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.     

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.     

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.     

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     

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.     

Application of near-infrared microscopy (NIRM) for the detection of meat and bone meals in animal feeds: A tool for food and feed safety

This paper reports on the development and validation of a method for detecting meat and bone meal (MBM) in compound feeds by near-infrared reflectance microscopy (NIRM) as an alternative in food and feed safety. A FT-NIR (Fourier transformer-near-infrared reflectance) instrument attached to a microscope was used to build up a spectral library containing reference feed particles identified as plant or animal origin, from various sources. Spectra were collected directly from particles in the NIR spectrum region (1112–2500 nm). The spectral library sample set was used to develop various discriminant models to classify spectra as MBM or plant material. The best discriminant model was obtained using partial least squares (PLS) discriminant analysis and standard normal variate and detrending (SNVD) and first derivative for spectrum pretreatment; this model had a coefficient of determination of 0.95 and a standard error of cross-validation of 0.133. The model was externally validated. The results confirmed NIRM as a valuable technique for detection of banned MBM.     

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。     

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.     

Use of a polymerase chain reaction for specific detection of the malolactic fermentation bacterium Oenococcus oeni (formerly Leuconostoc oenos) in grape juice and wine samples

A PCR method has been developed that enables rapid and direct identification of the malolactic bacterium Oenococcus oeni from grape must or wine samples. Two primers, based on unique, highly conserved regions within the 16S rRNA gene of O. oeni , were used to amplify a 995 bp fragment which is specific for O. oeni . Other species of bacteria from Lactobacillus, Pediococcus and Acetobacter which may be found in grape must or wine were not detected using this technique. This diagnostic test is able to specifically detect in the order of 10³colony forming units per mL of O. oeni in a wine sample, and can be used for monitoring bacterial growth during malolactic fermentation.     

A cotton‐specific gene,stearoyl‐ACP desaturase,used as a reference for qualitative and real‐time quantitative polymerase chain reaction detection of genetically modified organisms

Biotechnology has permitted the modification of agricultural materials in a very precise way to improve productivity and yields. Polymerase chain reaction (PCR)‐based methods have been the first choice of most analytical laboratories for routine use in the detection of genetically modified organisms (GMO) and their derived products. These methods rely on the amplification of transgenic sequences and quantification of the transgenic DNA by comparison with an amplified reference gene. This paper describes the specific primers and probe for the cotton stearoyl‐ACP desaturase (sad1) gene, and PCR cycling conditions suitable for the use of this sequence, which acts as an endogenous reference gene in both qualitative and quantitative PCR assays. The two methods were tested with 18 cotton varieties and identical amplification products were obtained with all of them. No amplification products were detected when DNA samples from other species, including soybean, rapeseed, tobacco, maize, tomato, potato, cucumber, pea, red pepper, sunflower, sesame, rice, peach, banana, apple, pumpkin, barley and carrot, were used as templates, which demonstrates that this system is specific for cotton. In real‐time quantitative PCR analysis, the detection limit was as low as 6 pg of DNA, which indicates that this method is suitable for application to processed food samples that contain very low copies of target DNA. Southern blot analysis confirmed that the sad1 gene was a single copy in the tested cotton varieties. Copyright © 2006 Society of Chemical Industry     

Determination of (E)‐5‐methylhept‐2‐en‐4‐one in deodorised hazelnut oil. Application to the detection of adulterated olive oils

The presence of (E)‐5‐methylhept‐2‐en‐4‐one (filbertone) in a hazelnut oil, deodorised in a laboratory system using nitrogen as stripping gas, is studied regarding its usefulness as a chiral marker for detecting adulterations of olive oil. The analytical method involves the use of both simultaneous distillation–solvent extraction as a sample concentration step and a chiral stationary phase in the subsequent gas chromatographic analysis. The procedure is simple, rapid, effective for detecting adulterations of olive oil with hazelnut oil and can reduce falsely negative results obtained if conventional parameters included in current regulations are exclusively considered when establishing purity in olive oils. © 2000 Society of Chemical Industry     

Isolation of obligate anaerobic rumen bacteria capable of degrading the neurotoxin β‐ODAP (β‐N‐oxalyl‐L‐α,β‐diaminopropionic acid) as evaluated by a liquid chromatography/biosensor analysis system

Six pure strains of obligate anaerobes capable of degrading the toxin β‐N‐oxalyl‐L ‐α, β‐diaminopropionic acid (β‐ODAP) contained in grass pea (Lathyrus sativus) have been isolated from cow rumen. The new isolates were identified as Megasphaera elsdenii (five different genotypes) and Clostridium bifermentans using 16S rDNA analysis. The β‐ODAP degrading efficiency of the isolates was evaluated by measuring the amount of β‐ODAP in the growth medium, which contained β‐ODAP as the only carbon source, before and after incubation with the microbes. The method of analysis was liquid chromatography employing bioelectrochemical detection. The biosensor is based on co‐immobilising two enzymes, glutamate oxidase (GlOx) and horseradish peroxidase (HRP), on the end of a spectrographic graphite electrode. β‐ODAP is oxidised by GlOx to form H₂O₂, which in turn is bioelectrocatalytically reduced by HRP through a mediated reaction using a polymeric mediator incorporating Os^{2 + /3+} functionalities rapidly shuttling electrons with the electrode_giving rise to the analytical signal. On the basis of this analysis system, the new isolates are capable of utilising β‐ODAP as sole carbon source to a maximum of 90–95% within 5 days with concomitant increase in cell protein. Copyright © 2005 Society of Chemical Industry