三引物PCR检测牛羊猪源性成分的方法

建立了三引物PCR鉴定牛羊猪源性成分的方法,为肉类及产制品掺假鉴定或肉品溯源提供了简单易行的方法。通过对比牛羊猪的线粒体DNA(mt DNA)全序列,在保守区设计通用引物,在特异性区域设计特异引物并结合三引物PCR技术,扩增3个物种的特异性片段,羊、牛和猪产生片段大小分别为570、549、420 bp,依据片段大小可以在一次PCR中鉴定3个物种。建立的方法简单易行,特异性和灵敏度好,可以在肉类及产制品鉴定和溯源中应用,提高了检测效率。     

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。     

大西洋三文鱼和虹鳟鱼双重PCR检测方法的建立及应用

为了实现大西洋三文鱼和虹鳟鱼的物种掺假快速检测,本研究建立了一种基于双重PCR（普通和荧光）的物种鉴定方法。通过序列分析,分别基于线粒体COⅠ和Cyt b基因设计大西洋三文鱼和虹鳟鱼特异性引物,验证引物的特异性并对双重PCR反应体系进行优化,建立大西洋三文鱼和虹鳟鱼的双重PCR快速鉴定方法。利用本研究设计的引物,仅大西洋三文鱼和虹鳟鱼可以分别扩增出108 bp和207 bp的特异性条带,而其他23种非目标物种均未有扩增条带。经验证,在双重PCR反应体系中,大西洋三文鱼和虹鳟鱼引物的最佳添加量分别为0.1和0.4 μmol/L,熔解温度分别约为81.5 ℃和85 ℃。利用该方法从29份市售“三文鱼”样品中检测到6份大西洋三文鱼和3份虹鳟鱼,且与DNA条形码比对结果一致。本研究建立的大西洋三文鱼和虹鳟鱼双重PCR（普通和荧光）鉴定方法具有良好的特异性和适用性,为大西洋三文鱼和虹鳟鱼物种鉴定提供了可靠的技术手段。     

阿胶的半巢式-多重PCR鉴别方法研究

采用半巢式-多重PCR(聚合酶链式反应)法鉴别阿胶中是否掺杂有马、猪和牛源性成分。首先采用液氮研磨法从阿胶中提取高质量的DNA(脱氧核糖核酸),以通用引物P1和P2进行第一轮扩增,并将扩增产物作为第二轮PCR的模板;再以包含通用引物P1和4种物种(驴、马、猪和牛)特异性引物A、B、C和D的混合引物进行第二轮多重PCR扩增;最后电泳检测第二轮扩增产物,根据电泳条带的分子量大小,判断阿胶产品的动物原材料是否掺假。结果表明,采用本方法可观察到不同动物DNA分子量之间的明显差异,通过DNA电泳结果直接判断阿胶产品中是否掺假。     

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。     

加工食品中动物源DNA的提取和多重PCR检测方法的建立

为了从深加工食品中提取高质量和数量的动物源性DNA片段,以优化后CTAB方法提取,用于PCR检测。根据鸭线粒体基因序列,设计合成检测鸭源性成分引物,进行PCR体系和反应条件的优化筛选,建立三重PCR方法;同时扩增出牛源性116bp、猪源性212bp和鸭源性322bp目的基因片段。建立的三重PCR方法特异地鉴定牛肉及其制品中掺假猪和鸭源性成分,对打击食品掺假伪造、审核食品标签、保障消费者知情权、维护消费者健康等方面有重要作用。     

运用PCR技术进行5种液态奶的鉴别

采用聚合酶链式反应(PCR)对牛奶、羊奶、水牛奶、骆驼奶、黄豆奶这五种液态奶进行鉴别。其中,羊奶以ATP6为目的基因设计其特异性引物,能扩增出大小为292 bp的DNA片段,水牛奶以12SrRNA为目的基因设计特异性引物对,扩增出大小为328 bp的DNA片段,牛奶、骆驼奶以细胞色素b(cyt b)为目的基因设计引物,可以分别扩增出大小为725 bp和363 bp的DNA片段,黄豆浆以细胞色素f(cyt f)为目的基因,扩增出大小为510 bp的DNA片段,各引物对对于其它物种奶类无特异性扩增,并采用两种限制性内切酶验证了该扩增的特异性。通过不同物种的基因序列差距设计的引物,能同时鉴别出这几种液态奶,是一种可靠、简单、快捷的液态奶鉴别方法。     

基于PCR多物种鉴定技术及其在肉类 鉴定中的应用

国内外的肉类掺假由来已久,法律上监管部门对此采取了严格监管措施,技术上应用了感官、显微检测和免疫学等相应的检测技术。近几年来,PCR及其衍生技术的快速发展大大推动了肉类鉴定技术的快速发展,尤其在多物种鉴定技术领域建立了如多重PCR、通用单引物多重PCR、通用引物特异性PCR和PCR-RFLP等多项技术。这些技术的发展为肉类及其制品的检测提供了重要手段,代表了检测技术领域发展的方向,但其也各有其优缺点。本文介绍了上述4种多物种鉴定技术及其在肉类鉴定中的应用,并分析了其优缺点,旨在为物种鉴定方法选择及其研究提供参考。     

多基因DNA条形码鉴定6 个鳗鱼物种

建立6?种鳗鱼的物种多基因DNA条形码精准鉴定方法。以鳗鱼DNA为模板,采用3?对通用引物对6?种鳗鱼的3?个基因（16S rRNA、Cyt b、COⅠ）部分DNA片段进行聚合酶链式反应扩增、测序,结果6?种鳗鱼各获得3?条16S rDNA（638～643?bp）、Cyt?b（464～466?bp）、COⅠ（705～707?bp）基因部分DNA序列,从中选取各物种序列同源的片段设计3 对新引物对6 种鳗鱼的16S rRNA、Cyt b、COⅠ基因部分DNA片段进行PCR扩增,其产物大小分别为504～507、400、609?bp,再从各片段中筛选出具有6?种鳗鱼物种特异性强的、碱基数分别为262、280、300?bp的3?条DNA片段序列,作为6?种鳗鱼物种的3?个基因的标准DNA条形码,应用DNAMAN?V6软件进行同源性分析,并通过GenBank数据库的比对验证,制定了供检测实践用的同源率判别指标,建立鳗鱼物种的多基因条形码检测方法。应用该方法对30?个待检鳗鱼样品进行检测,结果表明,各样品基于3?个基因DNA条形码的比对,符合同源率指标,物种判别结果互相吻合,从而精准判别样品的所属物种。该方法稳定、精准、易于操作,可应用于6?种鳗鱼的物种鉴定,值得推广。     

羊肉与鸭肉的分子生物学鉴别技术

本研究建立羊肉与鸭肉的分子生物学鉴别方法,以羊肉和鸭肉线粒体细胞色素b基因(cytb)为特征靶基因,设计1条通用上游引物F和2条特异性下游引物:R_羊、R_鸭,并按照一定浓度比例组成混合引物,进行一次聚合酶链式反应(polymerase chain reaction,PCR)扩增即可获得目的DNA片段,根据琼脂糖凝胶电泳的DNA条带大小来判断羊肉中是否掺杂鸭肉。结果表明:当被检样本在367 bp和480 bp处分别出现特异性条带时,说明该样本的羊肉中掺杂了鸭肉;当被检样本在367 bp处出现特异性条带,而在480 bp处未出现特异性条带时,则说明该样本的羊肉中没有掺杂鸭肉。该方法准确、可靠、简单快捷,可用于食品中肉类种属的筛选和肉类掺假的检测和监管。     

膜芯片技术对牛、羊、牦牛、驴肉源食品的掺伪鉴别

采用膜芯片技术,通过对牛、羊、驴、牦牛、鸡、鸭、兔、貂、狐、鼠、猪11 种目标物种的检测,实现对牛、羊、驴、牦牛物种的掺伪鉴别。结果表明：该方法具有良好的特异性和适用性,检测灵敏度和掺伪灵敏度均可达到0.1%,能快速、准确地同时鉴别牛、羊、驴、牦牛、鸡、鸭、兔、貂、狐、鼠、猪11 种动物源性成分,可满足肉类食品样品中对牛、羊、牦牛、驴等掺伪鉴别的检验需求。     

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.     

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.     

Sequence analysis of mitochondrial 12S rRNA gene can identify meat species

In this study, sequence analysis of mitochondrial 12S rRNA has been applied for meat species identification. The procedure involves polymerase chain reaction (PCR) amplification of a fragment of mitochondrial (mt) 12S rRNA gene and sequencing of amplicons. Amplified product of mt 12S rRNA gene was 456 bp in size. Species sequenced include cattle (Bos indicus), buffalo (Bubalus bubalis), sheep (Ovis aries), goat (Capra hircus) and mithun (Bos frontalis). Sequences were compared with the reported sequences of low land anoa (Bubalus depressicornis), yak (Bos grunniens) and pig (Sus scrofa). There was no effect of routinely used additives or cooking temperature (72, 90, 120 and 180?°C) on the efficacy of PCR amplification. The closely related species like cattle and buffalo, sheep and goat could also be differentiated decisively by sequence analysis. Sequencing and analysis of mt 12S rRNA gene was, hence, found to be an ideal, authentic and unambiguous qualitative method for meat species identification.     

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).     

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.     

A quick and simple method for the identification of meat species and meat products by PCR assay

The polymerase chain reaction (PCR) was applied to identify six meats (cattle, pig, chicken, sheep, goat and horse) as raw materials for products. By mixing seven primers in appropriate ratios, species-specific DNA fragments could be identified by only one multiplex PCR. A forward primer was designed on a conserved DNA sequence in the mitochondrial cytochrome b gene, and reverse primers on species-specific DNA sequences for each species. PCR primers were designed to give different length fragments from the six meats. The products showed species-specific DNA fragments of 157, 227, 274, 331, 398 and 439 bp from goat, chicken, cattle, sheep, pig and horse meats, respectively. Identification is possible by electrophoresis of PCR products. Cattle, pig, chicken, sheep and goat fragments were amplified from cooked meat heated at 100 or 120°C for 30 min, but horse DNA fragments could not be detected from the 120°C sample. Detection limits of the DNA samples were 0.25 ng for all meats.     

基于TaqMan实时荧光PCR检测鲜肉及加工肉制品中的驴源性成分

根据出入境检验检疫行业标准SN/T 3730.4—2013《食品及饲料中常见畜类品种的鉴定方法 第4部分：驴 成分检测 实时荧光PCR法》合成引物和探针,利用TaqMan实时荧光聚合酶链式反应（polymerase chain reaction, PCR）技术检测鲜肉及加工肉制品中的驴源性成分。首先对13 种不同动物鲜肉组织的DNA进行驴源性成分特异 性检测,然后对驴源性DNA模板原液进行梯度稀释,检测方法灵敏度,最后在加工肉制品中检测方法的适用性。 结果表明：本研究建立的方法特异性强,除驴肉外,牛、羊、猪、马、骆驼、鹿、狗、兔、鸡、鸭、鸽子、鹌鹑 12 种动物鲜肉组织均无特异性扩增;方法的灵敏度较高,驴组分DNA的检出限可达100 fg/μL,灵敏度可达0.01%; 方法的适用性较广,可以用于加工肉制品中驴源性成分的检测。     

Development of an assay for rapid identification of meat from yak and cattle using polymerase chain reaction technique

Yak meat is of good quality with fine texture, high protein and low fat content, and rich in amino acids compared with that of cattle, and it lacks anabolic steroids or other drugs. In general terms, however, the meat yield of yak is relatively low compared with that of the cattle. In order to prevent possible adulteration of yak meat with cattle meat, based on the sequence of mitochondrial 12S rRNA gene, a multiplex PCR-based approach was proposed for rapid identification of the meat from yak and cattle using three primers designed in this work. Through the combinatorial usage of three primers with a single reaction set, two fragments of 290 and 159 bp were amplified from the cattle meat DNA, whereas only a fragment of 290 bp was obtained from the yak meat DNA. Using the assay described, satisfactory amplification was accomplished in the analysis of raw and heat-treated binary meat mixtures of yak/cattle with a detection limit of 0.1% for cattle meat. The technique is fast and straightforward. It might be a useful tool in the quality control of yak meat and meat products.