基于微型DNA条形码的多种动物源性成分的鉴定

通过分析51?种动物的DNA条形码序列,新设计一对微型DNA条形码的通用引物,建立一种联合微型DNA条形码和克隆测序法对动物源性多组分样品进行准确、高效鉴定的方法。用微型DNA条形码分别鉴定11?种常见食用肉类,判断其对物种鉴定的准确性,并比较其与标准DNA条形码对物种的鉴定效果,然后将11?种常见商品肉类等比例混合,探究克隆测序法对混合肉样品的鉴定效果。结果表明：引物有较好的通用性,能对具有不同的引物同源性水平的11?个物种进行有效扩增并获单一条带。微型条形码能准确鉴定11?个肉类物种,与常规DNA条形码鉴定效果一致。经克隆测序法,把混合样品的9?个物种一并检出,总体上达到了较高的检出效率,同时深入讨论了未能检出全部物种的原因。微型条形码良好的物种鉴定能力的研究结果为联合微型DNA条形码与二代测序技术进行混合样品中物种的高效鉴定提供参考依据。     

Authentication of Small Berry Fruit in Fruit Products by DNA Barcoding Method

Small berry fruit products are gaining an expanded market due to their high nutrition value. However, the authenticity of products is challenged by adulteration and mislabeling. To establish an accurate and robust method for identifying both known and unknown fruit species in small berry fruit products, DNA barcoding technology based on Sanger sequencing was adopted. To overcome the influence of processing conditions on DNA recovery, mini‐barcodes of rbcL and ITS and a medium‐barcode of psbA‐trnH were applied. To identify ingredients in products containing mixed species, plasmid cloning was applied to separate mixed barcodes. The method established in this paper could detect 1% to 10% target species in mixed fruit juice.     

DNA条形码技术在肉及其制品中的应用

DNA条形码技术作为一种新的分子生物学检测技术在鉴定和区分各物种和物种间亲缘关系方面得到了广泛应用,该技术能实现对肉的快速、准确检测。DNA条形码已成为生物学领域发展最迅速的一种技术,该技术基于广泛的物种基因数据库信息,在肉品研究中具有良好的应用前景。本文简述DNA条形码技术的基本原理,并基于DNA水平上与其他相关技术进行比较,综述其在物种鉴定、肉品质量安全、商业欺诈等方面的应用,并对该技术在今后肉品科学研究中的应用进行展望。      

基于DNA条形码技术的储粮害虫碎片鉴定研究

储粮害虫(螨)个体微小,种类繁多,与人类生活紧密相关,具有重要经济意义,储粮害虫快速鉴定是进行储粮害虫综合防治的前提和基础。传统的形态鉴定技术难以实现粮食及食品中害虫的非成虫态和碎片的准确鉴定,DNA条形码是近年来出现的物种分子鉴定技术,能够实现对昆虫非成熟虫态及碎片的快速鉴定。本研究利用DNA条形码技术和联合开发的中国储粮害虫DNA条形码鉴定系统(GPDBIS),针对某食品有限公司送检的储粮害虫碎片样品进行了序列测定、比对和分析,最终实现碎片的快速准确鉴定。结果显示,在所检测的7个害虫碎片样品中,有4头为锈赤扁谷盗Cryptolestes ferrugineus Stephens,3头为赤拟谷盗Tribolium castaneum Herbst,DNA条形码技术是储粮害虫快速准确鉴定的有效手段。     

基于16SrRNA基因DNA条形码鉴定美洲鳗、欧洲鳗、日本鳗

为满足鳗鱼养殖、加工、贸易企业及执法部门的需求,建立我国主要养殖鳗鱼美洲鳗(Anguilla rostrata)、欧洲鳗(Anguilla anguilla)、日本鳗(Anguilla japonica)的物种DNA条形码鉴定方法。以扩增16S rRNA基因的通用引物扩增美洲鳗、欧洲鳗、日本鳗的16S rRNA基因片段,各获得1条16S rDNA片段,测序结果表明,前二者的长度均为638 bp、日本鳗的长度为640 bp,为了使物种鉴定方法更简便,结果更准确,从各自片段中截取具有物种特异序列的片段(243 bp),作为3种鳗鱼的标准DNA条形码,设计两端碱基数分别为22 bp与23 bp的正向与反向引物,建立聚合酶链式反应-DNA条形码检测方法。3年来的应用结果表明:建立的美洲鳗、欧洲鳗、日本鳗3种鳗鱼的DNA条形码鉴定方法,操作简便、准确、稳定,可应用于3种鳗鱼的物种鉴定。     

A comparative study of different DNA barcoding markers for the identification of some members of Lamiacaea

The objective of the present work is to evaluate the efficacy of a DNA barcoding approach as a tool for the recognition of commercial kitchen spices belonging to the Lamiaceae family that are usually sold as enhancers of food flavor. A total of 64 spices samples, encompassing six different genera (i.e. Mentha, Ocimum, Origanum, Salvia, Thymus and Rosmarinus) were processed with a classical DNA barcoding approach by amplifying and sequencing four candidate barcode regions (rpoB, rbcL, matK and trnH-psbA) with universal primers. Results suggest that the non-coding trnH-psbA intergenic spacer is the most suitable marker for molecular spices identification followed by matK, with interspecific genetic distance values ranging between about 0% to 7% and 0% to 5%, respectively. Both markers were almost invariably able to distinguish spices species from closest taxa with the exclusion of samples belonging to the genus Oregano. Moreover, in a context of food traceability the two markers are useful to identify commercial processed spice species (sold as dried plant material). We also evaluated the potential benefits of a multilocus barcode approach over a single-marker and although the most suitable combination was the matK + trhH-psbA, the observed genetic distances values were very similar to the discriminatory performance of the trnH-psbA. Finally, this preliminary work provide clear evidences that the efficacy of a DNA barcoding approach to the recognition of commercial spices is biased by the occurrence of taxonomic criticisms as well as traces of hybridization events within the family Lamiaceae. For this reason, to better define a more practical and standardized DNA barcoding tool for spices traceability, the building of a dedicated aromatic plants database in which all species and cultivars are described (both morphologically and molecularly) is strongly required.     

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。     

基于PCR技术的肉类成分溯源鉴定方法研究进展

近几年屡屡曝光的食品安全事故引起了社会的广泛关注,食品安全已经成为社会共同关注的问题,肉类掺假造假现象更是层出不穷,其中用低价鸡肉、鸭肉、猪肉等掺入、冒充牛羊肉成为主要的掺假方式。国内外进行肉类掺假鉴定主要以核酸作为靶标,核酸鉴定也是物种鉴别最常用、最核心的方法,以DNA检测为基础建立起来的DNA条形码、多重PCR、荧光定量PCR、荧光探针等技术也得到空前发展和广泛应用。我国针对动物源性成分检测也制定了相关国家标准和行业标准,但大多现行标准中基于DNA检测建立的PCR技术只能检测单一物种,滞后于技术的发展。目前,基于PCR发展起来的衍生技术凭借其高灵敏度、强特异性和高通量等优势在动物源性成分检测工作中显示出巨大潜力,也是肉类成分鉴定未来的重要方向。本文综述了PCR技术在肉类检测中的研究概况和现行标准的技术概况,以期为肉类成分鉴定研究提供信息。     

二维码技术的应用现状及用于食品添加剂 系统管理的可行性

二维码识别技术是一种具有汉字编码特性, 可以储存大量产品信息, 使用方式简单便捷的数据携带、传递的高科技手段。基于该技术, 本文探讨了食品添加剂可追溯系统的补充方案。该方案不仅可以为消费者提供详细的食品添加剂信息, 而且为生产企业和监管部门提供了良好的管理和监督平台。     

Tetrodotoxin detection and species identification of pufferfish in retail roasted fish fillet by DNA barcoding in China

This study identifies the pufferfish species and detects tetrodotoxin (TTX) in roasted fish fillet samples collected in Beijing, Qingdao and Xiamen, China. The cytochrome c oxidase I (COI) gene was used as the target gene for identification of the pufferfish species in the samples. Enzyme-linked immunosorbent assay (ELISA) screened the TTX levels in samples that had been detected as containing pufferfish by DNA barcode. A total of 125 samples were identified by DNA barcodes; 32 (26%) samples contained pufferfish composition and, among them, 26 (81%) were the highly toxic species Lagocephalus lunaris. All 32 samples containing the pufferfish composition were positive for TTX with levels ranging from 100 to 63 800 ng g^–1. Most of the 32 samples contained the highly toxic L. lunaris. Based on the results, we suggest that the monitoring of roasted fish fillet should be strengthened and the processing procedures should be standardised to minimise TTX poisoning caused by pufferfish.     

DNA条形码在肉制品掺伪中非定向筛查技术的研究

目的对基于DNA条形码的肉制品掺伪中非定向筛选技术进行探索。方法以线粒体基因组中COI基因为靶标,设计猪、牛、羊、马、鸡、鸭、鹅、鼠8种动物源物种间的通用引物和特异性引物,建立禽畜肉的DNA条形码检测方法。同时应用该技术对50份市售的肉制品进行检测。结果本研究建立了DNA条形码筛选技术,电泳条带通过基因测序能正确识别猪、牛、羊、马、鸡、鸭、鹅、鼠8种动物源成分,结合分子克隆技术能实现对混合肉的检测。50份市售肉制品的DNA条形码结果显示,16份掺杂了除牛羊肉以外的其他禽畜肉。结论 DNA条形码技术能打破传统标准中PCR法检测目标唯一性的局限,具有良好的应用前景。     

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.     

Potential use of DNA barcodes in regulatory science: applications of the Regulatory Fish Encyclopedia

The use of a DNA-based identification system (DNA barcoding) founded on the mitochondrial gene cytochrome c oxidase subunit I (COI) was investigated for updating the U.S. Food and Drug Administration Regulatory Fish Encyclopedia (RFE; http://www.cfsan.fda.gov/-frf/rfe0.html). The RFE is a compilation of data used to identify fish species. It was compiled to help regulators identify species substitution that could result in potential adverse health consequences or could be a source of economic fraud. For each of many aquatic species commonly sold in the United States, the RFE includes high-resolution photographs of whole fish and their marketed product forms and species-specific biochemical patterns for authenticated fish species. These patterns currently include data from isoelectric focusing studies. In this article, we describe the generation of DNA barcodes for 172 individual authenticated fish representing 72 species from 27 families contained in the RFE. These barcode sequences can be used as an additional identification resource. In a blind study, 60 unknown fish muscle samples were barcoded, and the results were compared with the RFE barcode reference library. All 60 samples were correctly identified to species based on the barcoding data. Our study indicates that DNA barcoding can be a powerful tool for species identification and has broad potential applications.     

COI序列应用于羊肉掺伪非定向筛查技术的研究

本项目研究了应用COI序列对羊肉中掺入的其他动物源材料进行非定向筛查,并对山羊肉及绵羊肉进行分辨。利用了DNA条形码技术对六个羊品种(系)的线粒体基因组细胞色素酶基因片段进行引物筛选,并利用筛选出的引物对七种动物源样本的COI片段进行扩增,以确定所筛选出的引物是否能将羊肉与其他动物源样品进行有效区分。以LCOI490/HCO2198、F1-1/R1-1、ITS3/ITS4为引物,通过对36份血液样本(山羊3个品种,绵羊3个品种)的基因组DNA进行PCR扩增,其中以F1-1/R1-1为引物得到的PCR产物电泳检测条带清晰,结果重现性好;利用F1-1/R1-1引物对鸡肉、猪肉、驴肉、牛肉、莱芜黑山羊肉、洼地绵羊肉和鸭肉等七种样本的COI片段进行扩增,并对扩增片段进行测序,山羊、绵羊之间的序列同源性仅为36.62%,其他各品种间的序列差异也很明显。结果表明:以F1-1/R1-1为专用引物可以有效区分羊肉与其他动物源样品,并能够对山羊肉及绵羊肉样品进行有效区别。     

EAN128条码在食用农产品流通中的应用

日益突出的食品安全问题已经影响到了我国的经济发展。介绍了EAN.UCC系统中的EAN128条码,并结合其特点应用于食用农产品的生产、流通、销售各环节,为食用农产品供应链提供有效的信息标识,从而建立紧随其物流的信息流,帮助缓解食品安全问题。     

DNA barcoding detects market substitution in North American seafood

Seafood authentication and food safety concerns are a growing issue in today’s global marketplace, although traditional morphology-based identification keys and existing molecular approaches have limitations for species identification. Recently, DNA barcoding has gained support as a rapid, cost-effective and broadly applicable molecular diagnostic technique for this purpose. However, the maturity of the barcode database as a tool for seafood authentication has yet to be tested using real market samples. The present case study was undertaken for this reason. Though the database is undergoing continual development, it was able to provide species matches of >97% sequence similarity for 90 of 91 samples tested. Twenty-five percent of the samples were potentially mislabeled, demonstrating that DNA barcodes are already a powerful tool for the identification of seafood to the species level. We conclude that barcodes have broad applicability for authenticity testing and the phylogeographic patterning of genetic diversity can also inform aspects of traceability.     

DNA barcoding reveals fraudulent substitutions in shark seafood products: The Italian case of “palombo” (Mustelus spp.)

In food safety and traceability, consumers are more and more demanding about composition and provenance of processed seafood products. In the trade of many species, manufacturing alterations usually bring to the loss of any morphological diagnostic features of the species, enhancing the possibility of fraudulent substitutions and incorrect product labeling. In this study, we used a DNA barcoding approach to identify species substitutions cases in shark slices sold in Italy under the vernacular name of “palombo” (that is referred to the triakiids Mustelus mustelus and Mustelus asterias for the Italian regulation). We produced the coxI barcode sequence (550 bp long) for all the analysed specimens, and we compared them with reference sequences from different databases (GenBank and BOLD), using two bioinformatic identification methods, one of them developed in our laboratory. Results showed a high amount of commercial frauds rising the 80% of analysed “palombo” slices and highlighting a relevant economical impact for consumers.     

Molecular identification of dried squid products sold in China using DNA barcoding and SYBR green real time PCR

ABSTRACT

Dried squid products are popular in China as a snack food, side dishes, or refreshments, and the market appeal can be reflected by the high price that occasionally reaches 497 RMB per kg. However, the absence of harmonisation around the definition of squid, as well as the problems with visual inspection for processed seafood products, make alternative species substitution for dried squid products a frequent occurrence. The aim of the present study was to apply a DNA barcoding approach for species identification of 48 dried squid products collected from the largest online shopping platform in China. Moreover, we also developed a novel SYBR green real-time PCR assay (simplex and duplex followed by a melting curve analysis) specific for Illex argentinus and Todarodes pacificus based on cytochrome C oxidase subunit I (COI) gene.

Results highlighted the successful DNA extraction and PCR amplification of a 655 bp COI gene fragment from all products. A maximum similarity value in the range of 98-100% was obtained for all readable sequences using the BOLD and BLAST public databases and four species (Dosidicus gigas, Uroteuthis edulis, I. argentinus, and T. pacificus) were identified. The specificity of the designed primer sets was confirmed against 23 non-target species, and the newly developed methods were successfully applied to screen I. argentinus and T. pacificus in dried squid products.

Overall, DNA barcoding is a robust tool for seafood species identification and the novel method is effective in screening I. argentinus and T. pacificus in food products.     

Effect of dietary macronutrients on aflatoxicosis: a mini‐review

Aflatoxin is a toxin produced by Aspergillus species of fungi. The main route of aflatoxin exposure is through the diet. Indeed, long‐term aflatoxin exposure is linked to the development of hepatocellular carcinoma (HCC). Aflatoxin causes aflatoxicosis, which can be affected by several factors and is prevalent in many developing Asian and African countries. This mini‐review discusses the effects of carbohydrate, fat and protein on aflatoxicosis based on findings from animal and human studies. It was found that high carbohydrate intake enhanced aflatoxicosis occurrence, while low ingestion of carbohydrate with caloric restriction slowed the symptoms associated with aflatoxicosis. Additionally, diets with low protein content worsened the symptoms related to HCC due to aflatoxin exposure. Nevertheless, a study reported that a high‐protein diet favored detoxification of aflatoxin in vivo. There were also conflicting results on the influence of dietary fat, as high ingestion of fat enhanced aflatoxicosis development as compared with a low‐fat diet. Moreover, the type of fat also plays a significant role in influencing aflatoxin toxicity. In regard to food safety, understanding the influence of macronutrients toward the progression of aflatoxicosis can improve preventive measures against human and animal exposure to aflatoxin. © 2017 Society of Chemical Industry     

DNA barcoding for the species identification of commercially important fishery products in Indonesian markets

The DNA barcoding approach was used for the species identification of 44 Indonesian commercial fishery products. Additionally, the intronless nuclear rhodopsin gene fragment (RH1) was added to the analysis to enable the identification of species not yet barcoded and possible hybrids. The 655‐bp cytochrome C oxidase subunit I (COI) gene fragment marker was successfully amplified and used to identify 86% of the total fish samples at the species level using the BOLD and BLAST public databases. Moreover, the RH1 marker was used to complete COI analysis. For a number of fish species, the COI sequences (six species) and RH1 sequences (eight species) were the first entries submitted to GenBank. This study demonstrated that COI barcoding is a promising tool for Indonesian fishery products and confirmed that it could be adopted in the future for regular seafood control as part of the Indonesian integrated food traceability system.