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.     

Rapid detection of Campylobacter jejuni in chicken rinse water by melting-peak analysis of amplicons in real-time polymerase chain reaction

Five DNA extraction protocols for the detection of Campylobacter spp. by polymerase chain reaction (PCR) were compared. A method involving Triton X-100 produced template DNA of sufficient quality to allow the detection of Campylobacter jejuni at levels of 100 CFU/ml in pure culture. Primers were designed on the basis of the cadF gene sequence. With a SYBR Green I real-time PCR assay, these primers amplified only sequences present in C. jejuni to produce a product with a melting temperature of 81.5 degrees C. None of the strains of Campylobacter coli, Campylobacter lari, or Campylobacter fetus tested produced this product during the PCR assay. Other noncampylobacter species tested were shown not to possess the cadF sequence. The real-time PCR combined with a rapid, simple Triton X-100 DNA extraction protocol made it possible to detect < 10 CFU of C. jejuni per ml of chicken rinse within 14 h.     

Detection of chicken and turkey meat in meat mixtures by using real-time PCR assays

In this study, TaqMan-based real-time Polymerase Chain Reaction (PCR) techniques were developed for the detection of chicken and turkey meat in raw and heat-treated meat mixtures. Primers and TaqMan probe sets were designed to amplify 86 bp and 136 bp fragments for the chicken and turkey species, respectively, on the mitochondrial NADH dehydrogenase subunit 2 gene. In the results, it was possible to detect each species at the level of 0.1 pg template DNA with the TaqMan probe technique without any cross-reactivity with nontarget species (bovine, ovine, donkey, pork, and horse) while the detection level was 1 pg template DNA using conventional PCR. The TaqMan probe assays used in this study allowed the detection of as little as 0.001% level of both species in the experimental meat mixtures, prepared by mixing chicken and turkey meat with beef at different levels (0.001% to 10%). In conclusion, TaqMan probe assays developed in this research are promising tools in the specific identification and sensitive quantification of meat species even in the case of heat-treated meat products, and suitable for a rapid, automated, and routine analysis.     

Rapid detection of bovine milk in yak milk using a polymerase chain reaction technique

Yak milk contains a greater percentage of protein and has better quality than bovine milk. There has been an increasing focus on yak milk and milk products during the last few years. In the present study, a PCR-based assay was developed for the specific identification of bovine milk in yak milk by designing 3 primers targeting the mitochondrial ND1 gene. The use of 3 primers in a single PCR reaction set yielded 2 amplification fragments of 293 and 190 bp from bovine milk DNA, whereas only 1 amplification fragment of 293 bp was obtained in yak milk DNA. The technique was applied to raw and heat-treated binary mixtures of yak and bovine milks and enabled the specific detection of bovine milk with a detection limit of 0.1%. The assay developed is sensitive, fast, and straightforward, and it might be useful in the quality control of yak milk and milk products.     

速测盒法快速测定肉及肉制品中亚硝酸盐

目的 了解亚硝酸盐速测盒的可靠性,为现场监督执法及基层快速检测提供有力的技术支撑。方法采用速测盒方法检测亚硝酸盐标准溶液、肉及肉制品中亚硝酸盐添加情况,并与《GB 5009.33-2016食品中亚硝酸盐与硝酸盐的测定》第二法(盐酸萘乙二胺法)进行对比。结果速测盒对亚硝酸盐最低检出限可达到0.1 mg/L;检测样品时,速测盒与盐酸萘乙二胺法阴性符合率为97.8%,阳性符合率为100.0%。不同环境温度下,只需将反应时间控制在5 min以上,则不会对检测结果产生影响。样品经简单处理后,显色剂滴加到样品提取液中,混匀后反应3～5 min,即可观察结果。检测单个样品20 min内即可出结果。结论速测盒法具有快速、准确、方便、灵敏等特点,适用于肉及肉制品中亚硝酸盐现场定性分析。     

Immunohistochemical detection of brain tissue in heated meat products

Immunohistochemical methods were used to determine whether brain tissue could be detected in test batches of meat products prepared with known levels of this tissue (0, 1, 5, 10, or 20% bovine brain tissue or 5% porcine brain tissue). Four different, commercially-available antibodies were examined: anti-Neurofilament (anti-NF), anti-MyelinBasicProtein (anti-MBP), anti-NeuronSpecificEnolase (anti-NSE) and anti-GlialFibrillaryAcidicProtein (anti-GFAP). Results obtained with the four antibodies differed with the heat treatment applied to the products (pasteurisation or sterilisation). The amount of immunoreaction product in the raw meat product varied with the antibody, even when the sample contained the same amount of brain tissue. The staining pattern also varied with the antibody. Overall, the anti-MBP antibody proved to be most useful in detecting brain tissue in finely comminuted heated meat products.     

Real-time polymerase chain reaction detection of bovine DNA in meat and bone meal samples

We describe a real-time polymerase chain reaction (PCR) assay for the detection of bovine DNA extracted from meat and bone meal (MBM) samples. PCR primers were used to amplify a 271-bp region of the mitochondrial ATPase 8-ATPase 6 gene, and a fluorogenic probe (BOV1) labeled with a 5' FAM reporter and a 3' TAMRA quencher was designed to specifically detect bovine PCR product. The specificity of the BOV1 probe for the detection of the bovine PCR product was confirmed by Southern blot hybridization analysis of the probe with PCR products generated from ovine, porcine, and bovine genomic DNA extracted from blood and with PCR products generated from genomic DNA extracted from single-species laboratory scale rendered MBM samples. The specificity of the BOV1 probe was also evaluated in real-time PCR reactions including these genomic targets. Both methods demonstrated that the BOV1 probe was specific for the detection of bovine PCR product. The BOV1 probe had a detection limit of 0.0001% bovine material by Southern blot DNA probe hybridization analysis and a detection limit of 0.001% bovine material in the real-time PCR assay. Application of the real-time PCR assay to six industrial samples that had previously tested positive for the presence of bovine material with a conventional PCR assay yielded positive results with the real-time PCR assay for four samples.     

多重荧光定量聚合酶链反应法检测肉制品中的3种食源性致病菌

摘要：目的 建立多重荧光定量聚合酶链反应法（quantitative polymerase chain reaction,qPCR）快速检测肉制品中沙门氏菌、单增李斯特菌、大肠杆菌O157:H7 3种食源性致病菌的方法。方法 根据沙门氏菌的invA基因、单增李斯特菌的hemolysin基因、大肠杆菌O157:H7的rfbE基因序列分别设计特异性引物及探针,通过优化反应体系,测定其灵敏度、特异性和重复性,建立了可同时检测上述3种食源性致病菌多重qPCR方法。结果 该方法只扩增3种靶细菌,对其他供试菌不扩增;沙门氏菌、单增李斯特菌、大肠杆菌O157:H7的检出限分别为101、102、102拷贝数/μL,并且拥有良好的重复性和特异性。人工污染的猪肉样品经SLE（Salmonella、Listeria monocytogenes and Escherichia coli 3种菌株的共增菌培养基）培养基富集8 h后,分别可以检测到初始菌液浓度为1.56×102 CFU/25 g的沙门氏菌,2.13×102 CFU/25 g的单增李斯特菌,2.32×102 CFU/25 g的大肠杆菌O157:H7。结论 所建立的多重qPCR灵敏度高、特异性强、重复性好,可同时检测肉制品中沙门氏菌、单增李斯特菌和大肠杆菌O157:H7 3种食源性致病菌,在提高食品安全和保护人类健康方面有重要意义。     

Study of food products containing gluten by using the method of polymerase chain reaction

The results of studies on the detection of gluten in the 7 products, members of the group of gluten-free, used for patients with celiac disease. Used the method of polymerase chain reaction detection in real time (by method of polymerase chain reaction) using a test system SureFood Allergen Gluten. With this method, fully meets the requirements of the Codex Alimentarius Commission, confirmed the compliance of the investigated food gluten-free category, used for patients with celiac disease.     

同步增菌结合实时荧光聚合酶链式反应快速检测食品中5种致病菌

摘 要：目的 实现食品中5种致病菌的同步增菌,并结合实时荧光聚合酶链式反应（real-time polymerase chain reaction,RT-PCR）技术,建立快速检测的方法体系。方法 比较金黄色葡萄球菌、单核增生李斯特氏菌、大肠埃希氏菌O157:H7、副溶血性弧菌和鼠伤寒沙门氏菌5种目标菌分别在营养肉汤和缓冲蛋白胨水中的生长曲线;优化DNA模板提取的方法;采用实时荧光PCR技术及熔解曲线分析,建立5种致病菌的快速检测方法。结果 缓冲蛋白胨水可以作为5种目标菌的同步增菌液,增菌3 h后5种目标菌能被检出,最低检出的DNA浓度范围为10-3~10-4 ng/μL。经熔解曲线分析,5种目标菌的特征熔解Tm值范围为79.51~87.39℃。结论 本研究建立的5种致病菌快速检测方法,检测总时间大约为6 h,且方法灵敏度和特异性均能满足检测要求。     

基于实时荧光聚合酶链式反应快速检测包装饮用水中铜绿假单胞菌

目的 建立一种基于实时荧光聚合酶链式反应快速检测包装饮用水中铜绿假单胞菌的检测方法。方法 针对选择铜绿假单胞菌gyrB基因设计特异性引物和探针。250mL水样经膜过滤后经过短时培养(36℃、4 h),优化提取菌体DNA流程,预冻干聚合酶链式反应预混液,采用实时荧光聚合酶链式反应进行检测。结果 样品中铜绿假单胞菌量为≥1 CFU/250 mL时,检测结果均呈阳性,整个检测用时6 h左右。该方法检测结果与国家标准方法(GB8538—2022)检测结果一致。结论 该方法特异性强、灵敏度高、用时短,可为预包装饮用水中铜绿假单胞菌监管和生产企业质控提供更为快速、精准的技术手段。     

Immunological detection of keratan sulfate in meat products with and without mechanically separated chicken meat

Keratan sulfate is a glycosaminoglycan found in the structure of cartilage proteoglycans, aggrecan and fibromodulin. This study was undertaken to detect this glycosaminoglycan in meat products containing mechanically separated chicken meat (MSCM) having cartilage particles. Dry-defatted samples of MSCM and meat products with or without MSCM were digested with papain, and a non-dialyzable fraction from each papain digest was examined by immunodiffusion analysis using anti-keratan sulfate monoclonal antibody (IgM). No precipitine line was formed with the antibody for all samples of meat products without MSCM, while a sample of MSCM and all samples of meat products with MSCM gave clear precipitine lines with the antibody. The immunodiffusion test described here appears to be a simple sensitive specific method for qualitative analysis of keratan sulfate, which in combination with other methods may be useful for detection of MSCM in meat products.     

Immunological detection of keratan sulfate in meat products with and without mechanically separated chicken meat

Keratan sulfate is a glycosaminoglycan found in the structure of cartilage proteoglycans, aggrecan and fibromodulin. This study was undertaken to detect this glycosaminoglycan in meat products containing mechanically separated chicken meat (MSCM) having cartilage particles. Dry-defatted samples of MSCM and meat products with or without MSCM were digested with papain, and a non-dialyzable fraction from each papain digest was examined by immunodiffusion analysis using anti-keratan sulfate monoclonal antibody (IgM). No precipitine line was formed with the antibody for all samples of meat products without MSCM, while a sample of MSCM and all samples of meat products with MSCM gave clear precipitine lines with the antibody. The immunodiffusion test described here appears to be a simple sensitive specific method for qualitative analysis of keratan sulfate, which in combination with other methods may be useful for detection of MSCM in meat products.     

The detection of chicken meat in meat products by means of the anserine/carnosine ratio

Summary A distinctive difference was found between the ratio of the anserine and carnosine contents (a/c ratio) in beef or pork and of that in chicken/meat. The a/c ratio for beef varies between 0.06–0.2 and for pork between 0.02–0.1 but for chicken meat can reach values as high as 2.2–5.5.The high a/c ratio for chicken meat proved to be sufficient to detect this ingredient at a 5% level in both cooked pork products and 1:1 beef-pork mixtures, this being independent of the heat treatment.The a/c ratio should be considered to be a suitable parameter for the presence of chicken meat in meat products.

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Nachweis von Hühnerfleisch in Fleischerzeugnissen mit Hilfe des Verhältnisses der Anserin- und Carnosingehalte

Zusammenfassung Es wurde ein wesentlicher Unterschied zwischen dem Verhältnis der Anserin- und Carnosin-Gehalte (a/c-Wert) in Rind- und Schweinefleisch einerseits und Hühnerfleisch andererseits gefunden. Der a/c-Wert schwankt für Rindfleisch innerhalb der Bereich von 0,06 bis 0,2, und für Schweinefleisch von 0,02 bis 0,1. Für Hühnerfleisch können hohe Werte erreicht werden, zwischen 2,2 und 5,5. Dieses Ergebnis ermöglicht den Nachweis von Hühnerfleisch von 5% in erhitzten Produkten aus Schweinefleisch oder Rind-/Schweinefleisch-Mischungen (1 + 1). Das Resultat ist nicht von einer Hitzebehandlung abhängig. Der a/c-Wert ist als Parameter für Hühnerfleischzusätze in Fleischerzeugnissen geeignet.

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The investigation was made possible by financial support received from the Commodity Board for Livestock and Meat, Rijswijk, The Netherlands.     

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.     

The detection of chicken meat in meat products by means of the anserine/carnosine ratio

A distinctive difference was found between the ratio of the anserine and carnosine contents (a/c ratio) in beef or pork and of that in chicken/meat. The a/c ratio for beef varies between 0.06–0.2 and for pork between 0.02–0.1 but for chicken meat can reach values as high as 2.2–5.5.The high a/c ratio for chicken meat proved to be sufficient to detect this ingredient at a 5% level in both cooked pork products and 1:1 beef-pork mixtures, this being independent of the heat treatment.     

Rapid detection of cows' milk in sheeps' and goats' milk by a species-specific polymerase chain reaction technique

A polymerase chain reaction (PCR) assay was developed for the specific identification of cows' milk in sheep's and goats' milk by using primers targeting the mitochondrial 12S rRNA gene. The use of a forward primer complementary to a conserved DNA sequence, along with a reverse primer specific for cow, yielded a 223-bp fragment from cows' milk DNA, whereas no amplification signal was obtained in sheep's and goats' milk DNA. The technique was applied to raw, pasteurized, and sterilized milk binary mixtures of cow-sheep and cow-goat, enabling the specific detection of cows' milk with a good sensitivity threshold (0.1%). The proposed PCR assay represents a rapid and straightforward method applicable to the authentication of milk and other dairy products in routine analysis.     

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 Leuconostoc strains at a meat processing plant using polymerase chain reaction

To simplify the labor-intensive conventional routine testing of samples to detect Leuconostoc at a meat processing plant, we developed polymerase chain reaction (PCR) primers specific for Leuconostoc from 16S rRNA gene sequences. These primers did not detect other common lactic acid bacteria such as Lactobacillus plantarum, Lact. sake, Lact. fermentum, Lact. acidophilus and Weissella viridescens. PCR with this primer detected all Leuconostoc species tested (Leu. mesenteroides subsp. mesenteroides, Leu. pseudomesenteroides, Leu. carnosum, Leu. lactic, Leu. citreum, Leu. amelibiosum, Leu. gelidum), except for Leu. fallax, and no other lactic acid bacteria on agarose gel electrophoresis. The method could identify areas contaminated with Leuconostoc in a large-scale industrial meat processing plant. Of 69 samples analyzed, 34 were positive for Leuconostoc according to the conventional culture method (isolation of LAB producing dextran) and PCR, whereas 29 were negative according to both. Six samples were culture-negative but positive by PCR. No false negative results were generated by PCR. The method is rapid and simple, is useful for routinely monitoring areas contaminated with Leuconostoc in meat processing plants, and could help to prevent the spoilage of meat products.