Zhao Liangjuan, Hua Marti Z, Li Shenmiao, Liu Jinyu, Zheng Wenjie, Lu Xiaonan
Tianjin Key Laboratory of Animal and Plant Resistance, Tianjin Normal University Tianjin 300387 China
Tianjin Customs District Tianjin 300387 China.
RSC Adv. 2019 Aug 23;9(46):26552-26558. doi: 10.1039/c9ra05060d.
Food authenticity is a global issue and has raised increasing concerns in the past decades. DNA-based methods are more favourable than the conventional protein-based techniques and have been applied to species identification and meat fraud detection. To effectively identify donkey meat for meat product authentication, a highly specific and robust method that coupled polymerase chain reaction (PCR) with lateral flow immunoassay (LFI) was developed. Donkey-specific PCR primers were designed by targeting at the mitochondrial D-loop gene and the specificity was verified and against 22 species involved in meat authentication. A limit of detection of 0.0013 ng μL DNA extract was achieved and as low as 0.001% w/w (raw) and 0.01% w/w (cooked) donkey meat in beef were successfully detected using the developed PCR-LFI. LFI strip-based visualization of PCR products allowed for a 10-fold higher sensitivity than conventional gel electrophoresis and significantly reduced the analysis time for the post-PCR analysis. This PCR-LFI is highly suitable for rapid identification of donkey or incorporating into multiplex screening protocol for other meat authentication in the laboratories of both regulatory agencies and commercial services.
食品真实性是一个全球性问题，在过去几十年中受到越来越多的关注。基于DNA的方法比传统的基于蛋白质的技术更具优势，已被应用于物种鉴定和肉类掺假检测。为了有效地鉴定用于肉类产品认证的驴肉，开发了一种将聚合酶链反应（PCR）与侧向流动免疫分析（LFI）相结合的高度特异性和稳健的方法。通过靶向线粒体D-loop基因设计了驴特异性PCR引物，并针对22种参与肉类认证的物种验证了其特异性。使用所开发的PCR-LFI实现了0.0013 ng/μL DNA提取物的检测限，并成功检测到牛肉中低至0.001% w/w（生肉）和0.01% w/w（熟肉）的驴肉。基于LFI条带的PCR产物可视化比传统凝胶电泳的灵敏度高10倍，并显著缩短了PCR后分析的时间。这种PCR-LFI非常适合在监管机构和商业服务实验室中快速鉴定驴肉或纳入用于其他肉类认证的多重筛选方案。
