Mitochondrial DNA enrichment for species identification and evolutionary analysis

 Nucleic acid-based species identification often targets the mitochondrial encoded cytb gene. However, polymerase chain reaction (PCR)- restriction fragment length polymorphism (RFLP) analysis using universal primers sometimes leads to ambiguous results, which are due to the presence of nuclear encoded pseudo-cytb genes. Such ambiguities were succesfully avoided using a newly developed method for the enrichment of mitochondrial DNA. In addition, a mitochondrial cytb-specific PCR system was designed allowing the unambiguous identification of game meat. Received: 24 February 1998     

Mitochondrial DNA enrichment for species identification and evolutionary analysis

 Nucleic acid-based species identification often targets the mitochondrial encoded cytb gene. However, polymerase chain reaction (PCR)- restriction fragment length polymorphism (RFLP) analysis using universal primers sometimes leads to ambiguous results, which are due to the presence of nuclear encoded pseudo-cytb genes. Such ambiguities were succesfully avoided using a newly developed method for the enrichment of mitochondrial DNA. In addition, a mitochondrial cytb-specific PCR system was designed allowing the unambiguous identification of game meat. Received: 24 February 1998     

Differentiation of animal species in food by oligonucleotide microarray hybridization

Oligonucleotide microarray hybridization analysis of polymerase chain reaction (PCR) products from the mitochondrial cytochrome b gene DNA was applied to identify different animal species in meat and cheese food samples. A pair of universal primers binding to conserved regions of the vertebrate mitochondrial cytochrome b gene was used to amplify a 377 bp fragment with internal regions of high inter-species variability. PCR products of cattle, pig, chicken, turkey, sheep and goat were unequivocally identified by hybridization with species-specific probe sequences immobilized on an oligonucleotide microarray. In meat samples, 0.1% admixtures of beef or chicken meat were still detectable. By using this new PCR-based DNA chip hybridization for the analysis of 24 commercial food samples from routine control, the simultaneous species composition of mixtures with up to four different species could be determined in a single experiment. The results agreed well with those from the reference methods performed at the local food control authority, which are a combination of enzyme-linked immunosorbent assay (ELISA), species-specific PCR and PCR–RFLP (restriction fragment length polymorphism). Thus, the DNA chip hybridization analysis of cytochrome b PCR products offers a new way for rapid and sensitive species differentiation in food.     

Determination of Porcine Contamination in Laboratory Prepared dendeng Using Mitochondrial D-Loop686 and cyt b Gene Primers by Real Time Polymerase Chain Reaction

The identifications of species in meat products have created interests since these foods became the target of forgery and fraud in the market. The presence of pork in food products is not allowed for the Muslim community. Hence, an analysis is necessary to detect the presence of pork in processed meat products, such as in dendeng (dried meat) product. Real time polymerase chain reaction using mitochondrial displacement loop686 and cytochrome b (cytb) gene primers was used to identify specific pork DNA among other four types of DNA species; namely beef, chicken, goat, and horse. This method was also used to identify pork DNA in the laboratory processed pork-beef dendeng as well as commercial dendeng from market. The results showed that real time polymerase chain reaction using displacement loop686 and cytb gene primers were able specifically to distinguish between pork DNA and the other species. The lowest concentration of 0.5% of pork DNA in a mixture of pork-beef processed products of dendeng was able to be detected by both primers with the product amplification of 114 and 134 bp (base pair) for the displacement loop686 and 149 bp for cytb gene, respectively. High sensitivity was also obtained when both primers were applied with the lowest detection limit of 5 pg/µL pork DNA. The results of the six commercial dendeng amplification using both primers showed no amplified products present, meaning that these products do not contain porcine DNA.     

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.     

IDENTIFICATION OF GOAT MEAT USING HIGHLY SPECIES‐SPECIFIC POLYMERASE CHAIN REACTION

ABSTRACT

A highly species‐specific polymerase chain reaction (PCR) assay was developed for the authentic identification of goat. A product of 436 bp was amplified using newly designed primers against mitochondrial D‐loop region. The possibility of cross‐amplification was ruled out by considering as many as 25 other animal species. Suitability of the developed goat species‐specific PCR assay was confirmed for in raw, cooked (60, 80 and 100C for 30 min) and micro‐oven‐processed meat samples (n = 20 each). A sensitivity of 0.1% was established for detection of adulteration and limit of detection of goat DNA was 0.1 pg. This investigation presents a novel PCR assay with its newly designed primers that could be used for the authentic identification of goat species.

PRACTICAL APPLICATIONS

This work details about a novel diagnostic polymerase chain reaction, which could be used for authentic identification of goat species. This approach could be used for the confirmation of goat tissues in raw, cooked, as well as adulterated samples. The developed technique has also applications in the forensic analysis of wild animal‐related disputes, where this work could solve the problem of goat‐related issues.     

Standard and Light-Cycler PCR methods for animal DNA species detection in animal feedstuffs

In this work four species-specific primers and probes were designed and evaluated for the detection and quantification of bovine, ovine, swine and chicken mitochondrial DNA in feeds. PCR primers were optimized using conventional and Real Time PCR, to detect short species-specific sequences amplifiable from heat treated material. Both methods confirmed the high specificity of the primers designed. Real time quantitative PCR assay allowed the detection of as few as 0.01 ng and 0.05 ng of ovine and bovine genomic DNA, respectively. The detection limit for swine and chicken genomic DNA was 0.5 ng. Sensitivity levels observed in DNA extracted from meat samples processed according to EU legislation were different compared to those in genomic DNAs previously described. They resulted in swine 5 fg of MBM DNA, in chicken 25 ng, in ovine and bovine 50 ng. We confirmed the efficiency and specificity of primers in RT-PCR to detect 0.5% of bovine, ovine, swine and chicken MBM in contaminated feedstuffs.Industrial relevanceThe variant Creutzfeldt Jakob disease is a rare and fatal human neurodegenerative condition clearly linked with the bovine spongiform encephalopathies (BSE) of cattle. The ban of using animal derived protein in animal feeds has efficiently controlled the development of the BSE epidemic. The work presented by Frezza and collaborators is an application of the real time polymerase chain reaction (a standard procedure used in molecular biology also known as RT‐PCR) to identify specific DNA of four animal species (bovine, ovine, swine and chicken). This method is applied to the analysis of feeds to detect and eventually estimate the amount of animal derived proteins. The difficult aim to detect DNA derived from heat‐treated material was successfully reached using as target short mitochondrial DNA sequences. The method presented could have important application not only in the control of feed production but also in many fields of the food industry as quality and process control.     

Mitochondrial markers for the detection of four duck species and the specific identification of Muscovy duck in meat mixtures using the polymerase chain reaction

A polymerase chain reaction (PCR) assay for the qualitative detection of four duck species in meat mixtures, and a second PCR assay for the specific identification of Muscovy duck, have been developed based on oligonucleotide primers targeting the 12S rRNA mitochondrial gene. The specificity of both assays was tested against a wide range of animal species. The technique was applied to raw and sterilized muscular binary mixtures, with a detection limit that ranged from 0.1% to 1.0% (w/w). The short length (less than 100 bp) of the DNA fragments amplified with these primer pairs was found to be essential for the successful amplification in samples with highly degraded DNA, and consequently, it could be very useful in inspection programmes to enforce labelling regulation of heat and pressure-processed products, for which other methods cannot be applied.     

PCR法检测鱼及其制品中的鱼源性成分

目的:建立一种快速、特异、灵敏的鱼源性成分聚合酶链式反应(polymerase chain reaction,PCR)检测方法。方法:根据鱼线粒体基因组12S r RNA中的保守序列设计鱼源性特异性引物,进行PCR扩增,建立鱼源性成分检测方法;对24种鱼及鸡、牛、羊、猪、鸭、虾6种常见的易混于鱼制品的动物源性成分进行特异性检测;将草鱼肉混入其他动物肉中,混合均匀后提取DNA进行PCR扩增,确定肉样水平的检测灵敏度;将草鱼DNA混入其他动物DNA中,以混合后的DNA为模板进行PCR扩增,确定DNA水平的检测灵敏度。结果:该方法能特异性的对鱼源性成分进行快速检测,检测灵敏度达0.5%。结论:该方法能对食品中是否含有鱼源性成分进行初筛,到达快速检测的目的,对防止食品掺假、维护消费者利益、规范市场秩序有重要意义。     

Application of polymerase chain reaction–restriction fragment length polymorphism analysis and lab‐on‐a‐chip capillary electrophoresis for the specific identification of game and domestic meats

BACKGROUND: The objective of this study was to adapt and improve previously published polymerase chain reaction–restriction fragment length polymorphism (PCR‐RFLP) methods aimed at the identification of game and domestic meats, by replacing the gel electrophoretic steps for DNA fragment analysis by a chip‐based capillary electrophoresis system. RESULTS: PCR amplification of a mitochondrial 12S rRNA gene fragment and subsequent digestion of the amplicons with either MseI or a combination of MboII, BslI, and ApoI endonucleases generated characteristic PCR‐RFLP profiles that allowed discrimination among ten relevant game and domestic meat species. The Agilent 2100 Bioanalyzer utilises capillary electrophoresis on a microchip device that is capable of rapidly sizing DNA fragments, offering a valuable recent development for the analysis of complex DNA banding patterns. CONCLUSION: Results obtained in this work indicated that banding resolution on the system was sensitive, with detection of some small DNA fragments that were not observed with the published conventional PCR‐RFLP gel‐based method. Therefore, the new faster and easy handling procedure provides an additional powerful tool that can be employed for meat speciation. Copyright © 2009 Society of Chemical Industry     

多重位点特异性PCR快速检测牛肉中常见掺假动物源性成分

目的:基于动物线粒体cytb基因的多态性位点,建立一种特异性多重PCR体系检测牛肉、猪肉和鸡肉的方法。方法:提取肉类的基因组DNA,利用不同物种mtDNA cytb基因序列的SNP位点的差异,设计特异性引物。进行多重PCR扩增,利用扩增产物片段大小不同,检测牛肉中常见的掺假动物源性成分。通过灵敏性试验,确定最低检测量。结果:实验设计的引物特异性良好,在同一反应体系中,在同一退火温度52℃条件下,牛肉DNA扩增后产生149 bp的特异性条带,猪肉DNA扩增片段为261 bp,鸡肉DNA扩增片段为554 bp,未发生非特异性扩增。且检测的最低浓度达到100 pg/μL,具有高度的灵敏性和适用性。结论:根据动物线粒体cytb基因的差异性位点,开发的多重PCR体系,可一次性地同时检测牛肉、猪肉和鸡肉,可快速、灵敏、高通量地分析食品中掺假动物成分的来源。     

Development of a Polymerase Chain Reaction System for the Detection of Dog and Cat Meat in Meat Mixtures and Animal Feed

The identification of species origin of meat represents a considerable problem for food and animal feed analysis. In the present study a PCR‐mediated method for the detection of dog and cat meat was developed. For this the cytochrome b gene sequence of both species was analyzed by restriction fragment length polymorphism (RFLP) analysis. The use of the restriction enzymes Alu I and Hae III yielded specific restriction profiles characteristic for each species. The meat of both species could additionally be differentiated with species‐specific oligonucleotide primers based on specific parts of the cytochrome b gene sequences characteristic for dog and cat. The use of these oligonucleotide primers allowed a direct identification of dog and cat meat in meat mixtures even after heat treatment.     

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.     

A fast and accurate method for controlling the correct labeling of products containing buffalo meat using High Resolution Melting (HRM) analysis

The substitution of high priced meat with low cost ones and the fraudulent labeling of meat products make the identification and traceability of meat species and their processed products in the food chain important. A polymerase chain reaction followed by a High Resolution Melting (HRM) analysis was developed for species specific detection of buffalo; it was applied in six commercial meat products. A pair of specific 12S and universal 18S rRNA primers were employed and yielded DNA fragments of 220 bp and 77 bp, respectively. All tested products were found to contain buffalo meat and presented melting curves with at least two visible inflection points derived from the amplicons of the 12S specific and 18S universal primers. The presence of buffalo meat in meat products and the adulteration of buffalo products with unknown species were established down to a level of 0.1%. HRM was proven to be a fast and accurate technique for authentication testing of meat products.     

实时荧光聚合酶链式反应检测肉制品中的鸭源性成分

通过鸭的肌动蛋白β-actin保守基因设计可特异检测鸭肉成分的引物和探针,建立实时荧光聚合酶链式反应（polymerase chain reaction,PCR）技术检测鸭肉的方法,并通过模拟肉样和市售样品检测方法的准确性和适用性。结果表明：该方法可以特异性检测出麻鸭和草鸭成分,而对猪、牛、羊、鸡等DNA均没有扩增,检测灵敏度可达到1 pg DNA;通过模拟肉样检测确定最低质量分数检测限为0.01%;市售样品的检测结果表明,该方法能很好地应用于市场,满足市场检测需求。     

Establishment and application of a polymerase chain reaction for the identification of beef

A polymerase chain reaction (PCR) based method for the identification of beef by amplification of bovine 1.709 satellite DNA was established. The method not only was able to amplify raw beef DNA, but also cooked or autoclaved meat DNA. The sequence selected for amplification consisted of a 218 bp DNA fragment lying in the 1.709 satellite DNA of bovine. A pair of synthetic oligonucleotides flanking this sequence were used as printers, and genomic DNA extracted from beef samples employed as templates. Each batch of reaction mix contained Taq DNA polymerase, a buffer component, deoxynucleotide triphosphates, genomic DNA template and a pair of bovine oligodeoxynucleotide primers in a final volume of 50 μl. The amplification of bovine DNA was performed by using 33 cycles of denaturation at 94°C (40 s), annealing at 53.5°C (50 s) and extension at 72°C (60 s), with a 7 min extension at 72°C in the last cycles. The amplified products were subjected to rapid electrophoresis in 3% agarose gel and visualized under ultraviolet illumination after ethidium bromide staining. A Hae Ш restriction endonuclease test was done to verify the specificity of the PCR amplification, and the expected DNA fragments were produced. The specificity test demonstrated that this method was positive for bovine, buffalo and yak meat DNA, but negative for equine, sheep, goat, camel, swine, deer and mouse meat DNA, etc. At least 33.6 fg of DNA from raw beef samples and 0.32 pg of DNA from cooked or autoclaved beef samples were detected, respectively, by PCR. We tested 103 beef samples by PCR and obtained 100% correct identification. The method needed only 6 h for detection of meat products of all kinds. The results showed that the PCR method was sensitive, specific, convenient and rapid, so it may be suitable for rapid identification of beef.     

Development and application of highly specific PCR for detection of chicken (Gallus gallus) meat adulteration

In order to prevent fraud in the sale and strengthen quality assurance, authentic identification of chicken meat is essential. In the present investigation, a chicken (Gallus gallus)-specific polymerase chain reaction (PCR) was developed for the unambiguous identification of chicken meat. The PCR assay employs pair of primers designed against chicken nuclear 5-aminolevulinate (ALA) synthase gene. Highly chicken-specific diagnostic amplicon of 288 bp was established upon PCR and was evident in all the nine breeds/strains of chicken species. Sensitivity of PCR in detecting chicken meat adulteration was established to be at 0.1 % in the foreign meat matrix, while limit of detection (LOD) of chicken DNA was 10 pg. Suitability of the developed chicken-specific PCR was validated and confirmed in raw, cooked/heat treated (60, 80, 100, and 121 °C), and micro-oven cooked meat samples. Possibility of cross-amplification of adulterating DNA was excluded by cross-checking the developed PCR assay with several animal and avian species. The PCR assay developed in this study is highly promising for applications involving circumstances that require authentic identification of chicken meat.     

Use of duplex polymerase chain reaction (duplex-PCR) technique to identify bovine and water buffalo milk used in making mozzarella cheese

Molecular biology techniques have been used for species identification in food of animal origin in relatively recent years. A polymerase chain reaction (PCR) based method, the multiplex PCR, was recently applied to species identification in meat and meat products. It allows co-amplification of separate regions of a single gene or specific fragments, each typical of a different animal species in a single PCR reaction, using different pairs of primers in the same reaction mix. In the present paper, the duplex-PCR technique is proposed to identify bovine and water buffalo DNA in a single PCR assay in milk and mozzarella cheese (a typical Italian cheese, originally made from pure water buffalo milk). Because of its lower cost, undeclared bovine milk is added to water buffalo milk for making different kinds of mozzarella cheese. The results of this experiment indicate the applicability of this method, which showed an absolute specificity for the two species and a high sensitivity even down to low DNA concentrations (1 pg). In bovine and water buffalo mixtures of both milk and mozzarella cheese, the minimum concentration tested was 1% of bovine in water buffalo milk and water buffalo in bovine milk. The importance of the somatic cell content in raw milk is also discussed with special reference to the evaluation of mixtures (milk or cheese) of the two species.     

肉制品中牛源性成分Taqman荧光定量PCR检测法的建立

本实验根据牛线粒体差异序列设计特异性PCR引物和Taqman探针,并基于16S rDNA内参基因设计通用引物及Taqman探针,绘制并通过标准曲线确定样品中总DNA浓度以及牛源性成分DNA浓度,采用相对定量法测定肉制品中牛源性成分的质量分数。并通过特异性、灵敏度实验,及混合肉样回收率及市售肉制品检测,对本方法进行验证。结果表明,方法特异性强,可对牛肉DNA进行特异性扩增。最低检出限为10 pg/μL,具有较高的灵敏度。并根据回收实验可知,平均回收率为98.62%。可以为肉制品中牛肉含量的测定提供技术参考。     

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.