PCR技术在肉类掺假检验中的应用进展

当前,对研究者、消费者、食品工业和政策制定者等各个方面来说,食品的真伪都是一个热点问题,尤其是肉类工业。PCR技术具有特异性强、敏感性高、操作简便、快速高效等特点,在肉类掺假检测方面具有巨大的应用价值。本文介绍了目前肉制品鉴定的方法,包括蛋白和核酸两个层次,用于肉制品鉴定的各种目的基因的选择。回顾了PCR技术在国内外肉类产品掺假鉴定中的重要应用。指出了PCR技术在肉制品鉴定中的不足,与各种新技术(基因芯片、蛋白质芯片等)有机结合将是以后的研究方向。     

PCR技术在肉类成分定量分析中的应用研究进展

PCR技术具有特异性强、灵敏度高、可同时分析大量样本的特点,在鉴别和定量分析食品中肉类成分上应用广泛。本文介绍了PCR扩增产物分析、实时荧光PCR和微滴数字PCR三种应用于肉类定量的技术,通过检出限、定量限和定量范围等数据分析这三种方法在肉类成分定量分析中的应用效果,并对其定量的局限性进行阐述,为进一步完善PCR定量技术提供参考。      

实时PCR技术在羊亚科肉类鉴定中的应用

根据线粒体cyt b基因的种间差异,建立羊亚科、绵羊、山羊肉类的实时荧光聚合酶链式反应检测技术,对方法进行系统的优化与完善,并进行特异性、灵敏度与扩增效率、检出限、试剂一致性分析与适用性考察,同时邀请5 家国内权威机构对方法进行复核。结果表明,方法的重复性好、适用性广,可用于不同基质的肉制品检测,扩增效率达95%以上,用于羊肉制品的动物源性成分检测准确率达100%,适于推广使用。     

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...     

pH值在鉴定肉类标准质量中的应用

通过测定胴体ｐＨ值，鉴定非正常肉，在公众判定肉质量的方法中，ｐＨ值不仅可提供极为有用的信息，并且方法简便可靠。     

6种肉类成分多重PCR鉴别方法的建立及应用

目的建立单管同步快速鉴别水牛、黄牛、牦牛、山羊、驴和鸭的物种成分的多重PCR鉴别方法。方法对水牛、黄牛、牦牛、山羊、驴和鸭的线粒体全基因组,采用Primer-blast在线软件,设计6对特异性引物,优化反应条件,建立PCR体系。结果该方法仅对水牛、黄牛、牦牛、山羊、驴和鸭肉有特异性扩增,与其他动物的核酸无交叉反应;同时检测6种物种成分的检测低限为10-4 ng/μL。在鸭肉中分别模拟掺入牛肉、羊肉和驴肉成分,方法对鸭肉中掺入牛成分、羊成分和驴成分中的检出限均小于0.5%。结论该方法特异性好、敏感度高,可以被应用于水牛、牦牛、黄牛、山羊、驴和鸭的物种成分鉴别。     

基于核酸分子学方法的肉类成分鉴别技术研究进展

近年来,肉类掺假问题频繁发生。基于核酸的分子生物学肉类成分鉴别技术已成为研究热点,其具有灵敏度高、特异性强、检测时间短以及成本低的优点。本文综述了基于核酸分子学的肉类成分种属鉴别技术在肉类掺假检验中的应用,着重于量化各种方法的检测限,并重点对实时荧光定量聚合酶链式反应(polymerase chain reaction,PCR)和数字PCR技术在动物成分鉴别定量分析的研究现状与前景做介绍。探讨不同来源的靶基因(核DNA和线粒体DNA)在动物成分鉴别中,定性和定量检测灵敏度与特异性的区别。     

实时荧光PCR技术定量检测肉类掺假的研究进展

肉类食品是人们饮食结构的重要组成部分,而近年来,肉类掺假现象频发,严重威胁到公众的利益。因此,建立快速准确的肉类鉴别检测方法逐渐成为一个热点问题。以PCR为基础的实时荧光PCR检测技术在实现定性定量分析的同时,与常规方法相比,具有自动化程度及动态范围更高的特点,在肉类掺假检测方面具有巨大的应用价值。该文从实时荧光PCR检测技术的检测原理出发,进一步探讨其操作流程,并对不同类别的实时荧光PCR检测技术在肉类掺假中的应用进行整合。在肉类鉴定中,实时荧光PCR技术可分为荧光探针法和荧光染料法2大类,近年来在不断发展完善的过程中仍存在着一些挑战,且将向提高检测效率和提高检测结果准确性的方向发展。因此该文提出在已有研究的基础上结合新技术建立标准化检验流程的想法,以期为肉类掺假检测技术的发展起到借鉴意义和指导作用。     

气调包装技术在肉类保鲜中的应用

肉类气调包装在欧洲较为普遍，它不仅能较大程度上抑制多数微生物引起的腐败变质，也能改善肉品品质，作者综述了近年来有关气调包装与肉品颜色，微生物之间关系的研究报道，以期为发展我国的气调包装参考。     

基于物种特异性PCR对海鳗鱼糜掺假检测技术的研究

为了对海鳗鱼糜掺假进行定性研究,从GenBank数据库下载海鳗及其他物种的线粒体12S rRNA,并通过BioEdit 7.0软件对该基因碱基序列进行比对。在此基础上,综合考虑引物设计原则,设计出关于海鳗的特异性引物。对引物的特异性、灵敏性以及主要加工过程进行实验,结果表明所设计的引物对海鳗DNA具有很强的特异性,灵敏性达到0.1%,加工过程也不影响检测过程,从而满足了对于海鳗鱼糜掺假的检测要求。     

Quantitative detection of poultry meat adulteration with pork by a duplex PCR assay

A species-specific duplex polymerase chain reaction (PCR) assay was developed for the simultaneous detection of pork and poultry meat species using the mitochondrial cytb and 12S rRNA as target genes for pork and poultry, respectively. By the amplification of binary reference meat mixtures, a linear normalised calibration curve was obtained using the fluorescence intensities of PCR products for pork (149 bp) and poultry (183 bp) species. The proposed method allowed the quantification of pork meat addition to poultry meat in the range of 1–75%, with a sensitivity of 0.1%. The in-house validation using samples with known amounts of pork meat (1.0%, 2.5%, 7.5%, 20.0% and 40%) evidenced a high reproducibility of the methodology (coefficient of variation from 4.1% to 7.6%). The successful application of the duplex PCR was also demonstrated by the high correlation (R² = 0.99) obtained from regression analysis between the predicted and the actual values of pork meat addition in blind meat mixtures. The suggested methodology presents a low cost, fast, easy and reliable alternative to estimate the level of poultry meat adulteration by the addition of pork meat.     

PCR amplification of repetitive sequences as a possible approach in relative species quantification

Both relative and absolute quantifications are possible in species quantification when single copy genomic DNA is used. However, amplification of single copy genomic DNA does not allow a limit of detection as low as one obtained from amplification of repetitive sequences. Amplification of repetitive sequences is therefore frequently used in absolute quantification but problems occur in relative quantification as the number of repetitive sequences is unknown. A promising approach was developed where data from amplification of repetitive sequences were used in relative quantification of species in binary mixtures. PCR LUX primers were designed that amplify repetitive and single copy sequences to establish the species dependent number (constants) (SDC) of amplified repetitive sequences per genome. The SDCs and data from amplification of repetitive sequences were tested for their applicability to relatively quantify the amount of chicken DNA in a binary mixture of chicken DNA and pig DNA. However, the designed PCR primers lack the specificity required for regulatory species control.     

A strategy for molecular species detection in meat and meat products by PCR-RFLP and DNA sequencing using mitochondrial and chromosomal genetic sequences

Molecular species detection in food has become common in the last 10 years. The methods are sensitive enough to detect small, but relevant, amounts of one species in composed food. We have developed a strategy for detecting different animal species in food by molecular means. This strategy uses a combination of published PCR systems and new developed PCR primer systems for the detection of porcine, bovine, ovine, avian, cervine and equine DNA by PCR followed by restriction analysis (PCR-RFLP). In some cases, analysis is completed by DNA sequencing. The species detection system includes an amplification control and so is in accordance with the relevant food standards.     

Authentication of meat from game and domestic species by SNaPshot minisequencing analysis

The aim of the present study is to develop an assay for the specific identification of meat from Capreolus capreolus, Cervus elaphus, Capra ibex, Rupicapra rupicapra, targeting sequences of the cytochrome b (cyt b) gene of mitochondrial DNA. The assay is also intended to enable differentiation between meat from these wild species as well as Ovis aries, Capra hircus, Bubalus bubalis, Bos taurus and Sus scrofa domestic species.The primers used in the preliminary PCR were designed in well conserved regions upstream and downstream of the diagnosis sites. They successfully amplified a conserved 232 bp region from the cyt b gene of all the species taken into consideration. The sites of diagnosis have been interrogated using a minisequencing reaction and capillary electrophoresis. All the results of the multiplex PER (primer extension reaction) test were confirmed by fragment sequencing. The assay offers the possibility of discriminating nine species at the same time.     

Identification of meat species by TaqMan-based real-time PCR assay

In this study, a convenient, sensitive and specific real-time PCR assay was described for the species identification and their quantification in raw and cooked meat products. Specific primers and TaqMan probes were designed on the mitochondrial ND2, ND5 and ATP 6-8 genes for donkey, pork and horse, respectively, and the performance of the method was tested. In the results, no cross-reaction was observed between the donkey and pork species specific primer-probe systems and non-target species (bovine, ovine, chicken and turkey). Only one cross reaction was observed between the horse species specific primer-probe set and 100 ng pork DNA at the ct 33.01 level (corresponding to 0.01 ng horse DNA). The real-time quantitative assay used in this study allowed the detection of as little as 0.0001 ng template DNA from pure meat for each species investigated and experimental meat mixtures. In conclusion, it can be suggested that the TaqMan probe assay used in this research might be a rapid and sensitive method for the routine meat species identifications studies in raw or cooked meat products.     

Identification of tuna species by a real-time polymerase chain reaction technique

Tuna are highly priced fishes that are often used in processed products. For effective fishery management and protection of consumers’ rights, it is important to develop a molecular method to identify the species of the tuna products. In this study we have developed a molecular method based on real-time polymerase chain reaction (real-time PCR) technology for the rapid identification of four tuna species. Four species-specific TaqMan probes were designed to identify bigeye tuna (Thunnus obesus), Pacific bluefin tuna (Thunnus orientalis), southern bluefin tuna (Thunnus maccoyii), and yellowfin tuna (Thunnus albacares). A SYBR green system was also designed to enhance the authentication of T. obesus. Both systems can distinguish target species from others in an efficient and high-throughput manner and can be applied to species identification of tuna products.     

Species determination – Can we detect and quantify meat adulteration?

Proper labelling of meat products is important to help fair-trade, and to enable consumers to make informed choices. However, it has been shown that labelling of species, expressed as weight/weight (w/w), on meat product labels was incorrect in more than 20% of cases. Enforcement of labelling regulations requires reliable analytical methods. Analytical methods are often based on protein or DNA measurements, which are not directly comparable to labelled meat expressed as w/w. This review discusses a wide range of analytical methods with focus on their ability to quantify and their limits of detection (LOD). In particular, problems associated with a correlation from quantitative DNA based results to meat content (w/w) are discussed. The hope is to make researchers aware of the problems of expressing DNA results as meat content (w/w) in order to find better alternatives. One alternative is to express DNA results as genome/genome equivalents.     

Survey of authenticity of meat species in food products subjected to different technological processes,by means of PCR-RFLP analysis

Polymerase chain reaction (PCR) coupled to restriction-fragment length polymorphism analysis (RFLP) was considered for exploring the incidence of incorrect labelling in food products containing one or more meat species. Universal primers CYT b1/CYT b2, which amplify a variable region of the mitochondrial cytochrome b of vertebrates, and endonucleases PalI, MboI, HinfI and AluI were used for this purpose. Fifty food products, nine of them raw or cured and the other 41 subjected to a variety of technological processes such as pre-cooking and freezing, cooking and smoking, dehydration or sterilisation, were investigated. Twenty of the 50 products declared mixtures of meat species on their labels. Fifteen (30%) of the 50 food samples investigated displayed an incorrect qualitative labelling. While this affected only one (11.1%) of the nine raw/cured products, 14 (34.2%) of the 41 products subjected to some type of heat-processing were not correctly labelled. The undeclared presence of turkey was the most frequent concern, since it was detected in seven food products. The complete absence of a declared species of high commercial value—such as beef or roe-deer—was observed in another four cases. The PCR-RFLP method used here proved to be a rapid and easy-to-perform two-step analytical approach to achieve qualitative meat species identification in raw and cooked food products containing one or more different species.