Multiplex Real-time PCR for the Simultaneous Detection of Salmonella spp. and Listeria monocytogenes in Food Samples

In this study, we have developed a rapid method for the simultaneous detection of Listeria monocytogenes and Salmonella spp. in foods, combining culture enrichment and a multiplex real-time polymerase chain reaction (PCR). The assay used two pre-existing primer-probe sets, labelled with different reporter dyes to enable the direct distinction of the original contaminating agent. Amplification efficiency and inclusivity/exclusivity of the combined assay was successfully assessed. The overall process included the culture enrichment based on the ISO standard, consisting of 24 h incubation in appropriate media (Half Fraser Broth for Listeria and buffered peptone water (BPW) for Salmonella), followed by a single DNA extraction of mixed enrichment aliquots, and real-time PCR detection of the hly and bipA genes of L. monocytogenes and Salmonella spp., respectively. An internal amplification control, co-amplified during the PCR run, was included in the assay to verify the results. The tool was evaluated with a variety of artificially inoculated samples of fresh products and ready to eat and cooked dishes, allowing the identification of the target pathogens down to 5 CFU/25 g of food sample. Moreover, the analysis saved a considerable amount of time compared to the ISO standard, being performed in less than 2 working days. Specificity, sensitivity and accuracy were satisfactorily tested by comparison to the standard methods ISO 11290-2:1998 and ISO 6579:2002, suggesting that the tool has a great potential as a reliable alternative for food safety assurance providing rapid detection of both pathogens in food samples.     

In-house validation of a real-time PCR method for rapid detection of<Emphasis Type="Italic"> Salmonella</Emphasis> ssp. in food products

A real-time polymerase chain reaction (PCR) method for Salmonella ssp. detection in food samples has been developed and validated in-house. The specificity of the assay was confirmed by tests with 295 different Salmonella strains, including four strains of Salmonella bongori. When tested with extracted Salmonella DNA the lowest detected amount was found to be 5 fg, which is equivalent to approximately one genome copy. The detection limit was further determined by artificial contamination of minced meat with S. Typhimurium cells and of pastry with S. enteritidis using the most probable number approach for cell dose dilutions. It was calculated that even one colony forming unit of Salmonella was still detectable in 25 g food after enrichment culture for 18 h. An additional PCR system for internal positive control, which was included in each reaction and detected in parallel via another reporter fluorescence dye, has no negative impact on the sensitivity of the assay. The method was evaluated with 1,293 naturally contaminated food samples and compared to the conventional cultural method. Of 55 positive PCR samples, 45 were confirmed by the cultural method. The statistical comparison revealed a correlation of 99.2% for specificity, of 100% for sensitivity and of 99.2% for trueness. The results of the comparative analysis and the advantages of the real-time PCR method for detection of Salmonella ssp. under routine laboratory conditions are discussed.     

Validation of a Duplex Real-Time PCR for the Detection of Salmonella spp. in Different Food Products

A 5′ nuclease duplex real-time polymerase chain reaction (PCR) assay was developed and validated with various food products for the specific and fast detection of Salmonella spp. in food. The assay used previously published primers in combination with a newly developed probe targeting the invA gene. An internal amplification control, which is coamplified in a duplex PCR, was included in the assay. The analysis of 1,934 natural food samples with real-time PCR and the cultural method in parallel resulted in a relative accuracy of 100% and 99.84% respectively, depending on the enrichment procedure in which buffered peptone water and selective enrichment in Rappaport–Vassiliadis (RV) broth were employed. The duplex real-time PCR assay has proven to be a specific, sensitive and fast screening method for Salmonella spp. in food. The overall analysis time of the PCR method was approximately 28 h, in contrast to 4 to 5 days with conventional Salmonella diagnostics. The developed assay has been shown to be a reliable diagnostic tool for use in routine analysis. It has been validated thoroughly and has become an official method in Germany for the detection of Salmonella spp. in food.     

Validation of Real-Time PCR and Enzyme-Linked Fluorescent Assay-Based Methods for Detection of Salmonella spp. in Chicken Feces Samples

A comparative study of enzyme-linked fluorescent assay (ELFA)-based methods and real-time polymerase chain reaction (PCR)-based methods using three and two different sample preparation protocols, respectively, and the standard culture-based method EN ISO 6579:2002/Amd1:2007, for the detection of Salmonella spp. in chicken feces, was performed on 20 artificially and 68 naturally contaminated chicken feces. Selectivity, relative specificity, relative accuracy, relative sensitivity, and relative detection level were determined. According to criteria established in the methods comparison study included in EN ISO 16140:2003 for validation of alternative microbiological methods, the ELFA-based methods (V1 and V2) as well as a real-time PCR method (PCR2) were comparable to the reference method for the detection of Salmonella in chicken feces. They provided results in 48 h and presented a high sensitivity (97% for all of them). The three methods showed a relative specificity of 94%, V1 being the method which presented the highest relative accuracy (96%). While detection level for V2 and reference method was between 3 and 13 CFU/25 g, PCR2 method was able to detect down to 3 CFU/25 g. In conclusion, both the real-time PCR and the ELFA-based assays can be used as rapid and user-friendly screening methods for detection of Salmonella spp. in chicken feces.     

Development of a fimY-based loop-mediated isothermal amplification assay for detection of Salmonella in food

In this study, we developed a rapid and sensitive fimY-based loop-mediated isothermal amplification (LAMP) assay on a real-time turbidimeter platform for the detection of Salmonella in food. Since turbidity of the reaction mixture would increase in correlation with the DNA yield, real-time monitoring of the LAMP reaction was achieved by real-time turbidimeter. Time threshold values which indicate positive results for 81 Salmonella strains of different serotypes ranged from 36 to 40 min. For the 20 non-Salmonella strains, turbidity did not increase in the reaction mixture. When testing 10-fold serial dilutions of Salmonella Typhimurium-ATCC 14128 DNA by LAMP, the time threshold ranged from 36 to 52 min on the real-time turbidimeter. The detection limit was 13 cells per reaction in pure culture, up to 10-fold more sensitive than that of PCR. When applied in deli food samples, the LAMP assay was able to detect Salmonella even though the sample was contaminated with very low concentration after 3 h enrichment culture. Increase in turbidity was observed on real-time turbidimeter. Additionally, the LAMP results detected by naked-eye or turbidity consistently matched with each other. Results from this study showed that the fimY-based LAMP assay is an effective method for the rapid detection of Salmonella.     

A Pilot Study for Identification of <Emphasis Type="Italic">Salmonella</Emphasis> in Food Processing Plants by Real-Time PCR Screening

Salmonella remains a major public health concern worldwide. Microbiological methods are the gold standard for Salmonella detection. These methods are highly specific, but their sensitivity is variable. Moreover, they are lengthy, labour intensive and not always consistent with the speed of food manufacturing processes. Thus, in the food industry, there is the need for more rapid, sensitive and accurate detection methods. The purpose of this study is to describe a Salmonella-monitoring scheme in different food processing plants based on a screening approach by a commercial real-time polymerase chain reaction (PCR) kit and subsequent confirmation of positive molecular results by the reference microbiological method. This scheme was tested on a total of 4,693 samples, 90 of which were positive with the real-time PCR screening; 52 of the positive samples were eventually confirmed by the microbiological method. The real-time PCR kit was tested in comparison to the microbiological method in order to evaluate its performances and drawbacks. The comparison between cycle threshold (Ct) values of real-time PCR and the microbiological results (Wilcoxon rank sum test) showed a statistically significant difference between the Ct values of bacteriological positive and bacteriological negative samples (p value, <0.05). Furthermore, receiver operating characteristic curve analysis was used to identify the Ct value ensuring the lowest level of misclassification between Salmonella-positive and negative samples. The present study confirms that the real-time PCR kit tested could be used as a screening tool, leading to a rapid and sensitive identification of Salmonella and confining bacteriological confirmation to samples previously identified as positive.     

Comparative Evaluation of RTi-PCR and Mini-VIDAS SLM System as Predictive Tools for the Routine Detection of Salmonella spp. in Naturally Contaminated Food Products

In the present work, we have evaluated a real-time polymerase chain reaction-based method (RTi-PCR) as a routine laboratory test, by comparing the results obtained in two laboratories with the mini-VIDAS SLM (bioMérieux), a standard accepted immunoenzymatic (enzyme-linked immunosorbent assay) method currently used in the food industry. To that aim, a set of 141 naturally contaminated food samples were analyzed after an enrichment step by conventional PCR and mini-VIDAS and prior to the enrichment by RTi-PCR. Results from both laboratories were statistically analyzed using the kappa coefficients, which indicated a perfect agreement between them. Out of the 141 samples, 11 were positive for Salmonella detection by mini-VIDAS, 35 by conventional PCR in Lab1, and 44 in Lab2. Twenty-eight of them tested positive by RTi-PCR in Lab1 and 31 in Lab2. In order to use the evaluated methods as a diagnostic test, their predictive capacity was analyzed on the basis of their positive and negative predictive values, calculated using the result obtained—after enrichment—by conventional PCR as the “gold standard.” Both positive and negative predictive values were higher for RTi-PCR than those obtained for mini-VIDAS, indicating good performance of the RTi-PCR technique—applied without enrichment—in the detection of Salmonella in food products. Overall, results obtained in this study on naturally contaminated food products highlighted that RTi-PCR without enrichment is a better predictive tool than mini-VIDAS.     

A loop-mediated isothermal amplification method targets the HisJ gene for the detection of foodborne Salmonella

A loop-mediated isothermal amplification (LAMP) assay was developed and validated for the specific detection of Salmonella in food. The four primers required for LAMP were designed using a conserved region in the histidine transport protein-coding region of Salmonella. Seventy-nine reference strains of 72 Salmonella serovars and 23 non-Salmonella strains were detected by LAMP. The detection limit of this assay is 16?CFU per reaction in pure culture, up to tenfold more sensitive than that of the PCR assay with the same target gene. When applied in raw food samples, a sample pretreatment protocol was performed that included a pre-enrichment step in 0.1?% buffered peptone water, followed by a selective enrichment in Rappaport–Vassiliadis medium. Using this method, 200 assorted food samples were investigated for Salmonella, including fresh pork, whole chickens, and green vegetables. The sensitivity of LAMP for the detection of Salmonella in food samples was 93.55?%, versus 87.10?% that tested positive using conventional PCR. The results from this study showed that the HisJ-based LAMP is an effective method for the detection of foodborne Salmonella.     

Application of magnetic immuno-polymerase chain reaction assay for detection of <Emphasis Type="Italic">Salmonella</Emphasis> spp. in chicken meats

Since contaminated chicken meats have been the principal foodborne source of the contamination of Salmonella to human beings and cultural detection methods are labor-intensive and time-consuming, a study evaluating the performance of the combination of two techniques that are immunomagnetic separation (IMS) and polymerase chain reaction (PCR) for the detection of Salmonella in chicken meats was conducted. The IMS and PCR assay combines selective extraction of Salmonella by specific antibodies with primer-specific (primer pair based on the sequence of invA gene) PCR amplification. Initially chicken meat samples, in which no Salmonella contamination had been determined by using ISO 6579 reference method, were inoculated with Salmonella Enteritidis culture and subsequently the shortest non-selective pre-enrichment time, that had been needed for the detection of approximately 1 or 10 CFU/mL chicken meat levels of target bacteria by magnetic immuno-PCR assay, was found by using 14, 12, 10 and 8-h periods. In conclusion, it was found that magnetic immuno-PCR assay was able to detect 1–10 CFU Salmonella/25 g chicken meat, after only incorporating a non-selective pre-enrichment period of 12 h. Therefore, an overall 16-h (magnetic immuno-PCR assay in conjunction with 12-h non-selective pre-enrichment) magnetic immuno-PCR assay statistically evaluated as sufficient (p = 0.182 > 0.05) for rapid and sensitive detection of approximately 1–10 CFU Salmonella from 25 g chicken meat samples. Accordingly, 16-h magnetic immuno-PCR assay can be promising for routine use in the detection of Salmonella in chicken meat samples, and it consequently may prevent the risk of Salmonella infections in regard to chicken meats.     

Development of a novel dual priming oligonucleotide system-based PCR assay for specific detection of Salmonella from food samples

In this study, a novel dual priming oligonucleotide (DPO) system-based polymerase chain reaction (PCR; DPO system-based PCR) assay, which detected the fimY gene of Salmonella, was developed for the fast food testing. The DPO system-based PCR assay allowed a wide range of annealing temperatures at 48–68°C to efficiently amplify fimY gene with an analytical detection limit of 1.2 × 10² CFU/ml for Salmonella in pure cultures and artificially contaminated food matrix. Significantly, the presence of a bubble-like polydeoxyinosine (polyI) linker in the DPO system brought an unparalleled high specificity in the identification of target bacteria, and consequently, the false positives and mismatches of PCR process can be eliminated in priming. Applying the DPO system-based PCR assay to 285 collected food samples revealed that 29 samples were positive in this assay, in accordance with the results of conventional culture-based method, indicating a potential diagnostic capability. The high specificity of the DPO system-based PCR indicates its great potential to be a quick, reliable and practical method for the detection of Salmonella in foods.     

Polymerase Chain Reaction for the Rapid Detection and Serovar Identification of Salmonella in Food and Feeding Stuff

The polymerase chain reaction (PCR) is one of the most important rapid methods for the sensitive and specific detection of pathogenic and spoilage microorganisms. The method is increasingly applied in surveillance and monitoring programs to detect pathogens, especially for ensuring the safety and quality of food. The food-borne pathogen Salmonella together with Campylobacter is the most predominant bacterial pathogen in Europe, causing approximately 160,000 confirmed human cases per year. During the last two decades, the importance of Salmonella for food safety triggered the development of dozens of PCR-based detection methods. They promise significant advantages compared to the traditional culture-based methods with respect to speed, easiness, reliability, sensitivity, cost effectiveness, and automation. However, many of them are not applicable because of lacking validation procedures. Meanwhile, PCR has internationally been standardized and guidelines as well as standards for the validation of alternative methods, such as PCR, were established. This review will give an overview of the historical development of PCR-based methods for the detection of Salmonella including validation aspects and summarizes the state-of-the-art for the detection of Salmonella in food and feeding stuff by real-time PCR. Furthermore, current multiplex PCR-based serovar-specific identification methods will be reviewed.     

Salmonella real-time PCR-Nachweis

Salmonella belongs to the most important bacterial pathogens worldwide causing disease in humans and animals mainly by the oral uptake of contaminated food. Consequently, detection methodologies for Salmonella from food items are meaningful for routine laboratories. Here, we describe two different real-time PCR based methods for the detection of Salmonella in food. The procedure begins with a cultural pre-enrichment in buffered peptone water for 18–24 hours at 37 °C followed by a selective enrichment step in Rappaport-Vassiliadis medium for at least six hours at 42 °C. Next, the microbial DNA is extracted and finally Salmonella-DNA is specifically detected by the real-time PCR. Both methods differ in the Salmonella target gene sequence. One assay amplifies a 285-bp-DNA-Fragment of the invA-gene, and the other a 95-bp-DNA-Fragment of the ttrC/ttrA-gene. An internal amplification control indicates the correct carrying out of the PCR. The duration of both detection methods is between 24 and 28 hours.

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Zusammenfassung: Salmonellen z?hlen zu den bedeutendsten bakteriellen Erregern weltweit, die meist durch die Aufnahme von belasteten Lebensmitteln eine Erkrankung bei Menschen und auch Tieren verursachen k?nnen. Verfahren für den Nachweis von Salmonellen in Lebensmitteln haben daher für die diagnostischen Routinelabore eine besondere Bedeutung. Hier beschreiben wir zwei verschiedene real-time PCR basierte Verfahren zum Nachweis von Salmonellen in Lebensmitteln. Das Verfahren beginnt mit einer kulturellen Voranreichung in gepufferten Peptonwasser für 18 bis 24 h bei 37°C und darauf folgender selektiver Anreicherung in Rappaport-Vassiliadis Medium für sechs Stunden bei 42 °C. Anschlie?end erfolgt eine mikrobielle DNA-Extraktion und der Nachweis der Salmonella DNA im Extrakt mittels der realtime PCR. Beide Verfahren unterscheiden sich in der Zielsequenz der nachzuweisenden Salmonella DNA. W?hrend der eine PCR Assay ein 285-bp-DNA-Fragment des invA Gens amplifiziert, weist der zweite Assay ein 95-bp-DNA-Fragment des ttrC/ttrA-Gens von Salmonellen spezifisch nach. Eine interne Amplifikationskontrolle, die in jeder PCR-Reaktion mitgeführt wird, zeigt den funktionsrichtigen Ablauf jeder PCR-Reaktion an. Die Gesamtdauer des Nachweises betr?gt 24 bis 28 Stunden.

Eingegangen: 18. Januar 2007     

Salmonella real-time PCR-Nachweis

Salmonella belongs to the most important bacterial pathogens worldwide causing disease in humans and animals mainly by the oral uptake of contaminated food. Consequently, detection methodologies for Salmonella from food items are meaningful for routine laboratories. Here, we describe two different real-time PCR based methods for the detection of Salmonella in food. The procedure begins with a cultural pre-enrichment in buffered peptone water for 18–24 hours at 37 °C followed by a selective enrichment step in Rappaport-Vassiliadis medium for at least six hours at 42 °C. Next, the microbial DNA is extracted and finally Salmonella-DNA is specifically detected by the real-time PCR. Both methods differ in the Salmonella target gene sequence. One assay amplifies a 285-bp-DNA-Fragment of the invA-gene, and the other a 95-bp-DNA-Fragment of the ttrC/ttrA-gene. An internal amplification control indicates the correct carrying out of the PCR. The duration of both detection methods is between 24 and 28 hours.     

A New Multiplexed Real-Time PCR Assay to Detect <Emphasis Type="Italic">Campylobacter jejuni</Emphasis>, <Emphasis Type="Italic">C. coli</Emphasis>, <Emphasis Type="Italic">C. lari</Emphasis>, and <Emphasis Type="Italic">C. upsaliensis</Emphasis>

A new rapid method based on real-time PCR was developed to detect four thermophilic Campylobacter species (Campylobacter jejuni, Campylobacter coli, Campylobacter lari, and Campylobacter upsaliensis) in food samples. The assay targeted the bipA gene for C. upsaliensis and C. lari, whereas the gene encoding the ATP-binding protein CJE0832 was used to detect C. coli and C. jejuni. These genes were chosen for this assay due to their low variability and mutation rate at a species level. The multiplex PCR showed 100% inclusivity for all 25 thermophilic Campylobacter strains tested and 100% exclusivity for 38 non-targeted strains belonging to closely related species. The newly developed real-time PCR could detect down to 10² genomes/reaction and displayed efficiency above 97% for all species except for C. upsaliensis (90.1%). The method proved to be a reliable tool for food analysis, showing 100% sensitivity, 96% efficiency, and 92.45% specificity when validated against the gold standard method UNE-EN ISO 10272:2006 using 200 diverse food samples (meat, fish, fruits and vegetables, and raw milk). In artificially spiked samples, the detection limit of the method was 10 cfu/g in salad, 5 cfu/g in turkey meat, and 1 cfu/g in the rest of meat samples tested. Consequently, the newly designed molecular tool represents a quick and safe alternative to obtain reliable results concerning the presence/absence of the main thermophilic Campylobacter in any food sample.     

Multiplex TaqMan® detection of pathogenic and multi-drug resistant Salmonella

Overuse of antibiotics in the medical and animal industries is one of the major causes for the development of multi-drug-resistant (MDR) food pathogens that are often difficult to treat. In the past few years, higher incidences of outbreaks caused by MDR Salmonella have been increasingly documented. The objective of this study was to develop a rapid multiplex real-time polymerase chain reaction (PCR) assay for simultaneous detection of pathogenic and MDR Salmonella spp. A multiplex TaqMan®real-time PCR was designed by targeting the invasin virulence gene (invA), and four commonly found antibiotic resistance genes, viz. ampicillin, chloramphenicol, streptomycin and tetracycline. To avoid false negative results and to increase the reliability of the assay, an internal amplification control (IAC) was added which was detected using a locked nucleic acid (LNA) probe. In serially diluted (5 ng–50 fg) DNA samples, the assay was able to detect 100 genomic equivalents of Salmonella, while in a multiplex format, the sensitivity was 1000 genomic equivalents. The assay performed equally well on artificially contaminated samples of beef trim, ground beef of different fat contents (73:27, 80:20, 85:15 and 93:7), chicken rinse, ground chicken, ground turkey, egg, spinach and tomato. While the detection limit for un-enriched inoculated food samples was 10⁴ CFU/g, this was improved to 10 CFU/g after a 12-h enrichment in buffered peptone water, with 100% reproducibility. The multiplex real-time assay developed in this study can be used as a valuable tool to detect MDR virulent Salmonella, thus enhancing the safety of food.     

The evaluation of a PCR-based method for identification of Salmonella enterica serotypes from environmental samples and various food matrices

The most commonly used method for serotyping Salmonella spp. is based on the Kaufmann–White scheme, and is composed of serological reactions using antibodies to LPS agglutinins. The multiplex PCR used in this investigation was established by Kim et al. to serotype the 30 most common clinical Salmonella serotypes, as determined by CDC. The PCR assay consists of two five-plex reactions and a single two-plex PCR reaction, based on six genetic loci from Salmonella enterica serotype Typhimurium and four loci from S. enterica serotype Typhi. In this investigation, we further evaluated the method for serotyping Salmonella spp. using a reference collection, environmental samples collected from a Mid-Atlantic region tomato farm study, four food matrices spiked with different Salmonella serotypes and a proficiency test. The PCR assay was first evaluated using DNA isolated from pure cultures of isolates obtained from various clinical and environmental samples, and then DNA isolated from broth cultures of food matrices of “Red round” and Roma tomatoes, Romaine lettuce, green onions and Serrano peppers spiked with serotypes Newport, Typhimurium, Javiana and Saintpaul, respectively. The results showed that the PCR assay correctly serotyped Salmonella spp. from the clinical, environmental, spiked food matrices, and proficiency test samples. These findings are significant because the PCR assay was successful in the identification of Salmonella in the spiked samples in a broth culture containing other non-salmonella organism. This method may be a useful resource for the food safety community.     

Rapid Conventional PCR and Real-Time-qPCR Detection of Low Numbers of Salmonella enterica from Ground Beef without Enrichment

Detection of low numbers of Salmonella in complex food matrices such as ground beef by polymerase chain reaction (PCR) without enrichment is particularly difficult because of the presence of PCR inhibitors and fat. This study used soluble starch for the removal of fat in ground beef followed by the use of activated carbon coated with milk proteins for the removal of PCR inhibitors prior to conventional PCR and RealTime qPCR. This methodology without pre-enrichment allowed detection with conventional PCR of 5 CFU/g and 1 CFU/g with the real-time qPCR in ground beef containing 7%, 15%, and 27% fat. The total assay time was 5 h from the seeding of a 25 g sample of ground beef to agarose gel detection of amplicons of a 284 bp invA gene fragment specific for Salmonella and 4.5 h for real-time qPCR detection.     

Development of a real-time PCR assay for detection and quantification of enterotoxigenic members of Bacillus cereus group in food samples

A highly sensitive real-time PCR (qPCR) procedure, targeting the phosphatidylcholine-specific phospholipase C gene (pc-plc), was developed for specific detection and quantification of strains belonging to Bacillus cereus group. The target region was selected based on the enterotoxigenic profiles of 75 Bacillus strains. The inclusivity and exclusivity of the RTi-PCR assay were assessed with 59 isolates of the B. cereus group, 16 other Bacillus spp., and 4 non-Bacillus strains. The assay was also used to construct calibration curves for different food matrices, and it had a wide quantification range of 6 log units using both serial dilutions of purified DNA and calibrated cell suspensions of B. cereus CECT 148^T. The detection limit for B. cereus in artificially contaminated liquid egg and reconstituted infant formula was about 3 CFU per reaction or 60 CFU/ml of food, with a relative accuracy of 86.27% to 116.12% in artificially contaminated liquid egg. Naturally contaminated food samples were tested for the presence of B. cereus with the standard method, a conventional PCR and the new developed RTi-PCR assay. Results showed that the new developed RTi-PCR assay is very suitable for detection and quantification of strains of B. cereus group in food samples without an enrichment step.     

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.