Method Comparison of DNA Isolation and Quantification for Fish and Seafood Authenticity Determination

Q3 Environmental Science
D. Dwiyitno, Stefan Hoffman, Koen Parmentier, C. V. Keer
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引用次数: 6

Abstract

Fish and seafood products has been commonly targeted for fraudulent activities. For that reason, authentication of fish and seafood products is important to protect consumers from fraudulent and adulteration practices, as well as to implement traceability regulation. From the viewpoint of food safety, authenticity is beneficial to protect public from serious food poisoning incidents, such as due to ingestion of toxic species. Since DNA based identification depends on the nucleic acid polymerase chain reaction (PCR), the quantity and quality/purity of DNA will contribute significantly to the species authentication. In the present study, different DNA extraction and purification methods (3 classical methods and one commercial kit) were compared to produce the better isolated DNA for PCR amplification. Additionally, different methods for the estimation of DNA concentration and purity which is essential for PCR amplification efficiency were also evaluated. The result showed that classical DNA extraction methods (based on TNES-Urea) yielded a higher amount of DNA (11.30-323.60 ng/g tissue) in comparison to commercial kit/Wizard Promega (5.70-83.45 ng/g tissue). Based on the purity of DNA extract (A260/280), classical DNA extraction method produced relatively similar on DNA quality to the commercial kit (1.79-2.12). Interestingly, all classical methods produced DNA with A260/280 ratio of more than 2.00 on the blue mussel, in contrast with commercial kit. The commercial kit also produced better quality of DNA compared to the classical methods, showing the higher efficiency in PCR amplification. NanoDrop is promising as cheap, robust and safe UV-spectrophotometer method for DNA quantification, as well as the purity evaluation.Keywords: seafood authenticity, DNA isolation, polymerase chain reaction, NanoDrop, Picogreen
方法鱼和海产品真伪鉴定中DNA分离与定量的比较
鱼和海鲜产品通常是欺诈活动的目标。因此,鱼类和海鲜产品的认证对于保护消费者免受欺诈和掺假行为的侵害以及实施可追溯性法规非常重要。从食品安全的角度来看,真实性有利于保护公众免受严重的食物中毒事件,例如由于摄入有毒物种。由于基于DNA的鉴定依赖于核酸聚合酶链反应(PCR),因此DNA的数量和质量/纯度将对物种鉴定起到重要作用。在本研究中,比较了不同的DNA提取和纯化方法(3种经典方法和1种商业试剂盒),以获得更好的分离DNA用于PCR扩增。此外,还评估了对PCR扩增效率至关重要的DNA浓度和纯度的不同估计方法。结果表明,传统的DNA提取方法(基于tes - urea)比商业试剂盒/Wizard Promega (5.70-83.45 ng/g组织)产生更高的DNA量(11.30-323.60 ng/g组织)。根据提取的DNA纯度(A260/280),经典的DNA提取方法产生的DNA质量与市售试剂盒(1.79-2.12)比较接近。有趣的是,与商业试剂盒相比,所有经典方法在蓝贻贝上产生的A260/280比大于2.00的DNA。与传统方法相比,商业试剂盒也产生了更好的DNA质量,显示出更高的PCR扩增效率。NanoDrop是一种廉价、可靠、安全的紫外分光光度计,可用于DNA的定量和纯度评价。关键词:海鲜真品,DNA分离,聚合酶链反应,NanoDrop, Picogreen
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来源期刊
CiteScore
1.40
自引率
0.00%
发文量
10
审稿时长
16 weeks
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