Real-time polymerase chain reaction detection of fishmeal in feedstuffs.

A SYBR Green PCR system was developed for detection of fishmeal in feedstuffs. The real-time PCR method combines the use of fish-specific primers that amplify an 87 base pair (bp) fragment of the mitochondrial 12S ribosomal RNA gene from fish species, and a positive control primer pair that amplifies a 99 bp fragment of the nuclear 18S ribosomal RNA gene in all eukaryotic organisms. The specificity of the primers was tested against 52 animal species and six plant species. Reference feedstuff samples were successfully tested for the presence of fishmeal, demonstrating the applicability of the assay to feedstuffs.

Specific PCR detection of Arcobacter butzleri, Arcobacter cryaerophilus, Arcobacter skirrowii, and Arcobacter cibarius in chicken meat.

An enrichment PCR assay using species-specific primers was developed for the detection of Arcobacter butzleri, Arcobacter cryaerophilus, Arcobacter skirrowii, and Arcobacter cibarius in chicken meat. Primers for A. cryaerophilus, A. skirrowii, and A. cibarius were designed based on the gyrA gene to amplify nucleic acid fragments of 212, 257, and 145 bp, respectively. The A. butzleri-specific primers were designed flanking a 203-bp DNA fragment in the 16S rRNA gene. The specificity of the four primer pairs was assessed by PCR analysis of DNA from a panel of Arcobacter species, related Campylobacter, Helicobacter species, and other food bacteria. The applicability of the method was then validated by testing 42 fresh retail-purchased chicken samples in the PCR assay. An 18-h selective preenrichment step followed by PCR amplification with the four Arcobacter primer sets revealed the presence of Arcobacter spp. in 85.7% of the retail chicken samples analyzed. A. butzleri was the only species present in 50% of the samples, and 35.7% of the samples were positive for both A. butzleri and A. cryaerophilus. A. skirrowii and A. cibarius were not detected in any of the chicken samples analyzed. The enrichment PCR assay developed is a specific and rapid alternative for the survey of Arcobacter contamination in meat.

SYBR-Green real-time PCR approach for the detection and quantification of pig DNA in feedstuffs.

A real-time polymerase chain reaction assay using primers targeting the porcine-specific mitochondrial 12S rRNA gene and universal eukaryotic primers amplifying a conserved fragment of the nuclear 18S rRNA gene has been developed for the detection and quantification of porcine DNA in food and feedstuffs. The 18S rRNA primers were used as endogenous control for the total content of PCR-amplifiable DNA in the sample. The assay was tested on DNA extracted from raw and heat-treated binary mixtures of porcine tissues in a plant matrix, and on DNA extracted from reference feedstuff samples. Analysis of experimental mixtures demonstrated the suitability of the assay for the detection and quantification of porcine DNA in mixtures containing as little as 0.1%.

Avian-specific real-time PCR assay for authenticity control in farm animal feeds and pet foods.

A highly sensitive TaqMan real-time PCR assay targeting the mitochondrial 12S rRNA gene was developed for detection of an avian-specific DNA fragment (68bp) in farm animal and pet feeds. The specificity of the assay was verified against a wide representation of animal and plant species. Applicability assessment of the avian real-time PCR was conducted through representative analysis of two types of compound feeds: industrial farm animal feeds (n=60) subjected to extreme temperatures, and commercial dog and cat feeds (n=210). Results obtained demonstrated the suitability of the real-time PCR assay to detect the presence of low percentages of highly processed avian material in the feed samples analysed. Although quantification results were well reproducible under the experimental conditions tested, an accurate estimation of the target content in feeds is impossible in practice. Nevertheless, the method may be useful as an alternative tool for traceability purposes within the framework of feed control.

High resolution TaqMan real-time PCR approach to detect hazelnut DNA encoding for ITS rDNA in foods.

A broad range of foods have been described as causing allergies, but the majority of allergic reactions can be ascribed to a limited number of food components. Recent extensive surveys showed how tree nuts, particularly hazelnut (Corylus avellana L.) seeds, rank amongst the most important sources of food allergy. In order to protect the allergic consumer, efficient and reliable methods are required for the detection of allergenic ingredients. For this purpose, we have developed a real-time polymerase chain reaction (PCR) for detection of hazelnut in commercial food products. In this way a specific hazelnut primer pair based on the ITS marker (70 bp) and a nuclease (TaqMan) probe labelled with FAM and BHQ were designed. Sensibility of real-time PCR was determined by analysis of raw and heat treated hazelnut-wheat flour mixtures with a range of detection of 0.1-100,000 ppm. Practical applicability of the real-time PCR assay developed for determining hazelnut in different food matrices was investigated by analyzing 179 commercial foodstuffs comprising snacks, biscuits, chocolates, bonbons, creams, nut bars, ice creams, precooked meals, breads, beverages, yogurts, cereals, meat products, rice cake and nougat. From the total of samples analyzed, 40 commercial food products that didn't declare hazelnut nor traces on the label were found to contain hazelnut. The real-time PCR method proposed herein due to its high sensitivity facilitates the detection of hazelnut traces in commercial food products and can also be useful for monitoring the effectiveness of cleaning processes and as consequence, can help to prevent the food allergic consumer from unintentional ingestion of hidden allergens.

Detection of banned ruminant-derived material in industrial feedstuffs by TaqMan real-time PCR Assay.

A ruminant-specific real-time PCR system was designed and applied for the detection of processed animal protein from ruminants in industrial feedstuffs. The assay includes a primer pair and a TaqMan probe selectively targeting mitochondrial 16S rRNA gene sequences from the ruminant group and another primer-probe set based on the eukaryotic nuclear 18S rRNA gene (positive amplification control). Both ruminant and eukaryotic PCR systems generated short PCR amplicons of 79 and 77 bp, respectively. To evaluate the suitability of the real-time PCR assay for the detection of banned by-products of ruminant origin, 126 feed samples subjected to rendering under current European legislation regulations were analyzed. The assay achieved 100% success in classifying the samples as positive or negative in terms of qualitative ruminant composition, with a detection limit of 0.1%. The quantitative ability of the assay is however restricted by variations in the composition and treatment of the feeds, which affect the amount and quality of amplifiable DNA.

PCR detection of Alternaria spp. in processed foods, based on the internal transcribed spacer genetic marker.

The genus Alternaria is considered one of the most important fungal contaminants of vegetables, fruits, and cereals, producing several mycotoxins that can withstand food processing methods. Conventional methods for Alternaria identification and enumeration are laborious and time-consuming, and they might not detect toxigenic molds inactivated by food processing. In this study, a PCR method has been developed for the rapid identification of Alternaria spp. DNA in foodstuffs, based on oligonucleotide primers targeting the internal transcribed spacer (ITS) 1 and ITS2 regions of the rRNA gene. The specificity of the Alternaria-specific primer pair designed (Dir1ITSAlt-Inv1ITSAlt) was verified by PCR analysis of DNA from various Alternaria spp., and also from several fungal, bacterial, yeast, animal, and plant species. The detection limit of the method was 10(2) CFU/ml in viable culture, heated culture, or experimentally inoculated tomato pulp. The applicability of the method for detection of Alternaria spp. DNA in foodstuffs was assessed by testing several commercial samples. Alternaria DNA was detected in 100% of spoiled tomato samples, 8% of tomato products, and 36.4% of cereal-based infant food samples analyzed.

Development of a real-time PCR assay to control the illegal trade of meat from protected capercaillie species (Tetrao urogallus).

A rapid and highly species-specific real-time polymerase chain reaction (PCR) assay has been developed for the detection of capercaillie DNA (Tetrao urogallus) in meat and meat mixtures. The method combines the use of capercaillie-specific primers, that amplify a 142bp fragment of the mitochondrial 12S rRNA gene, and a positive control primer pair that amplifies a 141bp fragment of the nuclear 18S rRNA gene from eukaryotic DNA. SYBR(®) Green dye or TaqMan(®) fluorogenic probes were used to monitor the amplification of the target genes. Results obtained with the use of TaqMan(®) probes as detection platform increased the specificity of the real-time PCR assay in comparison with the results obtained using SYBR(®) Green. The proposed real-time PCR assay represents a rapid and straightforward method for the accurate identification of capercaillie that could be used by law enforcement agencies as a tool for the control of poaching and illegal trade of meat from this protected species.

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.