CPA-核酸试纸条检测鸭肉源性成分方法的建立和优化

Cross Priming Amplification and Nucleic Acid Test Strip Analysis of Duck Meat in Meat Mixtures

为建立简单、有效的动物源性成分定性检测方法,根据交叉引物恒温扩增技术(CPA)原理,以鸭属线粒体D-loop基因序列设计引物和探针,优化反应体系与恒温扩增条件,并配合核酸试纸条显色,建立鸭肉源性成分的快速检测方法。结果表明,建立的鸭D-loop基因CPA扩增体系中交叉引物、内引物、外引物分别为0.6、0.3、0.1μmol·L-1,Mg SO4为8 mmol·L-1。在63℃、60min恒温扩增条件下,单一鸭源性DNA成分检出限为0.1 ng·μL-1,对混合肉制品中鸭肉成分的检出比例为1%(相当于1ng·μL-1)。本研究建立的鸭源性成分检测方法为肉制品掺假鉴定提供了有效的手段。

To establish a simple and effective method for the qualitative detection of animal-derived ingredients,the reaction system and isothermal amplification condition were optimized based on the principle of cross priming amplification（ cross priming amplification,CPA） and primers and probes were designed accroding to Anas platyrhynchos mitochondrial D-loop gene sequences,and the rapid detection method for duck derived ingredients was established with the nucleic acid test strip coloration. In this study, Duck-specific primers and probe were designed from the mitochondrial D-loop region,and CPA reaction mixtures and reaction condition were optimized. The results showed that the optimized CPA reaction mixtures were suggested to contain 0. 6 μmol·L- 1crossing primer,0. 3 μmol·L- 1inter primers,0. 1μmol·L- 1outer primers and 8 mmol·L- 1Mg SO4,and the nucleic acid test strips replaced gel electrophoresis leading to an quick result,simple data interpret and easy operation at 63℃ for 60 min. The detection limit of CPA-Strip assays were 0. 1 ng·μL- 1or 1% of duck meat in meat mixtures. The CPA-Strip detection methods established in this study offers a specific,sensitive,rapid,and simple detection tool for identifying duck meats,which can contribute to effectively control of presence of adulterated animal species in meat products.