Meat species identification and Halal authentication analysis using mitochondrial DNA

A method utilizing PCR-restriction fragment length polymorphism (RFLP) in the mitochondrial genes was developed for beef (Bos taurus), pork (Sus scrofa), buffalo (Bubalus bubali), quail (Coturnix coturnix), chicken (Gallus gallus), goat (Capra hircus), rabbit (Oryctolagus cuniculus) species identification and Halal authentication. PCR products of 359-bp were successfully obtained from the cyt b gene of these six meats. AluI, BsaJI, RsaI, MseI, and BstUI enzymes were identified as potential restriction endonucleases to differentiate the meats. The genetic differences within the cyt b gene among the meat were successfully confirmed by PCR-RFLP. A reliable typing scheme of species which revealed the genetic differences among the species was developed.     

Detection of porcine DNA in gelatine and gelatine-containing processed food products-Halal/Kosher authentication

A commercially available real-time PCR, based on a multi-copy target cytochrome b (cyt b) using porcine specific primers, has been validated for the Halal/Kosher authentication of gelatine. Extraction and purification of DNA from gelatine were successfully achieved using the SureFood® PREP Animal system, and real-time PCR was carried out using SureFood® Animal ID Pork Sens kit. The minimum level of adulteration that could be detected was 1.0% w/w for marshmallows and gum drops. A small survey was undertaken of processed food products such as gum drops, marshmallows and Turkish delight, believed to contain gelatine. Of fourteen food products from Germany, two samples were found to contain porcine gelatine, whereas of twenty-nine samples from Turkey twenty-eight were negative. However, one product from Turkey contained porcine DNA and thus was not Halal, and neither was the use of porcine gelatine indicated on the product label.     

Identification of pork genome in commercial meat extracts for Halal authentication by SYBR green I real‐time PCR

The identification of pork DNA in meat extracts is very important for Halal authentication and Muslim consumers demand protection from falsely labelled meat products. A pig‐specific SYBR green I real‐time PCR assay has been developed to address this issue. Using specific primers for pig mitochondrial DNA, successful amplification has been obtained by DNA extracted from control meat samples. With SYBR green I real‐time PCR, the specificity of the amplification was showed by Tm value. Detection limit of the real‐time PCR was down to 0.1 ng of porcine DNA. An appropriate linearity was obtained by construction of a standard curve based on Ct value and different concentrations of porcine DNA. By conventional PCR, no amplification was shown by porcine DNA less than 0.1 ng. The established method was conducted on commercially available meat extracts for detection and quantification of porcine DNA. The results showed the SYBR green I real‐time PCR could be considered a robust method for Halal authentication of meat extracts.     

Analysis of Porcine Gelatin DNA in a Commercial Capsule Shell Using Real-Time Polymerase Chain Reaction for Halal Authentication

The presence of pig derivatives, such as porcine gelatin, in any products is prohibited to be consumed by Muslim community. This study is intended to develop a specific primer from mytochondrial D-loop capable of amplifying DNA from porcine gelatin in commercial capsule shells. Two pairs of primers designed from mitochondrial D-loop region were tested in order to confirm the primer specificity in gelatin sources (pork, beef, and catfish) and fresh tissue (pig, cows, goat, chickens, and rat). Primers were then used to perform sensitivity test of six dilution series (1000, 200, 100, 10, 5, and 1 pg/µL) of porcine gelatin and porcine capsule shell. The amplification was also performed on capsule shell from porcine-bovine mixture gelatin at 0, 10, 20, 30, 40, 50, and 100% concentration. The repeatability test was performed by measuring amplification capsule shells from porcine–bovine gelatin mixture. Real time polymerase chain reaction method using primers designed was further applied to analyze capsule shells purchased from markets. From two primers have been designed specifically, only primer D-Loop 108 (forward: 5’-CGT ATG CAA AAA ACC ACG CCA-3’; reverse: 5’-CTT ACT ATA GGG AGC TGC ATG-3’) had the capability to identify the presence of porcine DNA in fresh tissue and gelatin sources at optimum annealing temperature of 58.4ºC. Sensitivity of the developed method expressed as limit of detection of DNA in gelatin and capsule shells is 5 pg.     

PCR assay for the identification of animal species in cooked sausages

A species-specific PCR assay was developed for the detection of low levels of pork, horse and donkey meat in cooked sausages. Oligonucleotid primers were designed for amplification of species-specific mitochondrial DNA sequences of each species and detected the presence of 0.01 ng of template DNA in water. When applying the assay to DNA extracts from sausages samples that were prepared from binary meat mixtures, it was possible to detect each species when spiked in any other species at the 0.1% level. In conclusion, it can be suggested that this assay can be used to determine mislabelled and/or fraudulent species substitution in comminuted meat products.     

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.     

Determination of Sex Origin of Meat and Meat Products on the DNA Basis: A Review

Sex determination of domestic animal's meat is of potential value in meat authentication and quality control studies. Methods aiming at determining the sex origin of meat may be based either on the analysis of hormone or on the analysis of nucleic acids. At the present time, sex determination of meat and meat products based on hormone analysis employ gas chromatography-mass spectrometry (GC-MS), high-performance liquid chromatography-mass spectrometry/mass spectrometry (HPLC-MS/MS), and enzyme-linked immunosorbent assay (ELISA). Most of the hormone-based methods proved to be highly specific and sensitive but were not performed on a regular basis for meat sexing due to the technical limitations or the expensive equipments required. On the other hand, the most common methodology to determine the sex of meat is unquestionably traditional polymerase chain reaction (PCR) that involves gel electrophoresis of DNA amplicons. This review is intended to provide an overview of the DNA-based methods for sex determination of meat and meat products.     

Identification of Species Origin of Meat and Meat Products on the DNA Basis: A Review

The adulteration/substitution of meat has always been a concern for various reasons such as public health, religious factors, wholesomeness, and unhealthy competition in meat market. Consumer should be protected from these malicious practices of meat adulterations by quick, precise, and specific identification of meat animal species. Several analytical methodologies have been employed for meat speciation based on anatomical, histological, microscopic, organoleptic, chemical, electrophoretic, chromatographic, or immunological principles. However, by virtue of their inherent limitations, most of these techniques have been replaced by the recent DNA-based molecular techniques. In the last decades, several methods based on polymerase chain reaction have been proposed as useful means for identifying the species origin in meat and meat products, due to their high specificity and sensitivity, as well as rapid processing time and low cost. This review intends to provide an updated and extensive overview on the DNA-based methods for species identification in meat and meat products.     

肉及肉制品中沙门氏菌活细胞的PCR检测研究

将荧光染料叠氮溴化丙锭(propidium monoazide,PMA)与普通聚合酶链式反应(polymerase chain reaction,PCR)结合,通过对PMA的曝光时间、浓度进行优化,确定PMA-PCR区别死活细胞的最佳条件,并制作活细胞定量标准曲线,建立肉及肉制品中沙门氏菌活细胞的PMA-PCR检测方法。结果表明：使插入死细胞DNA中的PMA活化并且光解溶液中游离PMA的最佳曝光时间为15min;不抑制沙门氏菌活细胞DNA扩增的最大PMA质量浓度为10μg/mL;完全抑制热致死细胞DNA扩增的最小PMA质量浓度为4μg/mL。经PMA处理,含有不同比例的沙门氏菌热致死细胞和活细胞的混合液中活的沙门氏菌能够通过PCR被选择性的检测,最小检测限为20CFU/PCR。而且,经研究发现在20～2×105CFU/PCR范围内,电泳条带相对荧光强度与活细胞数的对数具有线性关系。采集30份肉及肉制品样品,利用PMA-PCR方法检测出两份生肉样品中存在沙门氏菌,经过6h的富集培养后的活菌浓度分别为2.5×103CFU/mL和3.4×103CFU/mL。     

Application of the novel Droplet digital PCR technology for identification of meat species

The authenticity and traceability of meat products are issues of primary importance to ensure food safety. Unfortunately, food adulteration (e.g. the addition of inexpensive cuts to minced meat products) and mislabelling (e.g. the inclusion of meat from species other than those declared) happens frequently worldwide. The aim of this study was to apply a droplet digital PCR assay for the detection and quantification (copies μL⁻¹) of the beef, pork, horse, sheep, chicken and turkey in meat products. The analysis conducted on commercial meat showed the presence of traces of DNA from other animal species than those declared. We show that the method is highly sensitive, specific and accurate (accuracy = 100%). This method could be adopted by competent food safety authorities to verify compliance with the labelling of meat products and to ensure quality and safety throughout the meat supply chain, from primary production to consumption.     

Detection of central nervous system tissue as bovine spongiform encephalopathy specified risk material in traditional Turkish meat products

BACKGROUND: This study used enzyme‐linked immunosorbent assay kits to investigate the presence of central nervous system (CNS) tissue in commercial raw and processed traditional Turkish meat products offered for consumption in various markets. RESULTS: Ninety‐six raw traditional Turkish meat products (32 fresh raw beef patties, 32 cig kofta, 32 pastirma) and 64 processed traditional Turkish meat products (32 doner kebabs and 32 fresh processed beef patties) were analysed. CNS tissue was not found in pastirma, doner kebab, or fresh processed beef patty samples. The levels of CNS contamination in fresh raw beef patties were low (0.1% absorbance standard; 3.1%) and moderate (0.2% absorbance standard; 6.2%). The level of contamination in the cig kofta was low (0.1% absorbance standard; 18.8%). CONCLUSION: CNS tissue was present in all raw traditional Turkish meat products except for pastirma. Copyright © 2011 Society of Chemical Industry     

基于实时荧光定量PCR和数字PCR的肉制品中牛源性成分检测

为了能够快速有效的检测出肉制品中牛源性成分,采用实时荧光定量PCR和数字PCR检测方法,检测肉制品中牛源成分。先依据牛肉单复制基因组特异区域,通过多序列比对设计特异性引物与探针,再利用苯酚-氯仿法提取标准品基因组DNA,确立实时荧光定量PCR和数字PCR两种方法的反应条件,测定DNA模版纯度及浓度可得实验提取的DNA溶液不含杂质,在此基础上构建牛源性成分荧光定量PCR标准曲线,检测两种方法的特异性、抗干扰性与肉制品中牛源性成分。分析实验结果可知,引物与探针在两种方法中均对牛肉有荧光信号显示,两种方法具有牛源性特异性,检测数值与实际样品数值基本一致,且两种方法抗干扰性较好,当存在其他肉类干扰时,仍可准确监测出牛源性,两种方法的最大误差分别为3.8%和4.0%;可定量检测不同肉制品中牛源性成分,两种方法均未检测出样品10牛肉丸中牛源性成分,验证了市面上确实存在假肉情况。说明所用方法能够准确、快速地检测出肉制品中的牛源性成分,适用于市场中肉制品的检测,对保障消费者的权益和健康具有十分重要的意义。     

多重PCR技术检测肉品中致病菌的应用研究

快速检测和鉴别肉品中的致病菌是保障肉品质量安全,防止食源性疾病爆发的有效手段。多重PCR作为一种同时检测多种病原菌的PCR技术,具有高特异性,高灵敏度,低成本的特点。本文简要介绍了多重PCR原理,综述了PCR反应条件及检测肉品中致病菌的应用,并对其存在的问题做了简单阐述。     

食品中鲨鱼源性成分真实性PCR鉴别研究

建立食品中真实性鲨鱼源性成分PCR鉴别方法,该方法特异性强,灵敏度高。运用普通PCR技术对9种鲨鱼及42种常见非鲨鱼类动植物样品进行检测,9种鲨鱼中出现180bp的特异性扩增条带,其他非鲨鱼样品中均未出现扩增条带,实验表明,本检测方法具有特异性,检测限为0.1ng/μL鲨鱼DNA和0.1%(W/W)鲨鱼肉粉。运用建立的方法对20种常见的鲨鱼产品进行PCR检测,除仿鱼翅和鲨鱼肝油外,所有产品中均能检测出鲨鱼成分。该检测方法能够用于食品中鲨鱼源性成分的真实性鉴别。      

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.     

肉品微生态系统与肉类发酵剂研究

论述了鲜肉、腌制、熏制及传统自然发酵肉制品中主要微生态菌群的构成与气调贮藏、冷链系统中肉品微生态系的变化。介绍了国外商业化肉类发酵剂中酵母菌、霉菌、乳酸菌、微球菌、链霉菌等微生物的生理生化与分子特性。可以认为 ,以PCR为基础的分子检测技术将是对食品微生态系进行研究的有效手段。     

多重PCR法用于畜肉源性鉴定的研究

根据Genebank中猪、牛、羊的线粒体细胞色素b（Cyt-b）基因序列,设计了猪、牛、羊的通用上游引物和特异性下游引物,通过调节引物比例、反应体系、反应条件以及模板量等优化实验确立了多重PCR检测方法。研究结果显示,该多重PCR方法能够同时扩增样品中猪、牛、羊成分,对混合样品的检出限可达10%;并将该方法用于成都市猪、牛、羊肉的调查研究,得到了理想的结果。      

Chelex 法结合环介导间接聚合酶链式反应检测肉制品单增李斯特菌

目的：建立肉制品中单增李斯特菌环介导间接聚合酶链式反应检测体系,实现快速检测。方法：采用Chelex法从肉制品中分离模板DNA,根据单增李斯特菌hlyA基因的保守区设计两条探针,将探针标记于报告基因(大豆Lectin基因)的两端,此标记的报告基因与待测靶基因经杂交、缺口补平、环化后,采用反向聚合酶链式反应扩增报告基因,实现对靶基因的检测。结果：该检测体系检出限低于100CFU/g(mL),且与其他食源性致病菌的检测无明显交叉反应,对200份肉制品进行检测,阳性率为2.5%,与传统细菌分离检测结果完全相符。结论：环介导间接聚合酶链式反应可以快速、灵敏、特异检测肉制品中单增李斯特菌污染。     

Identification of pork in beef meatballs using Fourier transform infrared spectrophotometry and real-time polymerase chain reaction

A study on development of Fourier-transform infrared spectrophotometric method combined with principle component analysis as well as real-time polymerase chain reaction for determination of pork–beef mixture in meatballs has been performed. A lipid component extracted from pork and beef in meatballs is analyzed using Fourier-transform infrared spectroscopy, while DNA extracted from meatball was analyzed using real-time polymerase chain reaction. The correlation between actual and predicted concentration of lard using Fourier-transform infrared spectroscopy was performed by aid of partial least squares, while grouping of lard and beef fat components in meatball was carried out by Fourier-transform infrared spectra coupled with principle component analysis. The results showed that Fourier-transform infrared spectra at wavenumbers of 1000–1200 cm^?1 coupled with partial least square and principle component analysis are successfully used for quantification and classification of pork in beef meatballs. The relationship between actual value and predicted value of lard (lipid fraction obtained from meatballs containing pork) with Fourier-transform infrared spectrophotometric method revealed good correlation, with coefficient determination (R²) value of 0.997 and standard error of calibration of 0.04%. Principle component analysis is able to classify samples containing pork and beef meatballs. Fourier-transform infrared spectroscopy using normal spectra is fast technique for identification and quantification of lard extracted from pork in meatball. In addition, real-time polymerase chain reaction using Leptin Primer–AJ 865080 can be used for amplification of pork DNA specifically in meatballs containing pork.