基于微滴芯片数字PCR技术检测海水鱼糜中4种淡水鱼成分方法研究
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1.上海海洋大学食品学院;2.中国水产科学研究院黄海水产研究所;3.安井食品集团股份有限公司

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TS207.3

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国家重点研发计划课题(2016YFF0201805)和中国水产科学研究院基本科研业务费(2023TD76)共同资助


Detection of four freshwater fish components in marine fish surimi based on crystal digital PCR technique
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1.College of Food Science and Technology,Shanghai Ocean University;2.Key Laboratory of Testing and Evaluation for Aquatic Product Safety and Quality,Ministry of Agriculture and Rural Affairs,Yellow Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences;3.Anjoy Foods Group CO,LTD Xiamen

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    摘要:

    目的:建立海水鱼糜中青鱼(Mylopharyngodon piceus)、草鱼(Ctenopharyngodon idella)、鲢鱼(Hypophthalmichthys molitrix)和鳙鱼(Hypophthalmichthys nobilis)4种淡水鱼的微滴芯片数字PCR(crystal digital PCR)定量检测方法。方法:基于线粒体NADH脱氢酶亚基5(ND5)基因设计4种淡水鱼特异性引物/探针,通过正交试验优化反应条件,评估方法的特异性和灵敏度。对50份市售鱼糜制品进行检测,验证该方法的实用性。结果:该方法特异性强,使用浓度为0.2 ng/μL的样品鱼DNA进行扩增,得到青鱼、草鱼、鲢鱼和鳙鱼的拷贝数分别为638.8、402.5、785.3和627.5 copies/μL。最低定量限实验表明,该方法最低可检出青鱼、草鱼、鲢鱼和鳙鱼平均拷贝数分别为1.43 copies/μL、2.87 copies/μL、2.69 copies/μL、1.72 copies/μL。市售鱼糜样品分析表明,该方法可以准确地检测出样品中青鱼、草鱼、鲢鱼或鳙鱼源性成分的拷贝数,且定性结果与DNA条形码技术检测结果一致。结论:本研究可用于灵敏、快速地定量检测海水鱼糜中的青鱼、草鱼、鲢鱼和鳙鱼源性成分,为行业主管部门与检测机构监测海水鱼糜中淡水鱼源性成分是否合规标识提供有效的技术参考。

    Abstract:

    Surimi, a refined myofibrillar protein product, is high in protein and low in fat. It is made from raw fish through a series of processes. It has unique texture, delicious flavour and rich nutritional value. The global surimi industry has expanded in recent decades, but new challenges have emerged. As surimi is a source of myogenic fibrous protein derived from processed fish flesh, fish species identification through morphological and sensory methods is impossible. Moreover, the nutritional value and quality of surimi vary considerably depending on the fish species used to make surimi, so determining the species of fish used in surimi is essential for quality assurance. Additionally, whiteness and gel characteristics are key factors in determining surimi quality and grade. Improvements in these attributes increases surimi grade and market price. Black carp (Mylopharyngodon piceus), grass carp (Ctenopharyngodon idella), silver carp (Hypophthalmichthys molitrix), and bighead carp (Hypophthalmichthys nobilis) are the four major freshwater fish species in China, known for their low cost, white flesh, and effective gelatinization properties. Their use in surimi production can enhance whiteness and texture, increasing surimi’s grade and price. Therefore, driven by economic interests, some manufacturers add black carp, grass carp, silver carp, or bighead carp to surimi without labeling them, misleading consumers and disrupting market order. Therefore, developing an accurate, sensitive, and rapid method to identify these species in marine fish surimi is crucial. Digital PCR is a third-generation PCR technology with higher specificity and sensitivity, allowing quantification of the DNA template of the sample to be tested without the use of a standard curve. Crystal digital PCR combines both microtitre and microarray technologies, enabling sample partitioning, PCR amplification and fluorescence analysis to be performed on a chip, minimising handling time and cross contamination. In this study, we designed a pair of specific primers/probes based on the mitochondrial NADH dehydrogenase subunit 5 (ND5) gene for the simultaneous detection of four freshwater fishes, and developed a crystal digital PCR method. This method enabled the quantitative detection of black carp, grass carp, silver carp, and bighead carp in marine fish surimi. The reaction conditions were optimised via an orthogonal experimental design, and the method's specificity and sensitivity were subsequently validated. Fifty commercially available surimi products were tested to verify its practicality in detecting target species. The results indicated that the method exhibited high specificity. Furthermore, amplification of sample fish DNA (0.2 ng/μL) generated copy numbers of 638.8, 402.5, 785.3, and 627.5 copies/μL for black carp, grass carp, silver carp, and bighead carp, respectively, with no cross-reactivity to 24 other fish species in the crystal digital reaction. The determination of the lower limit of quantification demonstrated that the method could detect sample DNA at a minimum concentration of 0.32 pg/μL for black carp, grass carp, and silver carp, and 0.064 pg/μL for bighead carp. Additionally, the method detected the minimum of copy number of 1.43 copies/μL for black carp, 2.87 copies/μL for grass carp, 2.69 copies/μL for silver carp, and 1.72 copies/μL for bighead carp. Among 50 commercial samples, 31 tested positive and 19 tested negative by dPCR analysis. Analyses of commercially available surimi samples demonstrated that the method could quantify copy numbers of components derived from black carp, grass carp, silver carp, and bighead carp. Furthermore, the qualitative results were consistent with those obtained through DNA barcoding technology. This study developed a sensitive and rapid crystal digital PCR method for quantifying ingredients derived from black carp, grass carp, silver carp, and bighead carp in marine fish surimi. This approach provided an effective technical reference for industry authorities and testing agencies to monitor the compliance labelling of freshwater fish components in marine fish surimi, ensuring transparency in surimi product authentication.

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  • 收稿日期:2025-04-18
  • 最后修改日期:2025-05-06
  • 录用日期:2025-05-06
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