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.     

Detection of meat species using TaqMan real-time PCR assays

Species-specific real-time PCR (TaqMan) assays were developed for detection of beef, pork, lamb, chicken and turkey. Assays were developed around small (amplicons <150 base pairs) regions of the mitochondrial cytochrome b (cytb) gene. Speciation was achieved using species-specific primers. For detection purposes, two TaqMan probes were developed; the first was specific to the mammalian species (beef, lamb and pork), the second to the poultry species (chicken and turkey). Normal end-point TaqMan PCR conditions were applied; however, PCR was limited to 30 cycles. Applying the assays to DNA extracts from raw meat admixtures, it was possible to detect each species when spiked in any other species at a 0.5% level. The absolute level of detection, for each species, was not determined; however, experimentally determined limits for beef, lamb and turkey were below 0.1%.     

肉制品中鸭源性成分TaqMan探针实时荧光PCR检测方法的建立与应用

目的 建立TaqMan探针实时荧光PCR法快速检测肉制品中鸭源性成分的分析方法.方法 以鸭生长激素(growth hormone,GH)基因为靶基因,基于特异性保守序列设计引物和TaqMan探针,优化反应体系和反应条件,建立了鸭源性成分实时荧光PCR(real-time PCR)检测方法.结果 所建立的real-tim...     

Semiquantitative detection of male pork tissue in meat and meat products by PCR

Consumer awareness has increased concerning castration of piglets without analgesia or anaesthesia. On the other hand the occurrence of boar taint is not tolerated by consumers. Currently no reliable methods exist for the on-line detection of boar taint in the slaughterhouse or for genetic sexing of pigs. Therefore, as an alternative the detection of male pork meat was sought. Based on detection of a length polymorphism of the sex chromosomal amelogenin gene a reliable, specific and highly sensitive PCR method for qualitative and semi-quantitative determination of male pork tissue in meat and meat products was determined. A set of 25 male and 25 female meat samples could be correctly identified and mixtures with as little as 0.1% male meat content could be detected. Therefore the method can be used for production and control of specific meat products containing low amounts of male pork meat and thus avoiding boar taint.     

Detection of chicken and turkey meat in meat mixtures by using real-time PCR assays

In this study, TaqMan-based real-time Polymerase Chain Reaction (PCR) techniques were developed for the detection of chicken and turkey meat in raw and heat-treated meat mixtures. Primers and TaqMan probe sets were designed to amplify 86 bp and 136 bp fragments for the chicken and turkey species, respectively, on the mitochondrial NADH dehydrogenase subunit 2 gene. In the results, it was possible to detect each species at the level of 0.1 pg template DNA with the TaqMan probe technique without any cross-reactivity with nontarget species (bovine, ovine, donkey, pork, and horse) while the detection level was 1 pg template DNA using conventional PCR. The TaqMan probe assays used in this study allowed the detection of as little as 0.001% level of both species in the experimental meat mixtures, prepared by mixing chicken and turkey meat with beef at different levels (0.001% to 10%). In conclusion, TaqMan probe assays developed in this research are promising tools in the specific identification and sensitive quantification of meat species even in the case of heat-treated meat products, and suitable for a rapid, automated, and routine analysis.     

A SYBR Green real-time PCR assay to detect and quantify pork meat in processed poultry meat products

Species identification in meat products has grown in interest in recent years since these foodstuffs are susceptible targets for fraudulent labelling. In this work, a real-time PCR approach based on SYBR Green dye was proposed for the quantitative detection of pork meat in processed meat products. For the development of the method, binary meat mixtures containing known amounts of pork meat in poultry meat were used to obtain a normalised calibration model from 0.1 to 25% with high linear correlation and PCR efficiency. The method revealed high specificity by melting curve analysis, being successfully validated through its application to blind meat mixtures, which confirmed its adequacy for pork meat determination. The fully applicability of the method was further demonstrated in commercial meat products, allowing verification of labelling compliance and identification of meat species in processed foods.     

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。     

A simultaneous triplex TaqMan real-time PCR approach for authentication of caprine and bovine meat,milk and cheese

Goat foodstuffs are considered as healthy foods with high nutritional value. This study demonstrated the development and validation of a triplex real-time PCR on the basis of species-specific and species-conservative TaqMan probes for the simultaneous identification of caprine and bovine DNA in meats, milk and cheeses with a prerequisite designed endogenous control. In this research, caprine and bovine meat, milk and cheese were specifically identified via developed primers and probes, and the limits of detection of this methodology were 0.005 and 0.01 ng DNA of milk and cheese from goat, and 0.01 and 0.05 ng DNA of milk and cheese from cow. Taken together, this approach was elaborated to address dairy adulteration issues to eliminate the fraud of economically motivated goat milk and cheese adulteration by adding cow milk.     

Real-time TaqMan PCR for quantitative detection of cows’ milk in ewes’ milk mixtures

A real-time PCR based on the amplification of a fragment of the mitochondrial 12S ribosomal RNA gene was developed and evaluated for the detection and quantification of cows’ milk in raw and heat-treated cow/sheep milk mixtures. The method combines the use of cow-specific primers that amplify a 252 bp fragment from cow DNA, and mammalian-specific primers amplifying a 428 bp fragment from mammalian species DNA, which is used as an endogenous control. The method measures PCR product accumulation through a 6-carboxyfluorescein-labeled fluorogenic probe (TaqMan). A comparison of the cycle number at which mammalian and cow-specific PCR products were first detected, in combination with the use of reference standards of known bovine content, allowed the determination of the percentage of cows’ milk in mixtures. Experimental raw and heat-treated binary mixtures were analyzed, demonstrating the specificity and sensitivity of the assay for detection and quantification of cows’ milk in the range 0.5–10%.     

Monoclonal antibody sandwich ELISA for the potential detection of chicken meat in mixtures of raw beef and pork

A sandwich ELISA (enzyme-linked immunosorbent assay) has been developed successfully for the detection of defined amounts of chicken meat (1-100%) in beef and pork meat mixtures. The assay uses a monoclonal antibody (BC9) specific to a chicken muscle soluble protein to capture this protein from complex meat mixtures. Further immunorecognition of the captured protein was attained with rabbit polyclonal antibodies against chicken muscle proteins (anti-CHSP). A commercial goat anti-rabbit immunoglobulin conjugated to peroxidase was used to detect the anti-CHSP antibodies bound to the chicken protein. Subsequent enzymic conversion of substrate gave clear optical density differences when assaying mixtures of beef and pork meats containing variable amounts of chicken meat.     

TaqMan实时荧光聚合酶链式反应(PCR)技术定量检测婴幼儿配方食品中的金黄色葡萄球菌

靶向于耐热核酸酶基因nuc,研究建立了Taq Man实时荧光聚合酶链式反应(PCR)(qRT-PCR)法用于婴幼儿米粉及乳粉中金黄色葡萄球菌的快速定量检测。经优化的qRT-PCR体系特异性强,仅在金黄色葡萄球菌中产生典型扩增曲线;方法灵敏度高,对标准质粒、纯菌液、模拟染菌米粉及奶粉样液的检测限分别为17.94拷贝、1.2 CFU、0.42 CFU和0.74 CFU/PCR反应体系;10倍系列梯度稀释的标准质粒、纯菌及人工染菌样液的浓度对数与qRT-PCR的Ct值线性相关度良好,拟合方程的决定系数均≥0.99;平板计数法与qRT-PCR法对模拟米粉及乳粉盲样中金黄色葡萄球菌的定量检测结果间无统计学差异。文中所建立的qRT-PCR定量法特异性强、灵敏度高,简便快捷、可靠性佳,可为婴幼儿米粉及乳粉中金黄色葡萄球菌的快速、准确定量检测提供有效手段。     

PCR identification of beef, sheep, goat, and pork in raw and heat-treated meat mixtures

A PCR assay has been developed for the specific and qualitative detection of pork (Sus scrofa domesticus), beef (Bos taurus), sheep (Ovis aries), and goat (Capra hircus) in raw and heat-treated meat mixtures. A forward common primer was designed on a conserved DNA sequence in the mitochondrial 12S ribosomal RNA gene (rRNA), and reverse primers were designed to hybridize on species-specific DNA sequences of each species considered. The different sizes of the species-specific amplicons, separated by agarose gel electrophoresis, allowed clear species identification. Analysis of experimental meat mixtures demonstrated that the detection limit of the assay was 1% (wt/wt) for each species analyzed. This assay can be useful for the accurate identification of these species, avoiding mislabeling or fraudulent species substitution in meat mixtures.     

实时荧光定量PCR技术在肉及肉制品中的应用

实时荧光定量PCR技术作为一种DNA定量检测的工具,可应用于肉制品种类鉴别和定量分析中。相比于传统感官和理化的鉴别方法,该技术具有灵敏性高、特异性强、操作简便、省时省力等特点。使用Taq Man荧光探针和荧光染料可以直接测定PCR循环后产物的总量,确定扩增DNA片段的种类及数量,从而确定肉品种类及添加量。因此,可以将该技术应用到肉制品成分及安全的检测中。本文介绍了实时荧光定量PCR技术的基本原理,主要综述了该技术在肉品种类鉴定方面的应用,简述其在肉制品细菌污染检测的应用,并对该技术在肉类研究中的应用进行展望。      

多重实时荧光定量PCR检测肴肉中的特定腐败菌

本试验以真空包装水晶肴肉中的3株特定腐败菌屎肠球菌(Enterococcus faecium)、中间耶尔森菌(Yersinia intermedia)和清酒乳杆菌(Lactobacillus sakei)为研究对象,通过克隆3株菌的特异性DNA片段,构建重组质粒作为阳性模板,建立一种多重实时荧光定量PCR方法同时检测这3株特定腐败菌,结果表明:建立的多重实时荧光定量PCR具有较高的特异性和灵敏度,同时还具有很好的稳定性,可用于真空包装水晶肴肉贮藏期间特定腐败菌数量的检测,从而可以间接评价产品的货架期。     

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.     

Sensitive detection of porcine DNA in processed animal proteins using a TaqMan real-time PCR assay

A TaqMan real-time PCR method was developed for specific detection of porcine-prohibited material in industrial feeds. The assay combines the use of a porcine-specific primer pair, which amplifies a 79?bp fragment of the mitochondrial (mt) 12?S rRNA gene, and a locked nucleic acid (LNA) TaqMan probe complementary to a target sequence lying between the porcine-specific primers. The nuclear 18?S rRNA gene system, yielding a 77?bp amplicon, was employed as a positive amplification control to monitor the total content of amplifiable DNA in the samples. The specificity of the porcine primers-probe system was verified against different animal and plant species, including mammals, birds and fish. The applicability of the real-time PCR protocol to detect the presence of porcine mt DNA in feeds was determined through the analysis of 190 industrial feeds (19 known reference and 171 blind samples) subjected to stringent processing treatments. The performance of the method allows qualitative and highly sensitive detection of short fragments from porcine DNA in all the industrial feeds declared to contain porcine material. Although the method has quantitative potential, the real quantitative capability of the assay is limited by the existing variability in terms of composition and processing conditions of the feeds, which affect the amount and quality of amplifiable DNA.     

Sensitive detection of porcine DNA in processed animal proteins using a TaqMan real-time PCR assay

A TaqMan real-time PCR method was developed for specific detection of porcine-prohibited material in industrial feeds. The assay combines the use of a porcine-specific primer pair, which amplifies a 79?bp fragment of the mitochondrial (mt) 12?S rRNA gene, and a locked nucleic acid (LNA) TaqMan probe complementary to a target sequence lying between the porcine-specific primers. The nuclear 18?S rRNA gene system, yielding a 77?bp amplicon, was employed as a positive amplification control to monitor the total content of amplifiable DNA in the samples. The specificity of the porcine primers-probe system was verified against different animal and plant species, including mammals, birds and fish. The applicability of the real-time PCR protocol to detect the presence of porcine mt DNA in feeds was determined through the analysis of 190 industrial feeds (19 known reference and 171 blind samples) subjected to stringent processing treatments. The performance of the method allows qualitative and highly sensitive detection of short fragments from porcine DNA in all the industrial feeds declared to contain porcine material. Although the method has quantitative potential, the real quantitative capability of the assay is limited by the existing variability in terms of composition and processing conditions of the feeds, which affect the amount and quality of amplifiable DNA.     

Multiplex real-time PCR for the detection and quantification of DNA from beef, pork, chicken and turkey

Many meat products are composed of two or more meat species. To determine the proportion of these meat fractions, a quantitative multiplex PCR was developed for the quantification of beef, pork, chicken and turkey. This system proved its applicability, precision and accuracy in examining different meat products from the market. Thus it allows the efficient control of composed meat products in official food control and production control laboratories.     

Multiplex real-time PCR for the detection and quantification of DNA from beef,pork, horse and sheep

Meat products are often composed of more than one meat species. A quantitative multiplex PCR was developed to determine the proportion of meat fractions of beef, pork, horse and sheep. The precision and accuracy were investigated by dilutions of DNA from all four species and examining different meat products from the market. Application of this tetraplex quantitative real-time PCR system will enable official food control and production control laboratories to efficiently investigate the composition of meat products.