Qualitative PCR for the detection of chicken and pork adulteration in goose and mule duck foie gras

Polymerase chain reaction amplification of a conserved region of the α‐actin gene has been used for specific identification of chicken (Gallus gallus) and pork (Sus scrofa domesticus) adulteration in goose (Anser anser) and mule duck (Anasplatyrhynchos × Cairinamoschata) foie gras. The design of species‐specific forward primers, together with a reverse universal primer, allowed the generation of amplicons of different lengths in each species. The different sizes of the species‐specific amplicons, separated by agarose gel electrophoresis, allowed clear identification of the presence chicken and pork in goose and mule duck foie gras with a detection limit of 0.1% (w/w). The technique could be used in inspection programmes to enforce labelling regulation of foie gras and other meat products. Copyright © 2003 Society of Chemical Industry     

Identification of chicken,duck, pigeon and pig meat by species-specific markers of mitochondrial origin

In the present study, PCR based method for meat species identification of chicken, duck, pigeon and pig was achieved by developing species-specific markers. Using mitochondrial sequences species-specific primers were designed and the sizes of them were 256 bp, 292 bp, 401 bp and 835 bp for chicken, duck, pigeon and pig, respectively. The species-specific PCR products were sequenced to confirm the specificity of the product amplified. These markers were subsequently tested for cross amplification by checking them with beef, mutton, chevon, pork, rabbit, chicken, duck, turkey and pigeon meat. DNA markers developed in this study can help identify the species of fresh, cooked and autoclaved meat of chicken, duck and pigeon and fresh and cooked meat of pig. The process of identification is simple, economical and quick as compared to other methods such as RAPD, PCR-RFLP and sequencing method of species identification.     

液滴数字PCR定量检测鸭血制品中掺假成分

鸭血制品作为市场常见商品,掺假现象十分严重。选取鸡、鸭、鹅基因组中的特异性单拷贝基因为靶基因,设计引物和探针,建立鸡、鸭、鹅血微滴数字PCR定量检测方法。使用人工混合样品对该方法的准确性进行验证,同时通过检测市售样品来验证方法的适用性。实验结果表明,样品含量在1~1000 μg/mg时,靶基因拷贝数与样品质量之间具有良好的线性关系,检测限和定量限分别为5 μg/mg和1 μg/mg。通过人工混合样品和市售样品检测证明,该方法具有良好的准确性和适用性,可用于对鸡、鸭、鹅血制品的定量检测。     

基于单拷贝核基因的数字PCR定量检测肉制品中羊源性成分

GeneBank搜索牛、绵羊、山羊、猪、马、驴、鸡、鸭、鹅、火鸡、狗、猫、鼠、兔、貂等15种动物的单拷贝核基因组序列信息,应用生物信息学分析筛选15种动物共有的种间保守区域,设计一对可同时扩增15种动物源性DNA的内参基因引物和探针;同时分析筛选绵羊和山羊共有且和其余动物种内特异性区域,设计一对只能扩增羊源性DNA的特异性基因引物和探针。基于微滴数字PCR技术,引入内参基因校正羊种属特异性基因测定方法,建立科学准确的肉制品中羊源性成分量化判定方法。结果表明,所建立方法具有良好的特异性和通用性,内参基因和羊种属特异性基因的最低检出限分别为32和26 copies/μL,模拟添加样品的正确度偏差均值为8.66%,符合数字PCR方法制定指南要求不得大于25%,说明量化判定方法结果具有较高的准确性。     

DNA条形码COI序列在常见肉类鉴别中的应用研究

为了对常见的4种肉类及相关肉制品进行掺假鉴定,判别与产品标签是否相符,本研究以COI基因为靶基因,建立了4种动物源性食品DNA条形码鉴别技术。分别提取牛、羊、猪、鸭四大物种的基因组DNA为模板,以其COI基因的保守序列区设计6对通用引物,结合文献报道及数据库提供的7对通用引物进行PCR扩增,并将测序结果提交Gen Bank数据库Blast比对,评价不同DNA条形码的检测鉴别能力。筛选出COI-A为最优序列,在4个物种中扩增效率100%。对抽检的20个批次的肉加工品样品进行检测,鉴定结果约有90%的样品与产品标签标示的成分相符。其中1个批次的牛丸制品因肉类成分含量低未扩增成功,1个批次的牛丸制品检出鸭源成分,判定掺假。DNA条形码技术快速有效,本研究筛选的COI-A序列可直接用于牛、羊、猪、鸭及其肉制品的鉴定,并为其它常见动物源性食品的种类鉴定提供一定参考依据。     

A simple one-step PCR method for the identification between European and American razor clams species

A specific multiplex polymerase chain reaction (PCR) was applied to differentiate samples of razor clams Ensisarcuatus, Ensissiliqua, Ensisdirectus, and Ensismacha. Universal primers were used for the amplification of internal transcribed spacer 1 (ITS-1) in each species. The alignment of the obtained sequences was the basis for the specific design of species-specific reverse primers (ITSArSil-R, ITSDir-R, and ITSMa-R) located in the ITS-1 region. A multiplex PCR using each specific primer together with a common forward primer allowed identification of razor clam species by means of the different sizes of the species-specific amplicons separated in an agarose gel electrophoresis. This work provides a simple, reliable and rapid protocol for the accurate identification of Ensis species. The present methodology can be very useful for traceability of the species and to reinforce labelling regulations.     

An actin gene-related polymerase chain reaction (PCR) test for identification of chicken in meat mixtures

Using the polymerase chain reaction (PCR) and DNA extracted from muscle, a single pair of oligonucleotide primers can yield amplification products from several members of the actin multigene family simultaneously. These multiple PCR products form species-specific “fingerprints” on gel electrophoresis which may be useful for meat authentication. However, for analysis of meat mixtures, the presence of a single band unique to a species would have many advantages over a multi-component fingerprint. A procedure is described in which primers amplify at a single actin gene locus, giving a positive band with DNA extracted from chicken and turkey, but no reaction with duck, pheasant, porcine, bovine, ovine or equine DNA. The chicken signal was clearly detectable with DNA from meat admixtures containing 1% chicken/99% lamb and from meat heat-treated at 120°C. For further discrimination, the chicken PCR product could be differentiated from turkey by restriction enzyme digestion.     

Detection of Turkey, Duck, and Guinea Fowl Egg in Hen Egg Products by Species-Specific PCR

A simplex polymerase chain reaction (PCR) has been applied for the specific detection of hen, duck, turkey, and guinea fowl in egg products using species-specific primers targeting the mitochondrial cytochrome b genes. The species-specific PCR yielded excellent results for identification of duck, turkey, and guinea fowl eggs in hen egg products, since the detection of 0.1% of each species was achieved, in liquid egg products as well as in powders. The proposed method is then a potentially reliable and suitable technique in routine food analysis for the research of fraudulent species mixture practices.     

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 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.     

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。     

四重荧光定量PCR法鉴定肠炎、鼠伤寒以及伤寒沙门氏菌

目的建立四重荧光定量PCR体系鉴定肠炎沙门氏菌、鼠伤寒沙门氏菌以及伤寒沙门氏菌。方法针对沙门氏菌属特异性ompC基因、肠炎沙门氏菌sdf基因、鼠伤寒沙门氏菌STM4495和伤寒沙门氏菌STY2021序列设计引物和TaqMan探针,建立多重荧光定量PCR体系,进行特异性与敏感性研究。结果 28株不同血清型的沙门氏菌均扩增出ompC基因,其他13株非沙门氏菌均未出现ompC的非特异性扩增。sdf、STM4495、STY2021的探针和引物分别特异性扩增出肠炎沙门氏菌、鼠伤寒沙门氏菌以及伤寒沙门氏菌,而25株其他血清型沙门氏菌以及13株非沙门氏菌均未见扩增曲线。敏感性试验显示,该体系的最低检测限分别为48 pg/mL(ompC)、560 pg/mL(sdf)、530 pg/mL(STM4495)、35 pg/mL(STY2021)。结论该方法特异好、灵敏高、能够快速检测沙门氏菌并鉴定肠炎沙门氏菌、鼠伤寒沙门氏菌以及伤寒沙门氏菌。     

Identification of hare meat by a species-specific marker of mitochondrial origin

Meat species identification in food has gained increasing interest in recent years due to public health, economic and legal concerns. Following the consumer trend towards high quality products, game meat has earned much attention. The aim of the present work was to develop a DNA-based technique able to identify hare meat. Mitochondrial cytochrome b gene was used to design species-specific primers for hare detection. The new primers proved to be highly specific to Lepus species, allowing the detection of 0.01% of hare meat in pork meat by polymerase chain reaction (PCR). A real-time PCR assay with the new intercalating EvaGreen dye was further proposed as a specific and fast tool for hare identification with increased sensitivity (1 pg) compared to end-point PCR (10 pg). It can be concluded that the proposed new primers can be used by both species-specific end-point PCR or real-time PCR to accurately authenticate hare meat.     

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...     

Differentiation of sturgeon species by PCR-RFLP

A method for identification of sturgeon species in caviar has been developed based on the amplification of a region of the mitochondrial genome (tRNA^Glu/cytochrome b) using the polymerase chain reaction (PCR). To distinguish between several types of sturgeon caviar the obtained 462bp long PCR-products were cut with different restriction endonucleases (RE) resulting in species-specific restriction fragment length polymorphisms (RFLP). The method is suitable to differentiate between 10 species of Acipenser and Huso originating from Europe and Asia.     

常见肉类中鸭源性成分荧光PCR检测方法的建立

目的通过筛选引物、探针体系,优化PCR反应条件,建立常见肉品中鸭源性DNA的荧光PCR检测方法。方法针对线粒体基因设计50对引物探针,以鸭DNA为模板,利用PCR和荧光PCR方法筛选引物探针并确定反应条件,进行灵敏度、特异性和稳定性试验,最终确定荧光定量PCR方法的引物、探针及反应条件。结果当样本含量为0.1%时,本方法仍可特异、灵敏地检出鸭肉成分,检测Ct值可稳定在20以下。结论建立了常见肉品中鸭源性DNA的荧光PCR检测方法,可用于肉类制品中鸭源性成分的鉴定。     

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.     

Detection and Identification of S. Carnosus in Starter Cultures Using Real Time PCR and Subsequent HRM Analysis of Amplification Products

Nowadays, the necessity to control species composition of commercial starter cultures and their growth during the ripening stage of fermented sausage in order to monitor the technological processes has became topical in Russia. However, there can be some difficulties in identification of an isolated culture when using biochemical methods. Using primers for amplification of bacterial species-specific regions makes it possible to identify starter cultures with a high degree of probability even without using TaqMan probes. Furthermore, using intraspecies polymorphism of the gyrase B subunit gene, we managed to subtype two S. carnosus isolates with the use of HRM analysis.     

PCR法检测肉制品中鹅源性成分

目的 建立一种PCR法检测肉制品中鹅源性成分的方法。方法 根据鹅的线粒体DNA(mtDNA)序列设计引物, 以9种动物的DNA为模板, 利用新设计的鹅的特异性引物进行PCR反应, 并用琼脂糖凝胶电泳检测引物的特异性和敏感性。结果 通过PCR反应, 仅鹅的DNA扩增得到了194 bp的目的片段, 其余物种DNA和空白对照均无目的片段。扩增产物的核苷酸序列与GENBANK中检索到的相应序列基本相符合。 结论 该方法特异性强、灵敏度高, 适合检测肉制品中的鹅源性成分。     

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。