该研究建立了金枪鱼制品中的鲣鱼成分的环介导等温扩增(loop-mediated isothermal amplification,LAMP)检测方法。针对线粒体Cyt b基因设计了鲣鱼的特异性引物,着重对内引物FIP/BIP、dNTP Mix、缓冲液和Mg2+浓度等反应参数进行了优化,扩增产物采用恒温实时荧光仪和可视化核酸染料SYBR Green I进行检测,对方法的特异性和灵敏度进行了评价。结果表明,在0.2 μmol/L外引物(F3和B3)、1.6 μmol/L内引物(FIP和BIP)、1.6 mmol/L dNTP Mix、3.5 μL 10×ThermoPol缓冲液、6 mmol/L Mg2+等参数的优化条件下扩增效果最佳。该方法特异性强,灵敏度高。其检测限为50 pg/μL。采用此LAMP方法对59份市售样品进行检测,检出6份为阳性样品,与DNA条形码检测结果一致。该研究建立的LAMP检测方法可应用于金枪鱼制品中鲣鱼成分的快速特异检测,为监管机构提供技术支持。
The loop-mediated isothermal amplification (LAMP) assay for the detection of Katsuwonus pelamis was developed by using four primers including two inner primers and two outer primers that specifically amplify segments of the Cyt b gene. The optimized amplification conditions were determined as follows: Inner primers (FIP and BIP), 1.6 μmol/L; outer primers (F3 and B3), 0.2 μmol/L; MgSO4, 6 mmol/L; dNTP Mix, 1.6 mmol/L. The detection limit of the LAMP method was 50 pg/μL. Fifty-nine commercially samples were detected by using this LAMP method, in which 6 were positive, which consist with the DNA barcode detection result. Therefore, the LAMP detection method established in this study could be applied to the rapid and specific detection of bonito components in tuna products and provide technical support for regulatory agencies.
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