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.     

Quantitative detection of species-specific DNA in feedstuffs and fish meals

A sensitive and rapid method for the quantitative detection of bovine-, ovine-, swine-, and chicken-specific mitochondrial DNA sequences based on real-time PCR has been developed. The specificity of the primers and probes for real-time PCR has been tested using DNA samples of other vertebrate species that may also be present in rendered products. The quantitative detection was performed with dual-labeled probes (TaqMan) using absolute quantification with external standards of single species meat-and-bone meals. This method facilitates the detection of 0.01% of the target species-derived material in concentrate feed mixtures and fish meals.     

Detection of allergenic parvalbumin of Atlantic and Pacific herrings in fish products by PCR

The conventional polymerase chain reaction (PCR) method to detect the major allergenic protein parvalbumin beta 2 of Atlantic herring (Clupea harengus) and Pacific herring (Clupea pallasii) was developed. The specific set of primers for the amplification of the partial genomic sequence of the pvalb 2 gene encoding the main fish allergen of both herrings was designed and applied to the investigation of 24 commercial fish products. The targeted amplicon size was 189 bp of pvalb 2 gene of Atlantic herring and Pacific herring. As the internal amplification control, the DNA of 18S rRNA gene for eukaryotes (141 bp) was successfully used. The specificity of designed primer pair using 26 various fish species was assessed. The intrinsic detection limit was 10 pg µl^?1 of the present specific DNA. Atlantic herring or Pacific herring allergenic parvalbumins were detected in 22 investigated fish products in conformity with the package declaration. Two fish products were negative in spite of the declaration. The proposed PCR method is specific enough and can be used for the detection of Atlantic and Pacific herrings’ major allergen parvalbumin beta 2 in fish food products.     

Novel multiplex PCR-RFLP assay discriminates bovine,porcine and fish gelatin substitution in Asian pharmaceuticals capsule shells

Gelatin is widely used in pharmaceuticals as a protective coating, such as soft and hard capsule shells. However, the animal source of gelatin is a sensitive issue because certain gelatins such as porcine and bovine gelatins are not welcome in Halal, Kosher and Hindus’ consumer goods. Recently, we have documented DNA barcoding and multiplex PCR platforms for discriminating porcine, bovine and fish gelatins in various fish and confectionary products; but those assays were not self-authenticating and also not tested in highly refined pharmaceutical products. To address this knowledge gap, here we report a self-authenticating multiplex PCR-restriction fragment length polymorphism (RFLP) assay to identify animal sources of various gelatin in pharmaceutical capsules. Three different restriction enzymes, BsaAI, Hpy188I and BcoDI were used to yield distinctive RFLP patterns for gelatin-based bovine (26, 94 bp), fish (97, 198 bp) and porcine (17, 70 bp) DNA in control experiments. The specificity was cross-tested against 16 non-target species and the optimised assay was used to screen gelatin sources in 30 halal-branded pharmaceuticals capsule shells. Bovine and porcine DNA was found in 27 and 3 of the 30 different capsules products. The assay was suitable for detecting 0.1 to 0.01 ng total DNA extracted from pure and mixed gelatins. The study might be useful to authenticate and monitor halal, kosher, vegetarian and Hindu compliant pharmaceuticals, foods and cosmetics.     

Real-time PCR for quantitative detection of bovine tissues in food and feed

A real-time PCR approach with the SYBR Green detection system has been developed for the quantitative detection of bovine tissues in food and feedstuffs. The method combines the use of bovine-specific primers, which amplify an 84-bp fragment of the mitochondrial 12S rRNA gene, and universal primers, which amplify a 140-bp fragment of the nuclear 18S rRNA gene from eukaryotic DNA. The 18S rRNA primers are used as endogenous controls for the total content of PCR-amplifiable DNA in the sample. The specificity of the primers was tested against 18 animal species, including mammals, birds, and fish, as well as 6 plant species. Analysis of experimental bovine tissues-oats mixtures demonstrated the suitability of the assay for the detection of bovine DNA in mixtures containing as low as 0.1% of bovine tissues. The performance of the method is not affected by severe heat treatment (up to 133 degrees C for 20 min at 300 kPa). The reported PCR assay could be very useful for detecting bovine-derived ingredients in raw and heat-treated food and feedstuffs.     

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。     

A multiplex PCR method for the identification of commercially important salmon and trout species (Oncorhynchus and Salmo) in North America

Abstract: The purpose of this study was to develop a species-specific multiplex polymerase chain reaction (PCR) method that allows for the detection of salmon species substitution on the commercial market. Species-specific primers and TaqMan® probes were developed based on a comprehensive collection of mitochondrial 5′ cytochrome c oxidase subunit I (COI) deoxyribonucleic acid (DNA) “barcode” sequences. Primers and probes were combined into multiplex assays and tested for specificity against 112 reference samples representing 25 species. Sensitivity and linearity tests were conducted using 10-fold serial dilutions of target DNA (single-species samples) and DNA admixtures containing the target species at levels of 10%, 1.0%, and 0.1% mixed with a secondary species. The specificity tests showed positive signals for the target DNA in both real-time and conventional PCR systems. Nonspecific amplification in both systems was minimal; however, false positives were detected at low levels (1.2% to 8.3%) in conventional PCR. Detection levels were similar for admixtures and single-species samples based on a 30 PCR cycle cut-off, with limits of 0.25 to 2.5 ng (1% to 10%) in conventional PCR and 0.05 to 5.0 ng (0.1% to 10%) in real-time PCR. A small-scale test with food samples showed promising results, with species identification possible even in heavily processed food items. Overall, this study presents a rapid, specific, and sensitive method for salmon species identification that can be applied to mixed-species and heavily processed samples in either conventional or real-time PCR formats. Practical Application: This study provides a newly developed method for salmon and trout species identification that will assist both industry and regulatory agencies in the detection and prevention of species substitution. This multiplex PCR method allows for rapid, high-throughput species identification even in heavily processed and mixed-species samples. An inter-laboratory study is currently being carried out to assess the ability of this method to identify species in a variety of commercial salmon and trout products.     

Polymerase chain reaction (PCR) identification of Aspergillus niger and Aspergillus tubingensis based on the calmodulin gene.

Aspergillus niger and A. tubingensis, species belonging to section Nigri, are commonly found in plant products and processed food, such as grapes, cereals, coffee, and derived products. These two species are very difficult to differentiate by classical morphological criteria and some isolates are known to produce ochratoxin A. The exact identification of these two species is very important to avoid the overestimation of toxicological contamination and related risks. A polymerase chain reaction (PCR)-based identification and detection assay was developed as a tool to identify A. niger and A. tubingensis, using molecular differences obtained by sequencing the calmodulin gene. Two pairs of species-specific primers were designed and empirically evaluated for PCR identification of A. niger and A. tubingensis. Species-specific PCR products generated by each primer set were 505 bp (A. tubingensis) and 245 bp (A. niger) in length, which could be potentially useful for a multiplex PCR assay. The sensitivity of this assay was about 10 pg DNA in a 25-microl PCR reaction volume, using pure total DNA of the two species. The method described in this study represents a rapid and reliable procedure to assess the presence in food products of two ochratoxigenic species of section Nigri.     

Canine-Specific PCR Assay Targeting Cytochrome b Gene for the Detection of Dog Meat Adulteration in Commercial Frankfurters

This report described a cytochrome b (cytb)-based polymerase chain reaction (PCR) assay for the detection of canine tissues in commercial frankfurters. Discriminating detection of canine derivatives in processed food products has important application in halal authentication as well as in health, religions, and fare trades. The assay based on a pair of canine-specific primers that targeted a 100 bp region of canine mithochondrial-cytb gene which is present in multiple copies and highly conserved within the same species. The specificity of the assay was tested against dog and eight most common animal meat species as well as five plant species commonly found in frankfurter formulation. The stability and specificity of the assay were verified under different thermal processing conditions under pure and complex matrices. Three commercial brands of chicken and beef frankfurters were tested in triplicate, and specific PCR products were obtained only from deliberately contaminated formulations. The detection limit of the assay was 0.1 % (0.02 ng DNA) of canine meat spiked with other meats in a typical frankfurter formulation. Shorter amplicon length, superior stability, and higher sensitivity of the assay suggested its potential application in the screening of canine-origin biomaterials in processed food products.     

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。     

Identification of Ingredient in Mullet Roe Products by the Real-Time PCR Method

Mullet roe is a product of high economic value in a number of Asian countries, particularly Taiwan. However, actual mullet roe is commonly adulterated by the addition of other species, such as escolar and oilfish. The purpose of this study was to develop a method to detect the ingredient of mullet roe products. Based on the TaqMan real-time PCR assay, we designed the specific primers-probe set (mullet) that targets the mitochondrial 16S ribosomal RNA gene. Meanwhile, the positive amplification control is designed based on the eukaryotic 18S rRNA gene. The PCR amplicon used to identify fish species in processed roe products is smaller than 200 bp. Method specificity was evaluated by analyzing tissue samples of 29 food fish and 9 puffer fish species. No indications of cross-reactivity toward non-target species were observed. Sensitivity and linearity tests were conducted using five-fold serial dilutions of target DNA from processed mullet roe, and we determined that our proposed method has a sensitivity of 1.2 ng. Further tests on a random survey of commercial fish roe products demonstrated the efficacy of the technique in the detection of mullet DNA. The real-time PCR methods developed in this study could be used to verify the labeling of actual mullet roe products.     

Polymerase chain reaction-based analysis to detect terrestrial animal protein in fish meal

The recent European bovine spongiform encephalopathy crisis has focused attention on the importance of adopting stringent control measures to avoid the risk of the diffusion of mad cow disease through meat meal-based animal feedstuffs. Potential adulteration of such feedstuffs with bone particles from terrestrial animals is determined by microscopic examination by law before the release of these feedstuffs for free circulation in the European Community. This study describes a DNA monitoring method to examine fish meal for contamination with mammalian and poultry products. A polymerase chain reaction (PCR) method based on the nucleotide sequence variation in the 12S ribosomal RNA gene of mitochondrial DNA was developed and evaluated. Three species-specific primer pairs were designed for the identification of ruminant, pig, and poultry DNA. The specificity of the primers used in the PCR was tested by comparison with DNA samples for several vertebrate species and confirmed. The PCR specifically detected mammalian and poultry adulteration in fish meals containing 0.125% beef, 0.125% sheep, 0.125% pig, 0.125% chicken, and 0.5% goat. A multiplex PCR assay for ruminant and pig adulteration was optimized and had a detection limit of 0.25%.     

4组鱼类DNA条形码引物的筛选与优化

目的通过对DNA条形码氧化酶亚基I基因(cytochrome oxidase subunit I,COI)的4组常用引物进行比较筛选,选择适于舟山鱼类鉴定的最佳引物。方法以舟山主产大黄鱼、小黄鱼、带鱼和乌贼样品DNA为模板,对4组常用DNA条形码引物进行PCR体系优化。通过比较PCR产物量、特异性、灵敏度和测序成功率,综合分析筛选最佳引物组。结果 4组引物均能扩增大黄鱼、小黄鱼和带鱼的DNA,对于乌贼DNA的扩增具有明显差异。COI优化引物具有易于扩增、测序简单等优势,更适于作为海产品鉴定的DNA条形码引物。结论本研究为舟山海产品的市场监管和实验室大量样品检测提供了技术参考。     

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.     

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.     

Identification of tetrodotoxin and fish species in a dried dressed fish fillet implicated in food poisoning

There were five victims of neurotoxic food poisoning from a dried dressed fish fillet in Changhua County, Taiwan, in February 2000. The toxicity of the dried dressed fish fillets was 243 mouse units per g according to a tetrodotoxin bioassay. The partially purified toxin was identified as tetrodotoxin and anhydrotetrodotoxin. The sequence of the 376-nucleotide region in the cytochrome b gene of the mitochondrial DNA exhibited the same genotype as that of the toxic puffer fish Lagocephalus lunaris. The same single restriction site for Hinfl was found in the polymerase chain reaction (PCR) products from the dried dressed fish fillet and the muscle of L. lunaris, yielding two DNA fragments of 170 and 206 bp. However, no restriction site for Hinfl was found in the PCR products from other toxic puffer fishes, including Takifugu niphobles, Takifugu oblongus, and Takifugu rubripes. Therefore, the species of the dried dressed fish fillet was identified as L. lunaris and its causative agent was identified as tetrodotoxin.     

肉制品中羊源性成分的定性和定量检测

通过检测肉制品中DNA的来源进行动物源性检测是打击鲜肉及加工肉制品制假掺假的重要技术手段。依据GB/T 25165—2010《明胶中牛、羊、猪源性成分定性检测方法 实时荧光PCR法》合成用于TaqMan 实时荧光聚合酶链式反应的引物和探针,利用鲜肉及加工肉制品进行羊源性成分定性和定量检测方法研究。首先利用12 种不同动物鲜肉组织的DNA检测方法的特异性,然后以羊源DNA梯度稀释液为模板进行灵敏度实验,最后在加工肉制品中检测方法的适用性和定量检测能力。结果表明：此方法具有羊源性成分检测特异性,对其他动物来源的鲜肉DNA均无扩增信号,可以检出羊肉和猪肉混合样品中0.1%的羊肉;方法灵敏度高,可以检出10 pg的羊源DNA;方法适用性广,可以对加工肉制品（羊肉干）进行羊源性成分的定性和定量检测。     

一种基于实时荧光聚合酶链式反应的肉及肉制品中猪源性成分含量测定

为了建立一种基于实时荧光聚合酶链式反应的肉及肉制品猪源性成分含量测定方法,分别以猪线粒体DNA的NADH4基因和16S rRNA基因为靶位点设计猪特异性引物、探针和通用引物、探针,建立猪源性成分含量测定方法,通过特异性、灵敏性、线性、准确度及市售肉制品检测,对该方法体系进行检验和评价.结果表明:建立的猪源性成分含量实时荧光聚合酶链式反应测定方法体系具有良好的特异性及灵敏性,最低可检测到0.4pg/μL纯猪肉DNA;无论是猪特异性体系还是通用体系都呈现良好的线性关系(R2均达到0.999以上)和较宽的线性范围;通过含猪源性成分的模拟混合肉样检测,样品猪源性成分含量的回收率平均值为122.27％,说明该方法具有较高的准确度;通过市售肉制品检测表明该方法可以用来检测实际样品(包括生鲜样品和熟肉样品)中猪源性成分含量.     

鱼翅类食品中鲨鱼成分PCR鉴定方法研究

本文针对鱼翅中的鲨鱼成分进行检测鉴定开发了一种快速灵敏的PCR检测方法,可检测鱼翅类食品中是否存在鲨鱼成分。根据鲨鱼线粒体的细胞色素亚基I基因序列设计了鲨鱼特异性引物,扩增长度为228 bp;为了评价方法的特异性,将设计的引物分别针对22份鱼翅样品DNA和37种其它种类DNA进行PCR检测,结果显示,只在鲨鱼鱼翅中能检测出特异的228 bp条带,其它37种物种中均无条带检出。为了评价方法的灵敏度,将鱼翅DNA中掺入了不同比例土豆DNA的样品采用本方法进行了PCR分析,显示方法可检测灵敏度为0.1%(m/m)。随机抽取45份不同类型的鱼翅样品,检测出22份鲨鱼翅均含鲨鱼成分,而21份仿鱼翅均不含鲨鱼成分而含有植物成分。该样品前处理方法、DNA提取方法以及PCR检测方法可广泛应用于食品中鲨鱼成分检测鉴定。     

利用简并引物检测金黄色葡萄球菌肠毒素A、B基因

通过设计简并引物建立一种PCR技术同步快速检测金黄色葡萄球菌肠毒素A和B基因的方法。根据金黄色葡萄球菌肠毒素A、B基因编码序列,设计一对特异性简并引物SEAB来扩增靶基因片段,长度分别为105bp和135bp,通过对金黄色葡萄球菌肠毒素A、B菌株和4株对照菌株进行PCR检测,评价该引物的特异性;对金黄色葡萄球菌肠毒素B的DNA系列10倍稀释,对其灵敏性进行PCR检测。结果显示,金黄色葡萄球菌肠毒素A和B菌株的DNA检测结果呈阳性,4株对照菌株的检测结果呈阴性,通过基因测序证实了PCR产物的特异性,SEB的DNA最低检测浓度为3.58ng,整个检测过程不超过20h。建立了一个特异、快速灵敏的在同一条件下,金黄色葡萄球菌肠毒素A、B基因的PCR检测方法。