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 rapid polymerase chain reaction (PCR)-based assay for the identification of Listeria monocytogenes in food samples.

A rapid and simple assay has been developed which allows specific detection of Listeria monocytogenes within 3.5 h in cultures prepared from suspect food samples and propagated 48 h in selective medium. The assay is based on PCR technology, and uses a specific primer set derived from sequences located down-stream of the hlyA gene. The specificity of the primer set was confirmed by testing 115 L. monocytogenes, 14 L. innocua, 5 L. seeligeri and 4 L. ivanovii isolates. The assay was compared to standard microbiological tests and gave identical results for 83 food samples, including 32 positives. These field trials indicate that the assay developed provides an alternative detection system for L. monocytogenes in foods, which can be used by the food industry.     

Detection of pea in food by real-time polymerase chain reaction (PCR)

A qualitative 5′-nuclease real-time PCR-based method for the detection of pea (Pisum sativum) in food is described. The method consists of DNA isolation by chaotropic solid phase extraction and the subsequent PCR with pea-specific primers and a TaqMan fluorescent probe. The primers and the probe are oriented to the chloroplast DNA intron located between trnL and trnF exons encoding for tRNA. The analytical parameters of the method were inclusivity 100%, exclusivity 100% and the detection limit of 0.11±0.07 ng of pea DNA corresponding to 12±7 diploid pea genome copies. Using a set of model meat patés with defined pea contents, a matrix-related detection limit of 0.05% was determined and a linear calibration line was constructed. The presented analytical method was useful for qualitative detection or semiquantitative determination of pea in food products. The method was relatively fast because the analysis could be performed in one working day.     

A novel common single primer multiplex polymerase chain reaction (CSP‐M‐PCR) method for the identification of animal species in minced meat

BACKGROUND: Minced meat species adulteration is a severe problem. In this study a common single primer multiplex polymerase chain reaction (CSP‐M‐PCR) method was applied to the identification of five species (chicken, cattle, sheep, pig and horse) in minced meat products. CSP was designed as a common adapter at the 5′‐end of species‐specific reverse primers and employed in giving different length fragments from the respective meat species. RESULTS: Species‐specific DNA fragments could be identified in a single PCR reaction by mixing CSP, Chicken‐R, Cattle‐R, Sheep‐R, Pig‐R and Horse‐R primers in the ratio 1:0.01:0.01:0.01:0.01:0.10 (where ‘1’ represents 0.5 mmol L^?1). The specific DNA fragments could be efficiently amplified by CSP‐M‐PCR with > 5 µmol L^?1 species‐specific primers, except for 50 µmol L^?1 Horse‐R. A detection limit of 0.5 g kg^?1 was determined for both single species of minced meat and all species of five kinds of minced meat. CONCLUSION: The CSP‐M‐PCR method simplified the PCR reaction system and removed the inconsistent amplification efficiency from different primers. This highly sensitive, reproducible and rapid method could potentially be used as a screening or identifying assay to test for the presence of species or ingredients in minced meat and other meat products. Copyright © 2008 Society of Chemical Industry     

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.     

The detection of foodborne pathogens by the polymerase chain reaction (PCR)

Nucleic acid probes are being used increasingly in the food industry. These can be relatively insensitive but a new technique for amplification of a target sequence of nucleic acid has the potential of being rapid, sensitive and specific. The method, termed polymerase chain reaction or PCR, utilises a thermostable DNA polymerase from the bacterium Thermus aquaticus. Following thermal denaturation of double stranded DNA, small oligonucleotides, termed primers, are annealed to the single strands of DNA and these determine the starting point for replication of the DNA by the polymerase enzyme. Applications for food microbiology are being researched but problems have been encountered due to the complex nature of many foodstuffs.     

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.     

Effect of method of cooking on identification of heat processed beef using polymerase chain reaction (PCR) technique

Effects of various cooking methods including boiling, roasting, pressure cooking, and pan frying on species determination of beef by PCR was studied. The meat was cooked by boiling at 97.5°C for 140, 200 or 230min, by roasting at 200°C for 80, 120, or 150min or by autoclaving at 120°C for 30, 60, or 90min. The beef sample was pan fried until the meat was acceptable for sensory attributes (45min, meat temperature 115°C, fat temp 173°C) and further cooked until unacceptable. DNA was extracted from samples taken after cooking and a 271bp fragment of the mitochondrial DNA was amplified by PCR. The results indicated that with the exception of pan frying for 80min, beef was determined in all meat samples including the broth and sauce of the roasted meat.     

聚合酶链反应在融合子鉴定中的应用

以粟酒裂殖酵母和酿酒酵母为出发菌株,进行了原生质体融合试验,并对检出的融合子采用聚合酶链反应(PCR)扩增亲本的特定基因序列,以此达到鉴定融合子的目的.在检出的3株融合子中,均扩增出亲本特定基因,证明了该法在融合子鉴定中的可行性.     

A novel real-time polymerase chain reaction (PCR) method for the detection of hazelnuts in food

A real-time PCR (polymerase chain reaction)-based method for the detection of hazelnuts (nuts of Corylus avellana or C. maxima) in confectionery and bakery products is described. The method consists of DNA isolation by chaotropic solid phase extraction and the subsequent PCR with hazelnut-specific primers and a TaqMan fluorescent probe. The primers and the probe are targeted to the hsp1 gene encoding for a low molecular weight heat-shock protein. The method was positive for five hazelnut varieties approved in Slovakia and negative for all other tested plant materials used in food industry including peanuts, walnuts, almonds, pistachio nuts, cashews and chestnuts. The intrinsic detection limit of the method was 13 pg hazelnut DNA, which corresponds to approximately 27 genome equivalents (1C). Using a series of model pastry samples with defined hazelnut contents, a practical detection limit of 0.01% (w/w) hazelnut was determined. Practical applicability of the PCR method was tested by the analysis of 20 food samples (confectionery and bakery products) along with ELISA. For all of the food samples, identical results were obtained by both methods, which conformed to the labelling. The presented PCR method is useful for sensitive and selective detection of hazelnuts in food samples and can be performed in one working day.     

A novel real-time polymerase chain reaction (PCR) method for the detection of walnuts in food

A real-time polymerase chain reaction (PCR)-based method for the detection of the walnut (Juglans regia) component in food is described here. The method consists of DNA isolation by chaotropic solid phase extraction and the subsequent PCR with walnut-specific primers and a TaqMan fluorescent probe. The primers and the probe are targeted to the jug r2, a major allergen gene of walnut. The method was positive for 8 varieties of walnut and negative for all other tested plant materials used in food industry, including pecan nuts. The intrinsic detection limit of the method was 0.24 ng walnut DNA. Using a series of model pastry samples with defined walnut contents, a practical detection limit of 0.01% walnut content was estimated. Practical applicability of the PCR method was tested by the analysis of 13 food samples (bakery and confectionery products), out of which two cakes were found to contain walnuts although they were not adequately labelled. The presented PCR method is useful for sensitive and selective detection of walnuts in food samples and can be performed in one working day.     

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.     

Detection of wheat contamination in oats by polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA)

 It is well established that the consumption of wheat prolamins causes the characteristic symptoms of coeliac disease (CD) in subjects who are predisposed to it. There is currently much discussion about the role of oats in the pathogenesis of CD. Evidently, it is important that oats used for clinical studies are not contaminated with wheat. In this study, 38 oat samples were investigated by polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA): 30 samples consisted of flakes or grains and 8 probes were industrially processed oat diets. Wheat prolamin (gliadin) detection by ELISA showed that 16 of these samples contained less than the detection limit of 0.2 mg gliadin/100 g dry weight; 21 samples contained less than 2.8 mg gliadin/100 g dry weight and 1 sample reached 38 mg gliadin/100 g dry weight, clearly exceeding the allowed Swiss limit of 10 mg gliadin/100 g dry weight for gluten-free products. Spiking experiments showed that the wheat PCR system is about ten times more sensitive than the ELISA system, provided that the isolated DNA is fully amplifiable. Thus, wheat DNA could be detected by the wheat PCR system in ten samples with gliadin contents below the detection limit of the ELISA system used. Applying a eukaryote-specific 18S-PCR system the presence of amplifiable DNA was verified. Only two of eight samples of industrially processed oat products contained amplifiable DNA, the other six samples had no detectable DNA left. One sample was wheat-PCR positive. However, all eight samples contained detectable amounts of gliadin in the ELISA. Received: 7 November 1997     

Bias and artifacts in multitemplate polymerase chain reactions (PCR)

Polymerase chain reaction (PCR) is often used for the amplification of a mixture of homologous genes. PCR bias and artifact formation can occur in multitemplate PCR, and provide incorrect information on the abundance and diversity of genes. PCR bias and artifact formation occur at a higher rate during the last few cycles of the reaction, and therefore can be avoided by stopping the PCR earlier.     

Rapid polymerase chain reaction (PCR)-single-stranded conformational polymorphism (SSCP) screening method for the identification of Aspergillus section Nigri species by the detection of calmodulin nucleotide variations

Single-stranded conformational polymorphism (SSCP) analysis for genetic diversity studies has been widely applied to detect indirectly sequence differences up to a single base in amplified DNA fragments of the same length, representing an alternative to gene sequencing. In this study SSCP analysis was used to detect sequence variations contained in an about 180-bp region of the calmodulin gene in order to identify Aspergillus section Nigri species. The method described shows that fluorescence-based SSCP analysis by capillary electrophoresis is cheaper and faster than direct sequencing, and suitable for computer-assisted analyses allowing discrimination between the Aspergillus species belonging to the Nigri section: A. aculeatus, Aspergillus 'atypic uniseriate', A. brasiliensis, A. carbonarius, A. ellipticus, A. foetidus, A. heteromorphus, A. ibericus, A. japonicus, A. niger, and A. tubingensis.     

Rapid detection of chicken and turkey in heated meat products using the polymerase chain reaction followed by amplicon visualisation with vistra green

A rapid and highly specific assay suitable for the routine detection of turkey and chicken in processed meat products has been developed. Based on PCR amplification of species-specific amplicons with rapid visualisation using vistra green, the assay may be completed within 5 h of receipt of sample. DNA was isolated from meat samples by the use of Wizard DNA isolation technology and followed by DNA amplification in the polymerase chain reaction using species specific primers, chicken forward (CF), chicken reverse (CR), turkey forward (TF) and turkey reverse (TR): the production of an amplicon was detected after the end of the PCR in less than 5 min using vistra green and a fluorescence plate reader. The presence of fluorescence denoted the presence of the target species in the sample.     

食品中单核细胞增生性李斯特氏菌PCR快速检测方法

为建立食品中单核细胞增生性李斯特氏菌 (单增李斯特氏菌 )的快速、敏感、特异的PCR检测方法 ,选取hlyA基因作为靶序列设计一对引物 ,用该引物对 6 3株从国内食品中分离的李斯特氏菌 (进行传统方法验证 )和 2 0株非李斯特氏菌进行PCR扩增 ,并用此方法对人工污染食品进行检测 ,扩增片段表现出极好的单增李斯特氏菌种特异性 ,人工污染的生肉、冷冻虾仁、卷心菜的检出限为 39cfu g ,为食品中单增李斯特氏菌的快速、敏感并且特异的检测方法。     

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.     

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.     

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.