免疫磁珠结合PCR-试纸条法鉴定金枪鱼成分的研究

Identification of tuna ingredients by immunomagnetic beads combined with PCR-based lateral flow assay

中文关键词:  免疫磁珠  金枪鱼成分  PCR-试纸条

英文关键词:immunomagnetic beads  tuna ingredients  PCR-based lateral flow assay

中文摘要:

      目的 建立一种免疫磁珠结合PCR-免疫金标试纸条快速检测金枪鱼成分的方法。方法 使用免疫磁珠法提取金枪鱼样品的核酸, 针对金枪鱼基因组特异性基因片段设计引物, 优化反应体系和反应条件, 建立金枪鱼成分PCR产物试纸条检测方法, 验证方法的特异性、灵敏度和稳定性, 并对市售金枪鱼产品进行检测。结果 免疫磁珠法提取市售金枪鱼样品DNA的纯度高。所建立的PCR-试纸条方法特异性强, 仅金枪鱼能扩增出229 bp特异性DNA片段, 试纸条检测线变红, 其他鱼类均无非特异性扩增条带, 试纸条检测线未变红色; 灵敏度高, 达到0.01%; 方法快速, PCR产物的试纸条判读时间5 min。结论 该方法具有特异性强、灵敏度高、稳定性好、简便环保等优点, 适用于金枪鱼成分的快速检测。

英文摘要:

      Objective To establish a method for rapid detection of tuna ingredients by immunomagnetic beads combined with PCR-based lateral flow assay. Methods A nucleic acid of a tuna sample was extracted by using an immunomagnetic beads, a primer was designed aiming at a tuna genome specific gene fragment, a reaction system and reaction conditions were optimized, a tuna ingredients PCR-based lateral flow assay detection method was established, the specificity, sensitivity and stability of the method were verified, and a commercial tuna product was detected. Results The purity of DNA extracted from commercial tuna samples by immunomagnetic beads was high. The established PCR-based lateral flow assay method had strong specificity, only tuna samples could amplify 229 bp specific DNA fragments, and the detection line of the test strip turned red. There was no non-specific amplification band in other fish, and the detection line of the test strip did not turn red. The sensitivity was high, reaching 0.01%. The method was rapid, and the test strip interpretation time of PCR products was 5 min. Conclusion This method has the advantages of strong specificity, high sensitivity, good stability, simplicity and environmental protection, and is suitable for rapid detection of tuna ingredients.