Molecular detection of meat animal species targeting MT 12S rRNA gene

The efficacy of PCR-RFLP analysis of mt 12S rRNA gene in identification of animal species from meat samples of known and unknown origin and adulterated meat samples was evaluated. In PCR, all the samples generated an amplicon of 456 bp. Restriction enzyme digestion of the PCR product with AluI, HhaI, BspTI and ApoI revealed characteristic RFLP patterns. Of the samples of unknown origin few were identified as cattle, few as buffalo and some were admixtures of two, suggesting adulteration. The RFLP pattern of one did not match any of species included in the study, which on sequencing was confirmed as camel meat. Application of this technique on adulterated meat samples could detect both animal species in proportion of 50:50 and 75:25 (except in case of goat+cattle). The technique however could not detect any of the two species when proportion of mixture was 90:10 (except in case of cattle+buffalo).     

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.     

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.     

Detection of Adulteration of Meat and Meat Products with Buffalo Meat Employing Polymerase Chain Reaction Assay

The primer pair was designed based on mitochondrial d-loop gene for detection of adulteration of buffalo meat in admixed meat and meat products by polymerase chain reaction (PCR) assay. Amplification of 537-bp DNA fragments was observed from buffalo, without any cross-reaction with cattle, sheep, goat, pig, and chicken. The amplification was further confirmed by BamHI restriction enzymes. No adverse effect of processing was found on PCR amplification of buffalo meat DNA extracted from processed meat and meat products, even from meat emulsion autoclaved at 121 °C, 20 psi for 15–20 min. The detection limit for buffalo meat was found to be 1% in the admixed meat and meat products; however, very faint and inconsistent results were obtained in autoclaved meat emulsion at 1% level. The developed PCR assay was found to be specific for buffalo and could be a useful tool for detection of meat adulteration.     

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.     

一对同时鉴定8种动物源性成分的通用引物的制备及应用

肉类真假鉴定是食品检测工作的内容之一,目前已有多种基于PCR的肉类鉴定方法,但是鉴定种类和效率受限。本研究设计了一对基于普通PCR技术可同时鉴定8种动物源性成分的通用引物并建立了鉴定方法。该引物以线粒体DNA为靶标,利用扩增产物中不同物种间的插入缺失多态性片段大小即可鉴定山羊、绵羊、鹿、水牛、牛、牦牛、猪和骆驼8个物种,扩增后分别得到728 bp、704 bp、504 bp、453 bp、448 bp、431 bp、396 bp和326 bp的片段,每种PCR产物经SspI酶切后产生数量和大小不同的片段,可以进一步清晰鉴别8个物种。引物特异性测试表明和其他常见肉类动物DNA无交叉反应,DNA检测最低限度在0.01~0.05 ng。应用本方法对40份市场肉类及产品的检测表明,羊肉串、羊肉卷以及特色畜产品如驼肉、鹿肉和驴肉存在较多的掺假行为。与其他现有PCR检测方法相比,该方法具有简便易行和高通量的优点,可以作为肉类掺假筛选检测的常规方法。     

一种基于多重实时荧光聚合酶链式反应熔解曲线分析的肉及肉制品掺假鉴别方法

为实现肉及肉制品掺假快速鉴别,分别以猪、牛线粒体DNA的COXⅠ,绵羊、山羊、狗、狐狸、貉线粒体DNA的16S rRNA及鸡、鸭线粒体DNA的12S rRNA基因为靶位点,设计扩增产物熔解温度(T_m值)具有显著性差异的特异性引物,建立一种用于快速鉴别肉或肉制品中猪、牛、绵羊、山羊、鸡、鸭、狗、狐、貉9种源性成分的5重实时荧光聚合酶链式反应熔解曲线分析方法,通过特异性、灵敏度及市售样品的检测,对该方法进行检验和评价。结果表明:方法具有良好的特异性及灵敏度,单物种DNA检出限为0.001~1 ng,多物种混合DNA检出限均为0.1 ng,通过市售样品检测表明该方法可用于实际样品(包括生鲜样品和熟制样品)掺假的快速鉴别。     

Use of species-specific antisera to adrenal heat-stable antigens for the identification of raw and cooked meats by agar gel diffusion and counter immunoelectrophoretic techniques

Rabbit antisera to adrenal heat-stable and ethanol-precipitable antigens of buffalo, cattle, sheep, goat and pig were used to develop an agar gel precipitation test and a counter immunoelectrophoretic method for the identification of homologous species in raw, partially heated and boiled meat extracts. Immunoabsorption was necessary to make the primary antisera species specific. The specific antisera can be recommended for identification of the species of origin of meats and their mixtures (5-10% adulteration) in raw, partially heated and cooked states even in the case of closely related species, viz cattle and buffalo, sheep and goat.     

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.     

PCR identification of ruminant tissue in raw and heat-treated meat meals

To control the spread of bovine spongiform encephalopathy in cattle through contaminated animal feedstuffs, screening of feed products is essential. We designed five pairs of primers to identify specifically raw and heat-treated tissue from cattle, sheep, goat, deer, and ruminants in general. A forward common primer was designed based on a conserved DNA sequence in the mitochondrial 12S rRNA-tRNA(val)-16S rRNA gene, and reverse primers were designed to hybridize with a species-specific DNA sequence for each species considered. All primers were developed to create a specific PCR product small enough (less than 200 bp) to be suitable for heat-treated material. To evaluate the effect of heat treatment, a severe sterilization condition (133 degrees C at 300 kPa for 20 min) was chosen. Species-specific amplicons were obtained from all types of heat-treated meat meals. Analysis of laboratory-contaminated vegetable meals revealed that the detection limit of the assay was 0.05% for each species analyzed. This PCR-based analysis can be used as a routine method for detecting banned animal-derived ingredients in raw and heat-treated feedstuffs.     

利用12S rRNA基因的限制性酶切末端片段长度多态性鉴定动物种类

目的：建立一种精确可靠的鉴定常见的1 0 种动物( 猪、狗、牛、山羊、绵羊、马、鸡、鼠、三文鱼和鹿)的方法。方法：利用12S rRNA 基因的限制性酶切末端片段长度多态性(terminal restriction fragment lengthpolymorphism,T-RFLP)鉴别动物种类。将线粒体12S rRNA 基因通过引物的5＇端用FAM 荧光标记,从基因组DNA 中扩增450bp 的目的片段。引物对1(下游引物FAM标记,上游引物不标记)扩增的PCR 产物用限制性内切酶Alu Ⅰ酶切。引物对2(上游引物FAM标记,下游引物不标记)扩增的PCR 产物用限制性内切酶Tru9 Ⅰ酶切。得到的酶切产物分别在遗传分析仪ABI 3100 上进行毛细管电泳,片段大小用Peak Scanner 1.0 软件分析。结果：根据Alu Ⅰ酶切图谱能够区分鸡、马、猪和三文鱼,而鹿和牛、山羊和绵羊、鼠和狗因酶切图谱相同无法分开。根据Tru9 Ⅰ酶切图谱,能够进一步将鹿和牛、山羊和绵羊、鼠和狗分开。同一种动物不同个体的酶切图谱完全相同,结果具有可重复性。没有出现物种内多态的现象。大多数情况下,实际得到的末端片段长度与理论值非常接近,只存在2 ～5bp 的差异。结论：该方法操作简单、结果精确,适用于鉴定动物种类。     

Identification of meats from red deer (Cervus elaphus), fallow deer (Dama dama), and roe deer (Capreolus capreolus) using polymerase chain reaction targeting specific sequences from the mitochondrial 12S rRNA gene

Polymerase chain reaction (PCR) based on oligonucleotide primers targeting the mitochondrial 12S rRNA gene was applied to the specific identification of meats from red deer (Cervus elaphus), fallow deer (Dama dama), and roe deer (Capreolus capreolus). The use of a common reverse primer, together with forward specific primers for red deer, fallow deer, and roe deer, allowed the selective amplification of the desired cervid sequences. The specificity of each primer pair was verified by PCR analysis of DNA from various game and domestic meats. The assay can be useful for the accurate identification of meats from cervid species, avoiding mislabeling or fraudulent species substitution in meat products.     

COI序列应用于羊肉掺伪非定向筛查技术的研究

本项目研究了应用COI序列对羊肉中掺入的其他动物源材料进行非定向筛查,并对山羊肉及绵羊肉进行分辨。利用了DNA条形码技术对六个羊品种(系)的线粒体基因组细胞色素酶基因片段进行引物筛选,并利用筛选出的引物对七种动物源样本的COI片段进行扩增,以确定所筛选出的引物是否能将羊肉与其他动物源样品进行有效区分。以LCOI490/HCO2198、F1-1/R1-1、ITS3/ITS4为引物,通过对36份血液样本(山羊3个品种,绵羊3个品种)的基因组DNA进行PCR扩增,其中以F1-1/R1-1为引物得到的PCR产物电泳检测条带清晰,结果重现性好;利用F1-1/R1-1引物对鸡肉、猪肉、驴肉、牛肉、莱芜黑山羊肉、洼地绵羊肉和鸭肉等七种样本的COI片段进行扩增,并对扩增片段进行测序,山羊、绵羊之间的序列同源性仅为36.62%,其他各品种间的序列差异也很明显。结果表明:以F1-1/R1-1为专用引物可以有效区分羊肉与其他动物源样品,并能够对山羊肉及绵羊肉样品进行有效区别。     

Cloth-based hybridization array system for the detection and identification of ruminant species in animal feed

A cloth-based hybridization array system for the detection and identification of material derived from several ruminant species (cattle, sheep, goat, elk, and deer) in animal feeds has been developed. Primers targeting conserved mitochondrial DNA sequences amplified ruminant DNA in a universal PCR, and the digoxigenin-labeled amplicons were hybridized with an array of species-specific oligonucleotide capture probes on a polyester cloth support. The hybridized amplicons were detected on the cloth by sequential reactions with antidigoxigenin antibody-peroxidase conjugate and chromogenic substrate solution. This cloth-based hybridization array system provided sensitive and specific detection and identification of meat meal containing rendered cattle, sheep, goat, elk, and deer material blended in feeds.     

Analysis of raw meats and fats of pigs using polymerase chain reaction for Halal authentication

A method for species identification from pork and lard samples using polymerase chain reaction (PCR) analysis of a conserved region in the mitochondrial (mt) cytochrome b (cyt b) gene has been developed. Genomic DNA of pork and lard were extracted using Qiagen DNeasy^® Tissue Kits and subjected to PCR amplification targeting the mt cyt b gene. The genomic DNA from lard was found to be of good quality and produced clear PCR products on the amplification of the mt cyt b gene of approximately 360 base pairs. To distinguish between species, the amplified PCR products were cut with restriction enzyme BsaJI resulting in porcine-specific restriction fragment length polymorphisms (RFLP). The cyt b PCR-RFLP species identification assay yielded excellent results for identification of pig species. It is a potentially reliable technique for detection of pig meat and fat from other animals for Halal authentication.     

Cloth-based hybridization array system for expanded identification of the animal species origin of derived materials in feeds

A cloth-based hybridization array system (CHAS) previously developed for the detection of animal species for which prohibited materials have been specified (cattle, sheep, goat, elk, and deer) has been expanded to include the detection of animal species for which there are no prohibitions (pig and horse) in Canadian and American animal feeds. Animal species were identified by amplification of mitochondrial DNA sequences by PCR and subsequent hybridization of the amplicons with an array of species-specific oligonucleotide capture probes immobilized on a polyester cloth support, followed by an immunoenzymatic assay of the bound PCR products. The CHAS permitted sensitive and specific detection of meat meals from different animal species blended in a grain-based feed and should provide a useful adjunct to microscopic examination for the identification of prohibited materials in animal feeds.     

Identification of 12 animal species meat by T-RFLP on the 12S rRNA gene

The verification of authenticity of meat products is relevant for economical, religious or public health concerning reasons. A molecular approach using terminal restriction fragment length polymorphism (T-RFLP) was developed to distinguish 12 common economically important meat species. The partial 12S rRNA gene was amplified with double-fluorescently labeled primers. The amplified fragments were digested with two endonucleases and only the terminal restriction fragment containing labeled primer was detected on capillary electrophoresis system ABI3100. AluI and Tru9I generated differently-sized terminal fragments in different species. Pig and buffalo can be separated by 3′-terminal fragment of AluI digestion. Horse, turkey, goat, sheep, deer, and cattle can be further separated by 5′-terminal fragment of Tru9I digestion. Dog and chicken, sturgeon and salmon can finally be separated by 5′-terminal fragment of AluI digestion and 3′-terminal fragment of Tru9I digestion. Our results demonstrated the potential feasibility and applicability of T-RFLP method for rapid and accurate identification of animal species.     

Authentication of carabeef (water buffalo, <Emphasis Type="Italic">Bubalus bubalis</Emphasis>) using highly specific polymerase chain reaction

In order to ensure consumer satisfaction and fraud detection, correct identification of meat animal species becomes significant. Buffalo being one of the major meat animal species in Asia, a species-specific polymerase chain reaction (PCR) was developed for the accurate identification of carabeef (water buffalo, Bubalus bubalis) targeting mitochondrial D-loop region. Unique diagnostic PCR developed in this study employed novel primers to yield a 534-bp buffalo-specific PCR product, and chances of cross-amplification were excluded by including as many as 25 animal species. Applicability of PCR was established in raw, cooked (60, 80 and 100 °C for 30 min), autoclaved (121 °C for 30 min) and microoven-processed meats with a sensitivity of detection of 0.1% adulteration (10 pg bubaline DNA). Keeping in view adulteration, socio-economic, religious, quality assurance, forensic and legal issues, the novel buffalo-specific PCR developed in this study was found highly promising in authenticating buffalo meat, ensuring consumer satisfaction and labeling process.     

利用线粒体DNA Cyt b基因PCR-RFLP分析方法鉴别羊肉和鸭肉

建立了一种利用线粒体DNA(mtDNA) Cyt b基因PCR-RFLP分析来鉴别羊肉和鸭肉的方法。采用一对通用引物扩增绵羊、山羊和鸭的mtDNACytb基因,并对扩增产物用DNA限制性内切酶Bsu36I和SpeI进行酶切,电泳分析酶切产物的变化。结果表明通用引物可扩增羊和鸭472bp的PCR产物,经两种内切酶酶切后,绵羊、山羊和鸭的PCR产物分别被切为大小不同的片段,其中绵羊和山羊被SpeI切为213bp和259bp,而鸭则被Bsu36I切为95bp和377bp。利用PCR-RFLP分析mtDNA Cyt b基因的方法操作简单,是一种快速鉴别羊肉和鸭肉的可靠方法。     

Polymerase chain reaction assay for detection of sheep and goat meats

Polymerase Chain Reaction (PCR) was applied to a qualitative differentiation between sheep, goat and bovine meats. Oligonucleotide primers were designed for the amplification of sheep satellite I DNA sequence. The PCR amplified 374 bp fragments from sheep and goat DNA, but no fragment from bovine, water buffalo, sika deer, pig, horse, rabbit and chicken DNA. Sheep DNA (10 pg) was detected by 4% agarose gel electrophoresis following PCR amplification. Althoug cooking of the sample meats reduced the PCR products, sheep DNA was detected in the meat heated at 120°C. In order to differentiate between sheep and goat meats, nucleotide sequences of the PCR products were determined directly by cycle sequencing. The sequence of PCR products showed 92% of homology between sheep and goat. They were differentiated by ApaI digestion of the PCR products because sheep had one ApaI site and goat had no site in the PCR products.