Application of quantitative real-time PCR in the detection of prion-protein gene species-specific DNA sequences in animal meals and feedstuffs

This study describes a method for quantitative and species-specific detection of animal DNA from different species (cattle, sheep, goat, swine, and chicken) in animal feed and feed ingredients, including fish meals. A quantitative real-time PCR approach was carried out to characterize species-specific sequences based on the amplification of prion-protein sequence. Prion-protein species-specific primers and TaqMan probes were designed, and amplification protocols were optimized in order to discriminate the different species with short PCR amplicons. The real-time quantitative PCR approach was also compared to conventional species-specific PCR assays. The real-time quantitative assay allowed the detection of 10 pg of ruminant, swine, and poultry DNA extracted from meat samples processed at 130 degrees C for 40 min, 200 kPa. The origin of analyzed animal meals was characterized by the quantitative estimation of ruminant, swine, and poultry DNA. The TaqMan assay was used to quantify ruminant DNA in feedstuffs with 0.1% of meat and bone meal. In conclusion, the proposed molecular approach allowed the detection of species-specific DNA in animal meals and feedstuffs.     

肉制品中牛源性成分多重实时荧光PCR检测 方法的建立及初步应用

目的建立肉制品中牛源性成分的荧光PCR检测方法。方法根据牛特异性线粒体DNA片段,设计合成两对引物,以生、熟牛肉及超市牛肉加工品为材料,建立肉制品中牛源性成分的多重实时荧光PCR检测方法,并用该法与国标法同时对市售的25份肉制品同时进行检测,通过对其他种类的肉源DNA进行扩增验证方法的特异性;对含有不同比例牛肉成分的DNA样本进行检测确定检出限。结果该方法可成功检测出肉制品中的牛源性成分。在25份肉制品检测中,与国标法检测结果一致。该法的特异性为100%,灵敏度检测线为1%。结论本研究成功建立牛源性肉制品的检测方法,该方法快速简便,且具有较高的特异性和灵敏度,可用于市售肉制品中牛源性成分的鉴定。     

Polymerase chain reaction assay for identification of chicken in meat and meat products

The aim of this study was to develop polymerase chain reaction (PCR) assay for specific detection of chicken meat using designed primer pair based on mitochondrial D-loop gene for amplification of 442 bp DNA fragments from fresh, processed and autoclaved meat and meat products. The PCR result was further verified by restriction digestion with HaeIII and Sau3AI enzymes for specific cutting site in amplified DNA fragments. The specificity of assay was cross tested with DNA of cattle, buffalo, sheep, goat, pig, duck, guinea fowl, turkey and quail, where amplification was observed only in chicken without cross reactivity with red meat species. However positive reaction was also observed in quail and turkey. In this study, no adverse effects of cooking and autoclaving were found on amplification of chicken DNA fragments. Thus, the detection limits was found to be less than 1% in admixed meat and meat products. The developed assay was found specific and sensitive for rapid identification of admixed chicken meat and meat products processed under different manufacturing conditions.     

Novel TaqMan real-time polymerase chain reaction assay for verifying the authenticity of meat and commercial meat products from game birds

Species-specific real-time polymerase chain reaction (PCR) assays using TaqMan probes have been developed for verifying the labeling of meat and commercial meat products from game birds, including quail, pheasant, partridge, guinea fowl, pigeon, Eurasian woodcock and song thrush. The method combines the use of species-specific primers and TaqMan probes that amplify small fragments (amplicons <150 base pairs) of the mitochondrial 12S rRNA gene, and an endogenous control primer pair that amplifies a 141-bp fragment of the nuclear 18S rRNA gene from eukaryotic DNA. Analysis of experimental raw and heat-treated binary mixtures as well as of commercial meat products from the target species demonstrated the suitability of the assay for the detection of the target DNAs.     

Application of PCR and SYBR Green Q Rti-PCR Assays for the Identification and Quantification of Chicken Meat Under Different Cooking Conditions

Conventional and SYBR Green Q Rti-PCR assays using primers targeting the 12S rRNA of chicken mitochondrial genes were employed for the detection and quantification of chicken used in food stuffs. The assays were recruited to amplify different known concentrations of DNA in the mixtures. Different kinds of processed meats were prepared using various amounts of chicken that were heated at different temperatures in order to detect the chicken in the mixtures. The PCR amplification of DNA revealed that the assay can amplify the species-specific amplicons as little as 0.01 ng of DNA in PCR reactions. Different concentrations of raw chicken were detected based on the threshold cycle. The technique was able to detect from 5% to 90% ratios of the chicken materials in sausages. Analysis of the experimental meat mixtures revealed the usefulness of the assays in detecting and quantifying chicken mitochondrial DNA in the mixtures.     

Duplex PCR approach for the detection and quantification of donkey,horse and mule in raw and heat‐processed meat products

Donkey‐related products have been paid more attention for their high nutritional value to human beings. Due to donkey resource scarcity, coupled with gradually increasing market demand, adulterated donkey meat products with other low‐cost animal meat, especially with the similar species horse and mule, are often found in market. Therefore, detection of species fraud in donkey meat products is important for consumer protection and food industries. In this study, a simple and highly specific duplex PCR method, based on the simultaneous amplification of fragments of the mitochondrial ATP synthase subunit 8/6 and ND2, was developed and optimised for the identification of horse, donkey and mule species in raw and heat‐processed meat products. To the best of our knowledge, it was the first time for this strategy applied to these three genetically related animal meat products differentiation to date. The duplex PCR generated a 153‐bp and 83‐bp amplification products for horse and donkey, respectively. While for mule, both of the two length amplification products are appeared on the agarose gel. Target meat species could be detected at a level of 1%, and the results indicated that the duplex PCR assay could be used in the authentification of donkey‐related products with high specificity, cost‐effectiveness and simplicity.     

基于便携式荧光定量PCR仪定量检测驴肉中掺假鸭肉

为建立快速方便的驴肉制品分子鉴定方法,本文以驴肉和常见的掺假肉类(鸭肉)为研究对象,筛选特异性引物和TaqMan探针,利用便携式Mini8 Plus实时荧光定量PCR仪进行灵敏度和特异性实验,通过绘制扩增标准曲线及确定驴肉和鸭肉的质量与DNA比值常数,对不同掺入比例(加入定量的鸭肉制成含量分别为20%、40%、60%、80%)的模拟样品和实际驴肉样品进行检测。结果显示,该方法对驴、鸭肉均具有良好的特异性,可以与马、猪、山羊、梅花鹿、牛、鸡、狗肉明显区分;对驴源性DNA成分的检出限为0.01 ng/μL,鸭源性DNA成分的检出限为0.1 ng/μL,对驴肉与鸭肉混合物中鸭肉成分的灵敏度为0.1%(w/w);所建立的标准曲线线性关系良好,驴肉DNA扩增标准曲线:y=-3.584x+27.003,R²=0.9982;鸭肉DNA扩增标准曲线:y=-3.538x+30.907,R²=0.9991;采用已建立的方法对35份驴肉样本进行市场试点调查,发现6份(17.1%)驴肉样本中含有鸭肉成分。以上研究结果说明,该实时荧光定量PCR方法可用于驴肉产品中其他...     

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.     

肉制品中羊源性成分的定性和定量检测

通过检测肉制品中DNA的来源进行动物源性检测是打击鲜肉及加工肉制品制假掺假的重要技术手段。依据GB/T 25165—2010《明胶中牛、羊、猪源性成分定性检测方法 实时荧光PCR法》合成用于TaqMan 实时荧光聚合酶链式反应的引物和探针,利用鲜肉及加工肉制品进行羊源性成分定性和定量检测方法研究。首先利用12 种不同动物鲜肉组织的DNA检测方法的特异性,然后以羊源DNA梯度稀释液为模板进行灵敏度实验,最后在加工肉制品中检测方法的适用性和定量检测能力。结果表明：此方法具有羊源性成分检测特异性,对其他动物来源的鲜肉DNA均无扩增信号,可以检出羊肉和猪肉混合样品中0.1%的羊肉;方法灵敏度高,可以检出10 pg的羊源DNA;方法适用性广,可以对加工肉制品（羊肉干）进行羊源性成分的定性和定量检测。     

Rapid detection of chicken and turkey in heated meat products using the polymerase chain reaction followed by amplicon visualisation with vistra green

A rapid and highly specific assay suitable for the routine detection of turkey and chicken in processed meat products has been developed. Based on PCR amplification of species-specific amplicons with rapid visualisation using vistra green, the assay may be completed within 5 h of receipt of sample. DNA was isolated from meat samples by the use of Wizard DNA isolation technology and followed by DNA amplification in the polymerase chain reaction using species specific primers, chicken forward (CF), chicken reverse (CR), turkey forward (TF) and turkey reverse (TR): the production of an amplicon was detected after the end of the PCR in less than 5 min using vistra green and a fluorescence plate reader. The presence of fluorescence denoted the presence of the target species in the sample.     

Meat species identification by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of mitochondrial 12S rRNA gene

Adulteration of high quality meat and meat products with their inferior/cheaper counterparts is a problem in the meat industry. The present study investigated the use of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the mitochondrial 12S rRNA gene for identification of the origin of meats. PCR-RFLP was applied for species identification of beef, buffalo meat, mutton and chevon. PCR amplification yielded a 456-bp fragment in each of these species. The amplicons were digested with AluI, HhaI, ApoI and BspTI restriction enzymes resulting in a pattern that could identify and differentiate each of the above species. This technique did not yield satisfactory results with meat mixtures/meats. However, consistent results were obtained with both fresh and processed meat samples.     

实时荧光PCR定量检测肉制品中猪源性成分

为了准确可靠地对肉制品中猪源性成分进行定量检测,通过生物信息学方法筛选到猪细胞核单拷贝基因(carcinoembryonic antigen-related cell adhesion molecule 2-like,CACA).以CACA基因为扩增靶标,设计了特异性引物、TaqMan探针,建立了基于实时荧光定量PCR...     

一种基于实时荧光聚合酶链式反应的肉及肉制品中猪源性成分含量测定

为了建立一种基于实时荧光聚合酶链式反应的肉及肉制品猪源性成分含量测定方法,分别以猪线粒体DNA的NADH4基因和16S rRNA基因为靶位点设计猪特异性引物、探针和通用引物、探针,建立猪源性成分含量测定方法,通过特异性、灵敏性、线性、准确度及市售肉制品检测,对该方法体系进行检验和评价.结果表明:建立的猪源性成分含量实时荧光聚合酶链式反应测定方法体系具有良好的特异性及灵敏性,最低可检测到0.4pg/μL纯猪肉DNA;无论是猪特异性体系还是通用体系都呈现良好的线性关系(R2均达到0.999以上)和较宽的线性范围;通过含猪源性成分的模拟混合肉样检测,样品猪源性成分含量的回收率平均值为122.27％,说明该方法具有较高的准确度;通过市售肉制品检测表明该方法可以用来检测实际样品(包括生鲜样品和熟肉样品)中猪源性成分含量.     

4种动物源性成分多重real-time PCR检测方法的建立及其在驴肉制品检测中的应用

建立一种同时快速检测驴、马、猪及鸭源性成分的四重实时聚合酶链式反应(real-time polymerase chain reaction,real-time PCR)方法。分别以4种源性成分的Nad5、ATpase6、ATP8、cytb基因为靶基因设计特异性引物和Taq Man探针,以18S r RNA基因为内参基因,建立多重real-time PCR方法,并对该方法进行方法学验证,同时对不同掺入比例模拟样品、不同加工工艺模拟样品和实际驴肉样品进行检测。结果显示,该方法具有高通量、特异性强、灵敏度高等优点。当Ct值≤35.0时,方法对16种非目标源性具有良好特异性;灵敏度可检测到质量浓度为2×10^-4 ng/μL的模板DNA;对生肉的检出限为肉含量的0.001%,对熟肉制品的检出限为肉含量的0.01%;对100份实际样品进行检测,结果与标准方法一致,说明建立的多重real-time PCR法可用于肉及肉制品中常见掺假源性成分的检测。     

核酸检测技术检测肉类种源研究进展

肉及肉制品是人类重要的食物来源, 为人体提供必要的营养素。但是以经济为目的的肉制品掺假, 是食品安全中屡禁不止的全球性问题。快速、准确、有效的检测技术是有效监督肉类掺假的关键。本文综述了核酸检测技术中热循环扩增技术(如普通PCR、实时PCR、多重PCR)和等温扩增技术[如环介导等温扩增(loop-mediated isothermal amplification, LAMP)技术、重组酶聚合酶扩增(recombinase polymerase amplification, RPA)技术、滚环扩增(rolling circle amplification, RCA)技术、交叉引物等温扩增(cross prime amplification, CPA)技术等]的原理及在肉类种源鉴别中的应用。提出梯型熔解温度等温扩增(ladder-shape melting temperature isothermal amplification, LMTIA)技术, 以期推进核酸检测技术的研究及在肉制品领域的应用。在肉类种源的检测中, 实时荧光定量PCR技术、跨越式滚环等温扩增(saltatory rolling circle amplification, SRCA)技术等均能检出0.01%的掺伪, 可用于定量检测, 表明这些核酸技术在肉类种源检测中有很好的应用前景。     

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。     

Quantitative real‐time PCR assays to detect DNA degradation in soy‐based food products

BACKGROUND: Polymerase chain reaction (PCR)‐based DNA diagnostics have grown in importance for assessing food quality and safety. PCR diagnostic reliability and sensitivity depend on the quality of the DNA extractions, including the extent of DNA degradation and the presence of PCR inhibitors. RESULTS: An approach has been described that quantifies the extent of DNA degradation in soy food samples using anchored real‐time PCR (qPCR) assays that amplify target sequences ranging from < 100 to > 1000 bp, based on two endogenous soy sequences. DNA degradation was quantified for model foods produced in the laboratory (cooked soy meal, tofu) as well as purchased soy‐containing food products. Considerably less than 1% of the total DNA extracted from samples was available for amplification of the longest amplicons (830 and 1022 bp) from the most highly processed food products (e.g., soy‐based infant formula). CONCLUSION: The utility of anchored qPCR assays was demonstrated for characterizing the amount of DNA that is available for amplifying different‐length PCR products from a range of soy‐containing processed food products. This approach should be useful for estimating the amount of amplifiable DNA in food ingredients in cases where food processing has caused degradation of DNA. Copyright © 2009 Society of Chemical Industry     

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。      

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.