated a pre-amplification step, exhibited high specificity and no cross amplification was observed for Coho salmon, Atlantic salmon and other closely related species. The amplification efficiencies of the standard qPCR and the pre-amplified qPCR methods were 90.25% and 104.91%, respectively. The lowest limits of detection of the 2 kinds of methods were 2.99×10–3 ng/μL (0.83–8.87 pg/μL, 95% confidence interval) for the standard qPCR and 1.50×10–4 ng/μL (0.09–0.27 pg/μL, 95% confidence interval) for the pre-amplified qPCR. Conclusion The method established in this study has the characteristics of strong specificity, high sensitivity, and is suitable for authenticity and adulteration detection of Oncorhynchus mykiss source components in food products.