肉制品中动物源性成分DNA检测方法的研究进展

肉制品主要成分标识的真实性是全球重要的食品安全问题之一,特别是肉制品中动物源性成分的掺假和标识问题已引发全球关注。如何对肉制品中动物源性成分进行鉴定和标识已成为产品真实性鉴定的热点。基于DNA分子稳定性强的优点,DNA检测技术被广泛用于食品安全检测和监测诸多领域,体现出了灵敏度高、特异性强等优势。本文重点从动物源性检测的靶序列DNA选择和DNA分析技术研究2个方面,阐述了肉制品中动物源性成分定性、定量检测技术的研究和应用,并讨论动物源性成分定量分析的可能性,为我国实施动物源性成分量化监管提供思路。     

A PCR assay to detect trace amounts of soybean in meat sausages

Soybean proteins are the most widely used source vegetable proteins in the meat industry because of several interesting characteristics. As soybean is included in the group of ingredients potentially allergenic, if not declared, it can be considered a hidden allergen, representing a potential risk to sensitised individuals. The aim of this work was to optimise and apply DNA‐based techniques for soybean detection in meat products, as alternative to the currently used protein‐based methods. The optimised polymerase chain reaction (PCR) protocol targeting the soybean lectin gene enabled the detection of the addition of 0.1% and 0.5% of hydrated textured protein, which corresponded to 0.01% and 0.06% (w/w) of soybean protein in unprocessed and heat‐processed pork meats, respectively. The established PCR technique, when applied to commercial meat sausages (eighteen samples), confirmed the presence of soybean declared in nine samples and indicated the presence of soybean in four samples with no labelled information about soybean. Additionally, the event‐specific PCR detection of Roundup Ready^® soybean was also performed, enabling the detection of transgenic DNA in three samples.     

Quantitative detection method for Roundup Ready soybean in food using duplex real‐time PCR MGB chemistry

BACKGROUND: Methodologies that enable the detection of genetically modified organisms (GMOs) (authorized and non‐authorized) in food and feed strongly influence the potential for adequate updating and implementation of legislation together with labeling requirements. Quantitative polymerase chain reaction (qPCR) systems were designed to boost the sensitivity and specificity on the identification of GMOs in highly degraded DNA samples; however, such testing will become economically difficult to cope with due to increasing numbers of approved genetically modified (GM) lines. Multiplexing approaches are therefore in development to provide cost‐efficient solution. RESULTS: Construct‐specific primers and probe were developed for quantitative analysis of Roundup Ready ^® soybean (RRS) event glyphosate‐tolerant soybean (GTS) 40‐3‐2. The lectin gene (Le1) was used as a reference gene, and its specificity was verified. RRS‐ and Le1‐specific quantitative real‐time PCR (qRTPCR) were optimized in a duplex platform that has been validated with respect to limit of detection (LOD) and limit of quantification (LOQ), as well as accuracy. The analysis of model processed food samples showed that the degradation of DNA has no adverse or little effects on the performance of quantification assay. CONCLUSION: In this study, a duplex qRTPCR using TaqMan minor groove binder‐non‐fluorescent quencher (MGB‐NFQ) chemistry was developed for specific detection and quantification of RRS event GTS 40‐3‐2 that can be used for practical monitoring in processed food products. Copyright © 2010 Society of Chemical Industry     

食品添加剂在肉制品保藏中的应用

介绍了肉制品保藏中常用的食品的添加剂的功能特性及其应用。     

上海市肉制品中动物源性成分检测能力验证研究

目的了解上海市获得资质的各实验室动物源性成分检测的能力。方法按照能力验证活动的实施方案进行样品制备、样品均匀性和稳定性、发样检测、结果统计等,并对能力验证结果进行分析。结果共12家实验室参加了本次能力验证活动并及时反馈了检测结果,其中10家实验室的所有结果都满意,满意率为83.3%; 2家实验室的6个项次初测结果不满意,占参加总数的16.7%。复测结果均满意。结论通过本次能力验证了解了这12家实验室具备动物源性成分检测的基本能力。通过外部的现场核查和样品检测可大大提升实验室的能力。     

食用精炼大豆油DNA提取及单管巢式PCR检测

设计了针对转基因Roundup Ready大豆外源基因扩增的内外2对引物,分别位于CaMV35s启动子,CTP基因,CP4EPSPS基因区域,并成功对精炼大豆油中的外源基因进行单管巢式PCR检测。结果表明,用改良试剂盒法和冷冻干燥法提取大豆油中的DNA均可以满足单管巢式PCR检测要求;单管巢式PCR扩增对内外引物的Tm值有特殊要求,外引物的退火温度高于内引物。本实验建立的精炼大豆油转基因成分的单管巢式PCR检测方法特异性强,灵敏度高且快速方便。      

A survey of the use of soy in processed Turkish meat products and detection of genetic modification

To screen for possible illegal use of soybeans in meat products, the performance characteristics of a commercial polymer chain reaction (PCR) kit for detection of soybean DNA in raw and cooked meat products were established. Minced chicken and beef products containing soybean at levels from 0.1% to 10.0% were analysed by real-time PCR to amplify the soybean lectin gene. The PCR method could reliably detect the addition of soybean at a level of 0.1%. A survey of 38 Turkish processed meat products found only six samples to be negative for the presence of soybean. In 32 (84%) positive samples, 13 (34%) contained levels of soy above 0.1%. Of soybean positive samples, further DNA analysis was conducted by real-time PCR to detect whether genetically modified (GM) soybean had been used. Of 32 meat samples containing soybean, two samples were positive for GM modification.     

Accurate determination of meat mass fractions using DNA measurements for quantifying meat adulteration by digital PCR

The alarming problem of meat adulteration emphasises the demand for accessible analytical approaches for food regulatory agencies to detect and, specially, to measure altered meat fractions. This study proposes a novel cross-species triplex droplet digital polymerase chain reaction (ddPCR) assay to simultaneously identify and quantify the ratios of pork/beef meat fractions from a total DNA content, including processed and autoclaved meat, without requiring a standard, achieving high sensitivity with a limit of quantification estimated at 0.1% (w/w) and a limit of detection down to 0.01% (w/w). A single copy nuclear gene, β-actin, was employed as a target, accompanied with myostatin gene as a cross-species target to quantify the meat background. The duplex assay provided a simultaneous quantification of pork and myostatin, whereas the triplex assay was able to detect pork, beef and myostatin with a decrease of technical error, cost and time.     

PCR法检测肉制品中鹅源性成分

目的 建立一种PCR法检测肉制品中鹅源性成分的方法。方法 根据鹅的线粒体DNA(mtDNA)序列设计引物, 以9种动物的DNA为模板, 利用新设计的鹅的特异性引物进行PCR反应, 并用琼脂糖凝胶电泳检测引物的特异性和敏感性。结果 通过PCR反应, 仅鹅的DNA扩增得到了194 bp的目的片段, 其余物种DNA和空白对照均无目的片段。扩增产物的核苷酸序列与GENBANK中检索到的相应序列基本相符合。 结论 该方法特异性强、灵敏度高, 适合检测肉制品中的鹅源性成分。     

谷氨酰胺转胺酶改性大豆分离蛋白在低能量肉制品中应用的研究

利用谷氨酰胺转胺酶(MTG)对大豆分离蛋白(SPI)进行改性,并将改性SPI应用于低能量肉制品中。实验表明:除常温黏度外,改性SPI的其他功能性明显优于市售肉用SPI;改性SPI在低能量肉制品中适宜的添加量为5%,改性SPI使产品的固体脂肪乳化稳定性、得率分别比肉用SPI提高了14%和7%,产品的感官指标也明显好于肉用SPI。     

PCR法定性检测大豆食用油中转基因成分

针对大豆食用油中核酸含量低且被严重破坏、DNA难以提取的问题,对CTAB法进行优化,有效从食用油中提取到可用于PCR扩增反应的DNA模板。同时定性检测出大豆内源基因lectin,外源调控序列启动子CaMV35S、终止子Nos及目的基因CP4-EPSPS序列,成功建立了基于PCR技术在大豆食用油中快速检测转基因成分的方法。     

The determination of soya and meat protein in raw and processed meat products by specific peptide analysis. An evaluation

The detection and measurement of characteristic peptides formed on enzymatic hydrolysis of soya protein products, meats and offals is described. Samples were heated at 120°C for 3h prior to digestion with trypsin overnight, and the resultant peptide mixtures passed through an Amicon ultrafiltration membrane. After concentration the ultrafiltrates were analysed by ion exchange chromatography on Aminex A5 resin. Peptides were detected by post-column reaction with ninhydrin. Characteristic peaks designated SP 2 and MP 1 were seen in chromatograms of digests of soya protein isolate and beef respectively, and these peaks were well resolved in beef and soya protein isolate mixtures. The SP 2 peak was shown to contain peptides derived from soya 11 S globulin. The soya protein and beef contents of a series of mixtures of freeze-dried, defatted beef and soya protein isolate were determined by measurement of the SP 2 and MP 1 peaks respectively. Soya protein content could be determined within 2% of the true value over the range 30–70% soya protein isolate and beef content could be determined within 5% of the true value in the range 20–100% beef. Analysis of five soya protein isolates, four soya protein concentrates, six soya flours and 13 textured soya products indicated considerable interproduct variation in the yield of SP 2. The MP 1 peak was seen in a range of meats, both cooked and raw. It was also present in digests of offals which contained smooth or striated muscle but not in ‘non-muscle’ offals. The protein origin of the MP 1 peak was not established but the yield appeared lower in meat products which had been heated during manufacture than in those which had received no such treatment. Analysis of a series of laboratory prepared canned and heated pork and soya protein isolate mixtures enabled the pork content to be determined to within 8% of the true value, 2% soya protein isolate could be detected but not quantified accurately.     

Quantitative detection of soybean in meat products by a TaqMan real-time PCR assay

In the present work, we propose a normalised real-time quantitative PCR assay to determine the addition of soybean to meat products. The method proved to be a powerful tool for the quantification of soybean protein (dry basis) in the range of 0.01% to 6%, being successfully in-house validated. Its application was effective in the analysis of several meat products, indicating 2% of non-compliance with the food allergen labelling legislation, and some inconsistencies when comparing the declared with estimated amounts of soybean. This work highlights the importance of efficient tools to assess labelling statements of meat products, avoiding fraudulent practices.     

How meaty? Detection and quantification of adulterants,foreign proteins and food additives in meat products

The worldwide consumption of meat is increasing, especially in developing countries. Many studies have correlated a diet characterised by high intake of processed red meats with a risk of colorectal cancer, stroke, coronary heart diseases and diabetes. Moreover, the quality and safety of meat products may be compromised by several admitted and not admitted procedures (i.e. addition of food additives and/or foreign proteins). For these reasons, the topic ‘meat products’ quality and safety’ has gained much in importance during last few years. In this review, the recent advances in the field of analytical methods for the evaluation of meat adulteration due to the addition of foreign proteins and food additives are reported, compared and critically evaluated. Moreover, the most representative monitoring practices, developed worldwide, related to meats adulteration are described and discussed.     

不同添加量的大豆分离蛋白和淀粉对烤猪肉中杂环胺形成的影响

分别将2.5%、5.0%、7.5%和10.0%的大豆分离蛋白（SPI）和淀粉添加于猪肉中,采用UHPLC-MS/MS法测定其中16种杂环胺的含量,探讨其对杂环胺形成的影响。结果表明:添加2.5%的SPI和淀粉后,除喹喔啉类（Me IQx和4,8-Di Me IQx）外,各杂环胺的含量均显著增加（p<0.05）。随着添加量的增加,杂环胺的含量先升高后开始下降。当添加量达到10.0%时,SPI的添加对4,8-Di Me IQx的形成出现显著抑制作用（p<0.05）,而淀粉的添加对吡啶和喹啉/喹喔啉杂环胺的促进作用则变得不再显著（p<0.05）。由此可见,SPI和淀粉的添加对烤猪肉体系中多数杂环胺的形成具有低剂量促进,且随着剂量的增多促进效果逐渐减弱的影响规律。      

环介导等温扩增与qPCR用于检测肉制品中狗源性成分的比较

目的 建立基于实时荧光PCR和环介导等温扩增检测肉制品中狗源性成分的检测方法并比较2种方法的检测效能.方法 针对狗线粒体CYTB基因保守序列,采用Primer Explorer Version 4软件设计环介导等温扩增引物,Primer Express 3.0.1软件设计实时荧光PCR引物及探针.提取狗肉基因组DNA作...     

Detection of genetically modified organisms in foods by DNA amplification techniques

In this article, the different DNA amplification techniques that are being used for detecting genetically modified organisms (GMOs) in foods are examined. This study intends to provide an updated overview (including works published till June 2002) on the principal applications of such techniques together with their main advantages and drawbacks in GMO detection in foods. Some relevant facts on sampling, DNA isolation, and DNA amplification methods are discussed. Moreover; these analytical protocols are discuissed from a quantitative point of view, including the newest investigations on multiplex detection of GMOs in foods and validation of methods.