重组酶聚合酶扩增反应结合侧流层析试纸条技术快速鉴别大西洋鳕鱼制品真伪

建立了重组酶聚合酶扩增反应(recombinase polymerase amplification, RPA)结合侧流层析试纸条技术(RPA-lateral flow dipstick, RPA-LFD)快速鉴别大西洋鳕鱼制品真伪的方法。对比大西洋鳕鱼和常用来冒充鳕鱼的鱼类线粒体基因组序列,设计针对大西洋鳕鱼的特异性探针和引物,建立针对大西洋鳕鱼的RPA-LFD检测方法。该实验筛选最佳引物和探针并评估其特异性,优化反应温度、时间和现场检测条件,并对20份市售“大西洋鳕鱼”及常见的冒充鳕鱼的鱼类制品进行现场检测,评估检测效果。实验结果表明,RPA-LFD检测方法特异性强,在44℃、14 min条件下的检测效果最好。在现场开展快速检测,效果良好,20份鳕鱼制品检测结果经过基因测序证实和产品真实成分一致。该研究建立的大西洋鳕鱼RPA-LFD检测方法特异性强、反应耗时短、操作简便、结果可视,有良好的现场快速检测应用前景,为大西洋鳕鱼制品现场快速检测提供了新思路,为水产品市场监管提供了技术支持。     

鳕鱼产品属性鉴别及细鳞壮鳕检测方法建立

鳕鱼营养丰富,深受消费者喜爱,但国内外有关鳕鱼产品标签错误标示的报道较多。为了解目前鳕鱼产品标签标示情况,对46批次常见类型鳕鱼产品的进行检测。使用SN/T 3589.7—2013对鳕鱼DNA成分检测,发现在10批次冰鲜鳕鱼产品中无法检出。使用最新DNA条形码技术鉴定,结果显示均为细鳞壮鳕,该种鱼类属于鳕形目。鳕鱼为鳕形目鱼类,因此此次检测的所有产品的标签标示均符合要求。该次研究发现了标准方法存在的不足,并自行设计了可用于细鳞壮鳕DNA成分检测的实时荧光PCR法。最终构建的检测体系特异性好,可检测低至0.1%的掺伪量。且在10批次样品中均可检出细鳞壮鳕DNA成分。此次研究对鳕鱼产品属性进行鉴别,同时发现了标准方法的不足,并针对此问题建立了细鳞壮鳕DNA成分检测方法,可作为标准方法的补充,为监管部门提供技术支持。     

TaqMan荧光PCR法快速检测太平洋鳕鱼源性成分

目的建立TaqMan荧光PCR法检测太平洋鳕鱼源性成分的方法。方法以太平洋鳕鱼线粒体细胞色素氧化酶CoxⅠ检测靶标,设计特异性引物和探针,进行特异性和灵敏度试验,并对市售样品进行检测。结果该方法引物探针特异性和灵敏度较好,可以良好区分近属鳕鱼和其他科鱼类,灵敏度达到0.04 ng/μL。市售样品中, 90.9%太平洋鳕鱼切片及71.4%太平洋鳕鱼罐头中检出太平洋鳕鱼成分。结论该方法的特异性和灵敏度较好,适合用于大多数制品中太平洋鳕鱼成分的真实性甄别。     

主要进出口经济鱼类及其制品分子鉴定技术的研究进展

随着我国进出口贸易的不断发展,进出口鱼类品种繁多,数量巨大。但部分国内外企业受高额利润驱使,以假充真、以劣充好事件时有发生,鱼类制品更是真伪难辨,掺假状况极其普遍。通过形态学和生物学方法已经不能满足对鱼类种属鉴定。近年来,随着分子生物技术的迅猛发展,分子检测技术在鱼类品种鉴定领域得到广泛应用。本文针对不同分子鉴定技术进行阐述,包括普通聚合酶链反应(polymerase chain reaction,PCR)技术、多重PCR技术、实时定量PCR技术、DNA条形码技术、环介导等温扩增检测技术(loop-mediated isothermal amplification,LAMP)、聚合酶链反应-限制性酶切多态性技术(polymerase chain reaction-restriction fragment length polymorphism,PCR-RFLP)以及随机扩增多态性DNA检测技术(random amplified polymorphic DNA),并根据不同检测技术的特点进行分析,为进出口鱼类鉴定提供技术支撑。     

运用DNA条形码技术分析市售鱼类及制品的物种真实性

目的:运用准确快速的鱼类品种鉴定方法,对上海市售鱼类制品标识符合性进行调查。方法:利用DNA条形码技术,以动物线粒体细胞色素C氧化酶(Cytochrome C Oxidase Subunit I,COI)基因序列为鉴定靶标,对采集的63种市售鱼类样本进行序列分析比对后,分析样品的标注名称与真实物种名的一致性。结果:经序列测定和比对,13份样品的鱼类品种名称与标注名称不一致,占20.63%,此外有19份样品标注名为一类鱼的总称或俗称,占30.16%。结论:目前,我国对鱼类及其制品物种真实性鉴定研究亟待发展,鱼类标注物种名称混乱,分类不清,概念模糊,急需规范化。     

3组常见马源性成分鉴定引物特异性和灵敏度比较

比较筛选基于聚合酶链式反应(polymerase chain reaction, PCR)技术的马源性成分鉴定方法中高灵敏度和特异性引物。方法 选取来自国标法、专利和文献报道的3组马源性成分鉴定引物, 比较其灵敏度和特异性, 筛选高灵敏度和特异性引物。结果 3组引物的灵敏度和特异性存在差异, 以线粒体DNA为靶标的Ⅱ组马源性成分鉴定引物的特异性和灵敏度优于分别以线粒体和核基因组DNA为靶标的马驴通用鉴定引物, 检出最低限度均满足检测需求。结论 通过比较马源性成分鉴定的3组PCR引物, 以线粒体DNA为靶标的Ⅱ组马源性成分特异性鉴定引物可作为马成分鉴定的首选, 线粒体和核基因组靶标联合可提高马成分鉴定的准确性并可用于杂交种鉴定, 为马源性成分及产制品鉴定提供了一定基础。     

太平洋无须鳕鱼及其制品的PCR鉴定

目的建立一种SYBR Green荧光PCR法定性检测太平洋无须鳕鱼成分的分析SYBR Green荧光PCR定性检测方法。方法根据太平洋无须鳕鱼线粒体细胞色素氧化酶CoxⅠ基因中的保守基因序列设计一对太平洋无须鳕鱼特异性引物,通过SYBR Green荧光PCR法进行PCR扩增反应,可对DNA提取试剂盒提取的太平洋无须鳕鱼成分DNA进行特异性检测。结果该SYBR Green荧光PCR定性检测方法的引物对于太平洋无须鳕鱼成分的检测特异性良好;方法的灵敏度分别为0.06 ng/μL太平洋无须鳕鱼DNA和质量分数为0.01%太平洋无须鳕鱼肉粉。通过对市售太平洋无须鳕鱼片、太平洋鳕鱼片和太平洋无须鳕鱼肝油的检测,该方法可检测出鱼片中的太平洋无须鳕成分。结论该检测方法特异性强,灵敏度高,能够用于食品中太平洋无须鳕鱼成分的真实性鉴别。     

高通量二代测序基因条形码技术在动物源性制品物种鉴定中的应用

该研究利用二代测序和DNA条形码技术对生物制品中动物源性成分鉴定方法进行探索性研究。首先对常见的哺乳动物、禽类、鱼类物种以及多种混合样本进行了核酸提取,并利用PCR技术对其线粒体基因16S rRNA区域354 bp的检测位点进行扩增。使用Illumina Miseq二代测序技术获取所有序列信息,并与Gen Bank数据库比对分析样本中的物种组成。结果显示,混合样品中所有动物源性成分均得到正确鉴定;鹅和鸭核酸的最低检测下限为0.5ng/μL;市售商业化动物源性制品经检测发现2起标签不符问题。该检测方法在一个高通量测序和结果比对分析实验周期内,实现了混合DNA片段序列信息的一次性获取。检测结果准确,适用范围广,有望为动物制品标识符合性检查、打击走私等方面提供技术保障。     

5种海鱼挥发性风味成分分析

采用顶空固相微萃取-气相色谱-质谱联用技术,对黄鱼、鳗鱼、鳕鱼、鲳鱼、马鲛鱼等5种海鱼进行挥发性成分分析。研究结果表明:在黄鱼、鳗鱼、鳕鱼、鲳鱼、马鲛鱼中分别鉴定出45、41、62、53、39种挥发性成分,其中5种鱼类样品中均被检出的成分有1-戊烯-3-醇、1-辛烯-3-醇、戊醛、已醛和4-庚烯醛。     

三引物PCR检测牛羊猪源性成分的方法

建立了三引物PCR鉴定牛羊猪源性成分的方法,为肉类及产制品掺假鉴定或肉品溯源提供了简单易行的方法。通过对比牛羊猪的线粒体DNA(mt DNA)全序列,在保守区设计通用引物,在特异性区域设计特异引物并结合三引物PCR技术,扩增3个物种的特异性片段,羊、牛和猪产生片段大小分别为570、549、420 bp,依据片段大小可以在一次PCR中鉴定3个物种。建立的方法简单易行,特异性和灵敏度好,可以在肉类及产制品鉴定和溯源中应用,提高了检测效率。     

Species Identification and PCR-RFLP Analysis of Cytochrome b Gene in Cod Fish (Order Gadiformes) Products

ABSTRACT: Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) profiles using the PCR product tRNA^Glu-cytochrome b of 9 standard cod fish, all of which are imported into Japan under the name "cod fish," were investigated as a means of rapid species identification of imported cod fish products. Endonuclease digestion using 4 restriction enzymes ( Alu I, Fok I, Mbo I, and Mse I) generated different digestion patterns for the standards, enabling species identification in imported cod fish products by comparison to the standards'RFLP profiles. Additionally, we conducted a more strict check by phylogenetic analysis on imported cod fish products with 13 standards (including 4 additional cod species from GenBank) using the mitochondrial cytochrome b gene (385 nt). Consequently, it was found that the PCR-RFLP method was sufficient for rapidly screening cod products, and in cases that require conclusive identification, phylogenetic analysis was the most suitable.     

Nuclear DNA barcodes for cod identification in mildly-treated and processed food products

Gadoids are a group of fish with historical importance in the fishing industry. The high demand for cod is one of the reasons why cod products are often mislabelled, and numerous observations have been made on the replacement of Atlantic cod (Gadus morhua) by cheaper species or its illegal capture in contravention of fish quotas. Fish species identification is traditionally based on morphological features, but this may be difficult in case of heat-treated or processed products, or where the species look similar, as in the Gadoid group. DNA-based approaches (using either nuclear or mitochondrial DNA) are most commonly used in this case, due to their high specificity and to the high resilience of the target molecules to food processing techniques. In this article, we identified, using an automated screening approach, novel barcode regions and their associated primers in the nuclear genome, to be used for the efficient identification of Gadoids. The barcode regions were tested on official and commercial samples, raw or mildly treated products, like frozen, or salted, as well as pre-cooked complex mixtures and processed samples, using next-generation sequencing (NGS) technique. The method proposed could complement existing fish identification strategies in establishing an efficient framework to detect and prevent frauds along the food chain.     

A short‐cut DNA extraction from cod caviar

Caviars represent the most consumed form of fish roe products. Due to high demand, ingredient roes of fish are often susceptible to illegal substitution with those of related fish. This study developed a simple and inexpensive protocol enabling the rapid extraction of DNA of acceptable quality and amount to PCR amplification from both cod caviars and their ingredient pollack roes. The protocol was based on extracting total genomic DNA from eggs using urea and a Chelex 100 chelating resin, and could be completed in less than 15 min. Approximately 8 µg of DNA were reproducibly obtained from single eggs of cod caviars and pollack roes in eight individual experiments, and the quality and amount of DNA were sufficient to serve as template for hundreds of PCR reactions of polymorphic DNA markers for phylogenetic analysis. Being applicable to various caviars, this protocol can be useful to detect illegal substitution among ingredient roes of related fishes in PCR‐based food inspection. Copyright © 2005 Society of Chemical Industry     

Molecular Identification of Commercial Spicy Pollack Roe Products by PCR-RFLP Analysis

ABSTRACT: Spicy pollack roe products are a popular seafood item made from fish eggs that should be made with salt-cured mature roes of walleye pollack Theragra chalcogramma . Because of high demand and poor catch of walleye pollack, however, spicy pollack roe products are often susceptible to substitution with roes of closely related codfish. In this study, a simple method identifying the ingredients of commercial spicy pollack roe products was developed to differentiate walleye pollack from codfish substitutes such as gray cod Gadus macrocephalus using PCR-RFLP (Restriction Fragment Length Polymorphism) analysis. PCR amplification of the mitochondrial cytochrome b gene yielded single fragments commonly from pollack and cod. Direct digestion of the PCR products with Mph 11031 restriction enzyme showed an unique restriction fingerprint only in pollack. This PCR-RFLP analysis enabled the reliable identification of commercial spicy pollack roe products made by only pollack roes from products padded with cod roes. It thus can be useful to expose substitution of pollack roes with lower valued codfish roes in commercial spicy pollack roe products.     

Mitochondrial cytochrome b DNA sequence variations: an approach to fish species identification in processed fish products

The identification of fish species in food products is problematic because morphological features of the fish are partially or completely lost during processing. It is important to determine fish origin because of the increasing international seafood trade and because European Community Regulation 104/2000 requires that the products be labeled correctly. Sequence analysis of PCR products from a conserved region of the cytochrome b gene was used to identity fish species belonging to the families Gadidae and Merluccidae in 18 different processed fish products. This method allowed the identification of fish species in all samples. Fish in all of the examined products belonged to these two families, with the exception of one sample of smoked baccalà (salt cod), which was not included in the Gadidae cluster.     

基于近红外光谱结合机器学习的鳕鱼品种二分类方法研究

目的 鳕鱼假冒伪劣事件层出不穷,为保障鳕鱼肉制品购买者能够买到放心的鳕鱼产品,探索适合分析鳕鱼近红外光谱数据的机器学习模型,实现鳕鱼品种的快速二分类。方法 选取挪威大西洋真鳕、冰岛黑线鳕等8种鳕鱼,对其研磨物进行傅里叶变换近红外光谱测试,并采用Min-Max归一化和独立成分分析法对近红外光谱数据进行预处理和降维,进一步分别使用9种机器学习模型进行二分类,通过6项指标对比各个模型的预测效果,从中选出最适合鳕鱼二分类的模型。结果 本文提出的独立成分析法结合支持向量机的鳕鱼品种二分类模型的预测准确率可达到97.2%,F1分数可达到97.3%,召回率达到99.4%。结论 本研究可实现较为准确的大西洋鳕鱼和非大西洋鳕鱼品种的分类,为鳕鱼品种鉴别提供了方法依据。     

Extending shelf life of desalted cod by high pressure processing

Salted fish need to be rehydrated before eaten, but rehydrated fish have a relatively short shelf life. To increase the shelf life the products can e.g. be frozen, packed in modified atmosphere or processed by high pressure (HPP). Here, rehydrated cod was packed with different packaging regimes; in vacuum, with CO₂ emitter or in modified atmosphere [MAP], either alone or in combination with HPP. A shelf life study was performed, and headspace gas composition, drip loss, pH, colour, texture and microbial counts were assessed in the packaged and processed portions. The results showed that a shelf life of minimum 49 days can be obtained by treating the rehydrated cod by HPP or by combining HPP with modified atmosphere or in combination with a CO₂ emitter. The results of this study have shown that different packaging and processing methods can increase the shelf life of desalted cod.Industrial relevanceThe growing trend and availability of “ready to cook” products are forcing the food industry to increase production of convenience products. Today desalted saltfish or clipfish need to be consumed immediately, stored chilled for a few days, stored frozen or being packed and/or processed to extend its shelf life. The use of high pressure processing represents a promising strategy to enhance the shelf-life of fish products. This study shows that HPP alone or combined with CO₂ can extend the shelf life of rehydrated cod. Hence, these results can open up for new products.     

Biological value and assimilation of sarcoplasmic protein concentrates from commercial fish products and the effect of their consumption on rats]

The biological value of 3 concentrates of sarcoplasmatic proteins from industrial fish (minthal, cod, putassu) was assayed in the 4-week experiment on growing male Wistar rats by the methods of counting the coefficients of protein effectiveness and pure protein effectiveness that comprised 100, 110 and 110% in relation to the corresponding parameters obtained with casein. The actual assimilation of the fish product proteins proved to be significantly higher than that of casein. Hypocholesterolemic and hypolipidemic effects of the protein products studied are, probably, caused by the presence of pectin.     

Effect of Fish and Oil Nature on Frying Process and Nutritional Product Quality

ABSTRACT: The modifications on a lean fish (cod—Gadus morhua) and a fatty fish (farmed salmon—Salmo salar) after the application of pan-frying using 2 types of oil with different lipid profile (extra virgin olive oil and sunflower oil) was the aim of this study. Fat content and total energetic value increased significantly after the frying process only in the lean fish, without relevant changes in the fatty fish. Extra virgin olive oil led to a higher fat absorption rate than sunflower oil in both fish. Frying hardly affected the lipid profile of farmed salmon regardless the oil used, however it drastically changed in fried cod compared to raw cod. Omega-6/omega-3 ratio increased from 0.08 in raw cod to 1.01 and 6.63 in fried cod with olive oil and sunflower oil, respectively. In farmed salmon, the omega-6/omega-3 ratio was 0.38 (raw), and 0.39 to 0.58 in fried salmon. The amount of EPA + DHA slightly decreased with frying in salmon, and increased in cod. The type of oil has more influence in the nutritional fish quality for the lean fish compared to that of the fatty fish. The use of extra virgin olive oil was efficient to avoid a significant increase of the lipid oxidation intensity during frying in cod but not in salmon. Practical Application: Food modifies its composition and nutritional value with the application of cooking technologies. As most food table composition tables are based on raw food products, this article contributes with interesting data on pan-fried fish composition, which may improve the approach to achieve a real intake of healthy nutrients as omega 3 fatty acids.     

Process control of lightly salted wild and farmed Atlantic cod (Gadus morhua) by brine injection, brining, and freezing--a low field NMR study

The aim of this study was to use low-field nuclear magnetic resonance (NMR) and traditional chemical methods to investigate the physical and chemical differences in wild and farmed cod processed pre- and postrigor, and how these properties were affected by brine injection, brining, and freezing. In prerigor processed farmed or wild cod, brine injections followed by brining for 2 d, with brine concentrations up to 5.5% and 4%, respectively, were not sufficient to reach a muscle salt concentration of 2% as aimed for, while wild cod processed postrigor had sufficient salt uptake after the same processing. Low-field NMR gave valuable information about the differences in the muscle structure between wild and farmed cod as well as the state of the water in the muscle during brine injection, brining, and during rigor tension. Low-field NMR is, therefore, a valuable tool that can be used to optimize the salting and storing processes of lightly salted cod products from both wild and farmed cod. For farmed cod to be used in the production of lightly salted products further research is needed. Practical Application: Optimal processing of lightly salted cod products is important to the fish industry, due to an increasing market for this product in southern Europe. Farmed cod, which is seen as a potential steady raw material source for this production, differs considerably from its wild counterparts by having other chemical and physical muscle properties, such as lower water content and lower pH. With the processing procedures used today the farmed cod can, therefore, only be used in some of the products, where wild cod is currently used as raw material. It is, therefore, important that the processing of these products is optimized with regard to these differences in the raw material. This study gives a valuable contribution to further studies about optimal combinations of brine injections, brining, and freezing of pre- and postrigor processed farmed compared to wild cod.