可视化跨越式滚环等温扩增技术在猪肉掺假检测中的应用

胡学佳; 卢鑫; 张蕴哲; 徐慧; 杨倩; 赵峥山; 李子英; 苏玲玲; 张伟

doi:10.13386/j.issn1002-0306.2020040328

可视化跨越式滚环等温扩增技术在猪肉掺假检测中的应用

Application of Visual Saltatory Rolling Circle Amplification Method in Detection of Adulterated Pork

摘要

摘要: 目的:建立了一种跨越式等温扩增技术(SRCA)结合荧光染料SYBR Green I检测肉制品中猪肉掺假的方法。方法:根据NCBI中猪的线粒体序列,通过DNAMAN等软件设计SRCA的特异性引物,分别采用普通SRCA和荧光可视化SRCA的方法建立肉制品中猪肉成分的检测方法,对9个不同物种的新鲜肌肉组织样本进行实验从而验证SRCA方法的特异性,并对66份未标识猪肉成分商业肉制品进行猪肉成分的检测。结果:SRCA荧光可视法检测猪肉DNA的灵敏度为6.5 fg/μL,与传统PCR方法相比,SRCA荧光可视法的灵敏度提高了1000倍。在人工添加猪肉的混合样品中,SRCA方法检测猪肉含量为0.01%(w/w)。与NY/T 3309-2018行业标准相比,SRCA方法检测肉制品中猪肉的敏感性、特异性和符合率分别为100%、96.66%、98.48%。结论:SRCA是一种检测肉制品中猪肉掺假的灵敏、直观的方法,具有很大的应用潜力。

Abstract: Objective:A saltatory rolling circle amplification(SRCA)combined with the fluorescent dye SYBR Green I was developed to detect the adulteration of pork in meat products. Methods:According to the DNA sequences of pork from NCBI database,pork-specifie primers for conventional SRCA and fluorescence visualization SRCA were designed by DNAMAN software. Fresh muscle tissue samples of nine different species were used to confirm the specificity of SRCA. Moreover,66 commercial meat products with unlabeled pork ingredients were tested for pork ingredients. Conclusion:The detection sensitivity of SRCA method for pork DNA was 6.5 fg/L by visualization with fluorescence,with conventional PCR method,the SRCA achieved 1000-fold higher sensitivity. In artificially added pork samples,the minimum detectable proportion of pork component in food samples was 0.01%(w/w). The results were 100% sensitivity,96.66% specificity and 98.48% accuracy compared with the industry standard NY/T 3309-2018. Conclusion:SRCA would be a sensitive and visual detection method for the detection of pork adulteration in meat products with great application potential.