摘要
目的建立一种高效、精准的DNA条形码技术准确快速鉴定鱼胶物种。方法以细胞色素C氧化酶I(cytochrome C oxidase subunit I,COI)基因为靶标,筛选并优化DNA条形码引物,通过聚合酶链式反应(polymerase chain reaction,PCR)扩增和Sanger测序评估其鉴定效果。对市售鱼胶样品进行特异性扩增,结合生命条形码数据系统(barcode of life data systems,BOLD)和美国国家生物技术信息中心(national center for biotechnology information,NCBI)数据库进行物种比对,并采用邻接法(neighbor-joining,NJ)和系统发育树分析验证鉴定结果。结果通过筛选8对COI基因微型DNA条形码引物,最终确定了一组扩增目标片段约190 bp的引物组合,其PCR测序成功率达100%,物种鉴定准确率达98.15%。基于该引物对16份鱼胶样品的鉴定结果显示,仅56.25%的样本与标注物种一致,其中不符样品主要是鳘鱼胶和白花胶,鉴定阈值设为95%。进一步通过NJ分析和系统发育树构建,证实鱼胶样品的系统发育关系与BOLD/NCBI数据库比对结果高度一致,验证了鉴定结果的可靠性。结论本研究成功筛选出适用于鱼胶微型DNA条形码鉴定的引物,为鱼胶产品的真伪鉴别和质量控制提供了高效、可靠的技术手段。
Objective To develop an efficient and accurate DNA barcoding technology for rapid identification of fish maws species.Methods Targeting the cytochrome C oxidase subunit I(COI)gene,DNA barcode primers were screened and optimized.Polymerase chain reaction(PCR)amplification and Sanger sequencing were employed to evaluate identification efficacy.Commercial fish maw samples underwent specific amplification,followed by species comparison using the barcode of life data systems(BOLD)and the national center for biotechnology information(NCBI)database.Neighbor-joining(NJ)method and phylogenetic tree analysis were utilized to validate identification results.Results The 8 pairs of mini-DNA barcode primers of COI gene were screened,and finally a set of primer combinations were determined to amplify the target fragment by about 190 bp.The success rate of PCR sequencing was 100%,and the accuracy of species identification was 98.15%.The identification results of 16 samples of fish maw based on this primer showed that only 56.25% of the samples were consistent with the labeled species,among which the non-conforming samples were mainly silver fish maw and white flower maw,and the identification threshold was set to 95%.Further,through NJ analysis and phylogenetic tree construction,it was confirmed that the phylogenetic relationship of fish maw samples was highly consistent with the comparison results of BOLD/NCBI database,and the reliability of the identification results was verified.Conclusion This study successfully screens primers suitable for fish maw mini-DNA barcode identification,providing an efficient and reliable technical approach for authenticity verification and quality control of fish maw products.
作者
潘影俊男
苗雅倩
陈凤明
冯峰
王云帆
PAN Ying-Jun-Nan;MIAO Ya-Qian;CHEN Feng-Ming;FENG Feng;WANG Yun-Fan(Chinese Academy of Quality and Inspection&Testing,Beijing 100176,China;School of Pharmacy,China Medical University,Shenyang 110122,China;College of Life Sciences,China Jiliang University,Hangzhou 310018,China;Chinese Academy of Inspection and Quarantine Greater Bay Area,Zhongshan 528437,China)
出处
《食品安全质量检测学报》
2025年第18期298-305,共8页
Journal of Food Safety and Quality
