DNA条形码技术在食品鉴定中的应用

近年来,国内外不断出现有关食品掺假造假等食品安全问题的报道。产品与标签不符,肉类掺假,以次充好等商业欺诈问题影响着人们的利益与健康。食品掺假造假问题越来越受到重视,而传统的检测方法已不能满足食品鉴定的需要。DNA条形码技术是从分子水平上对食品进行鉴定,弥补了传统鉴定方法的不足,其准确、高效、简单的特点为食品鉴定领域带来了新的革命。本文在简要介绍DNA条形码研究现状的基础上,主要总结目前国内外关于DNA条形码技术在食品鉴定中的应用情况,包括在渔类产品、肉类产品、可食用植物、加工食品鉴定中的应用,最后讨论DNA条形码技术在食品鉴定中的优缺点以及发展趋势。     

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。     

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。      

DNA条形码技术在石斛分类鉴定中的应用进展

由于石斛属植物种间、种内形态相似,地域分布范围广泛、杂交种众多,市场上混乱,现亟需一种简单、高效的鉴定方法对其进行准确地鉴定。DNA条形码技术利用标准的一个或多个DNA片段对物种进行鉴定,是近年来生物学研究的热点领域,也是生物学发展最迅速的方向之一。DNA条形码技术可以从分子水平弥补传统鉴定方法的一些不足。该技术具有良好的通用性,使得物种鉴定过程更加快速,已经广泛应用于石斛的鉴定研究中。本文综述了DNA条形码技术及其原理,同时讨论了基于核基因片段(ITS、ITS2)以及叶绿体基因片段(matK、rbcL、psbA-trnH)在石斛分类鉴定中的应用,以期为加大石斛分类鉴定的力度和精度,以及为DNA条形码技术在石斛分类鉴定领域拓展和应用提供一定的理论指导依据。     

宏条形码技术在食品物种鉴定中的应用及展望

食品物种鉴定是食品真伪鉴别中的重要研究内容之一。聚合酶链式反应、基因芯片等方法均被应用于食品 物种鉴定。近几年,基于高通量测序的宏条形码技术发展迅速,与基于传统测序方法的条形码技术相比,该技术具 有成本低、通量高、速度快等优点,且可以实现同时检测复杂样品中多个物种的目的,因而在食品物种鉴定方面显 现出很大的优势。本文介绍了宏条形码的含义和研究方法,综述了近年来宏条形码技术在食品物种鉴定领域的应 用,总结和讨论了宏条形码技术应用于食品物种鉴定领域面临的挑战,并对该技术的发展方向进行了展望。     

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.     

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。     

Authentication technologies using DNA-based approaches for meats and halal meats determination

Food authenticity and safety are major concerns for researchers, consumers, and particularly the meat industry. Meat products are targets for species substitution and adulteration due to their market value. Presently, the demand for halal products is witnessing a substantial increase. Therefore, it is essential to use appropriate science-based methods for determining the species origin of halal meat. DNA barcoding is a useful technique for the molecular identification of biological specimens, and raw and processed foods. The potential of using DNA barcoding is increasingly applied as an authentication tool for halal animal and meat products. Our review will bring together all DNA-based techniques that have been developed for the authenticity of meat derived from halal and non-halal animals and also their derivatives. Additionally, the present paper will highlight the possibility of using the DNA barcoding approach for halal meat authenticity.     

乌贼DNA条形码的筛选与验证

目的筛选适于鉴定乌贼的DNA条形码,建立鉴定舟山常见乌贼种类的DNA条形码技术体系。方法用聚合酶链反应(polymerase chain reaction, PCR)对现有5组DNA条形码引物组合进行了筛选,设计一对新的DNA条形码引物以供筛选备用。结果现有引物与部分乌贼的DNA模板存在错配而缺乏通用性,在部分乌贼DNA的扩增受阻。本实验设计的12S rRNA基因引物可以特异性扩增乌贼的DNA,提高了乌贼鉴定的准确度。结论本研究设计的12S rRNA基因引物可以作为现有乌贼DNA条形码鉴定的补充。     

基于DNA条形码技术的海参物种鉴定

DNA条形码技术(DNA barcoding)是一种新型高效的物种鉴别方法。本研究基于DNA条形码技术,以线粒体细胞色素氧化酶I基因(COI)和16S核糖体RNA(16S rRNA)基因作为靶基因对海参物种进行鉴别,结果表明COI基因或16S rRNA基因均能实现大部分海参的物种鉴定,部分样品需结合两个靶基因鉴定出来。将所建立的DNA条形码方法用于市售海参样品的物种鉴定,24份市售海参样品中10份市售海参样品的物种鉴定结果与标签名称相符,6份样品与标签名称不符,存在将低价海参品种标为高价海参的现象;其余8份样品的标签只有商品名但没有明确的物种信息,利用DNA条形码技术对其鉴定可得到明确的海参种名。本研究结果证实DNA条形码技术可应用于市售海参的物种鉴定,为海参的监管提供技术支撑。     

线粒体细胞色素b基因在鱼唇制品物种鉴定中适用性的研究

目的 探讨线粒体细胞色素b基因(cytochrome b, Cyt b)作为DNA条形码在鱼唇制品物种鉴定中的适用性。方法 对全国31个城市购买的252份鱼唇样品进行聚合酶链式反应(polymerase chain reaction, PCR)测序,同源基因比较分析,构建系统发育树,鉴定制作鱼唇产品的鱼种,并对其进行濒危评价分析。结果 成功鉴定250个样品,一致性物种基因序列相似性在99%以上,涉及8个鲨鱼物种,最多样品为大青鲨(Prionace glauca),占样品65.5%,其余还有镰形真鲨(Carcharhinus falciformis)、路氏双髻鲨(Sphyrna lewini)、锤头双髻鲨(Sphyrna zygaena)等7类鲨鱼物种。结论 Cyt b可以作为对鲨鱼物种进行鉴定的一种DNA条形码,在对鲨鱼种鉴定时可以使用Cyt b基因及细胞色素氧化酶亚基I基因联合鉴定条形码,为深加工海产品物种鉴定提供更多的技术支撑。     

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.     

Potential DNA markers as a rapid tracing tool for animal adulterants in vegetarian food

DNA-based methods are rapid, cost-effective and broadly applicable approaches for food authentication. Recently, the requirements for food safety and food integrity have increased with improved quality of life. Methodologies regarding food authentication based on DNA analysis are more commonly being used. With the increasing number of vegetarians, searching for markers for blind identification across kingdom species, such as an ingredient of animal origin in vegetarian food, would be valuable and attractive. Using bioinformatic analysis of an existing data source composed of 481 ultraconserved sequences, we selected 6 new candidate DNA segments that exist in most vertebrates but that do not exist in plants. Then, primers were designed for all of the candidate DNA markers, and DNA samples isolated from cow, pig, chicken, duck, soy bean, rice, pepper, wheat, sunflower and colza were amplified using each primer pair. None of the plant DNA samples generated a PCR product, while the DNA samples of animal origin were amplified successfully using 5 of the candidate segment primers; the 6th segment primer failed to amplify the DNA and was discarded. Moreover, a simulation experiment containing a plant product contaminated by an animal component indicated that the candidate DNA markers can be used for the rapid detection of animal adulterants in vegetarian products with a promising 5% detection limit. The identified candidate DNA markers for the blind identification of animal adulteration in vegetarian food may be highly desirable in the vegetarian food market, and these markers may facilitate the study of molecular technology for food authentication.     

食品组学技术在食品真伪鉴别和溯源方面应用进展

食品真伪鉴别和溯源技术是保障食品安全的重要手段。食品鉴别和溯源技术是对食品的真伪、产地和来源等相关信息进行分析及追溯,在食品质量与安全领域中占据强有力的地位。随着食品掺假频发、掺假手段多样,传统检测方法已不能满足食品的质量与安全管理要求,因此亟须发展新型快速准确的食品鉴别和溯源技术。食品组学是近年新兴的采用基因组学、转录组学、蛋白质组学及代谢组学等组学技术系统研究食品的营养与安全的一类技术,因其强大的物质鉴定功能,可以为食品鉴别和溯源提供科学依据和技术支撑。本文对食品组学技术在食品真伪鉴别和溯源方面的应用进展进行了综述,并对未来食品组学技术的发展趋势进行了展望,以期为食品质量与安全控制提供技术参考,保障人民身体健康和生活质量。     

肉制品异源基因检测技术研究进展

近年来,食品掺假逐渐成为消费者关注的重要食品安全问题之一。由于利益的驱使,在肉制品行业异 源肉质掺假现象尤其严重。目前,用于肉制品异源基因检测的技术包括普通聚合酶链式反应（polymerase chain reaction,PCR）技术、DNA指纹技术、实时荧光定量PCR技术、微滴式数字PCR技术、DNA条形码技术等。本文 综述了肉制品中异源基因检测技术的研究进展,并对每种方法的优缺点和发展趋势予以讨论。     

Applying COI Barcode to Identify Animal Origin of Food

DNA barcoding possesses advantages of high resolution, high sensitivity, and capability in capturing as much identity information as possible. However, highly varying sources of food materials and a complicated supply chain bring about challenge to the application of barcoding methods. In this study, different barcode systems were compared to establish a robust method for tracing animal species in food. Experiments on food samples from mammal, poultry, and fish proved that a mini barcode system targeting a 192 bp COI gene fragment was able to accurately identify both raw and highly processed animal food. In order to distinguish species in a mixed food sample, cloning technique was used by which as low as 10% target animal ingredient could be detected. Testing of marketed food products verified the capability of the mini barcoding method in identifying illegally claimed product.     

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.     

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.     

Applicability of DNA barcode for identification of fish species

DNA barcoding is a species identification technique, which uses a very short DNA sequence from a region of approximately 650 base-pairs in the 5'-end of the mitochondrial cytochrome c oxidase subunit I gene as a marker to identify species of mammals and fishes. The applicability of DNA barcoding for identification of fish species consumed in Japan was studied. Among thirty-one fresh or processed fishes were obtained from the market, two samples could not be identified due to lack of data in the Barcode of Life Data (BOLD) database. However, BLAST-search of 16S rRNA genes in the National Center for Biotechnology Information (NCBI) database and the PCR-RFLP method published by the Food and Agricultural Materials Inspection Center (FAMIC) were found to be applicable to identify these 2 fishes. The results show that the DNA barcoding technique is potentially useful as a tool for confirming the proper labeling of fish species in the Japanese market.     

Application of real-time PCR (qPCR) for characterization of microbial populations and type of milk in dairy food products

Dairy foods represent an important sector of the food market for their nutritional qualities and their organoleptic characteristics, which are often linked to tradition and to region. These products are typically protected by labels such as PDO (Protected Designation of Origin) and PGI (Protected Geographical Indication). Real-time PCR (qPCR) is a fundamental tool in “Food Genomics;” a discipline concerned with the residual DNA in food, which, alongside traditional physical and chemical methods, is frequently used to determine product safety, quality and authenticity. Compared to conventional or “end-point” PCR, qPCR incorporates continuous monitoring of reaction progress, thereby enabling quantification of target DNA. This review describes qPCR applications to the analysis of microbiota, and to the identification of the animal species source of milk from which dairy products have been made. These are important aspects for ensuring safety and authenticity. The various applications of qPCR are discussed, as well as advantages and disadvantages in comparison with other analytical methods.