No abstract

european union regulations (e.g. Commission Regulation (eu) no 56/2013) set restrictions on the use of poultry meals in animal nutrition, which requires the species composition of manufactured feeds to be constantly monitored. The aim of this study is to develop a method for qualitative analysis of poultry meals, enabling the four main poultry species (chicken, turkey, duck, goose) to be detected using one pair of primers, and a method for quantitative determination of chicken meals in poultry meals. The qualitative identification method was developed using PCR technology, whereas qPCR and TaqMan probes were used for quantitative identification. The study was performed with samples of feed mixture containing poultry and chicken meal. The limit of determination was 0.08% and 0.02% for qualitative and quantitative identification, respectively. The results of quantitative determinations obtained for independent dna isolations from the same samples are repeatable (RSDcT ≤0.46%). The determined concentrations are accurate (Dc ≤11.23% for c ≥0.06). The identification of target sequences in both tests is good enough for commercial applications.

The objective of the study was to demonstrate how mitochondrial DNA (mtDNA) can be used to determine the species origin of animal microtraces. The study included pieces of cat and dog hair without the root, a fragment of cooked chicken bone (0.1g), three goose down samples (0.028 g), a pork swab, a pork scratching (5×5×5 mm), and pork lard (0.22 g). DNA was isolated from all of these samples using the method appropriate for the particular source material. The extracts had DNA concentration exceeding 5.4 ng/µl with A purity range of 1.14-1.88. Next, the samples were subjected to PCR and real-time PCR with species-specific primers and primers complementary to mitochondrial DNA (mtDNA). Control reactions based on the amplification of eukaryotic-specific fragment (18S rRNA) were additionally performed. PCR and real-time PCR products for detection of species-specific mtDNA were obtained for all templates, whereas during the detection of eukaryote DNA no product was obtained for dog and cat hair only. The poor quality of the obtained DNA did not prevent the analysis. The results showed that mitochondrial DNA is suitable for identification of small or highly processed samples, in which genomic DNA often cannot be analyzed.

Monitoring food quality is an important and constant element of the food market. This need is connected with health issues, religious beliefs of consumers, and economic considerations. For analysis, mtDNA is most commonly used because it is resist to physical factors such as temperature and pressure, which very often accompany food processing. Nowadays, scientific publications present a number of methods describing species identification from both farm animals and also less common animals. The most effective methods for determining species are based on PCR, real-time PCR, and sequencing. The methods are very sensitive, limit of detection (LOD) is 0.001% for many of them. An indispensable element of performing the described research is the strict application in the laboratory of several principles, which are intended to improve the work and make it safe for the lab technician, as well as guarantee the quality and effectiveness of the experiments carried out. The high work requirements set for the crew naturally shape the quality system from which the most popular is ISO/IEC 17025. Modern methods based on mtDNA are a good tool for food analysis, creating great opportunities for the researcher, at the same time causing challenges for the contemporary laboratory.

This study developed a method to identify the species of duck and goose DNA extracted from various matrices. The research material included meat, down, whole feathers, and blood. The analysis was performed with real-time PCR using minor groove binder probes and primers that bind to cytochrome b (duck) and 12S ribosomal RNA (goose) sequences. For all matrices, the reactions were species-specific. Cross-reactions either did not occur or occurred after 36 cycles, corresponding to an amplification for the species content of 10 − 5 %, i.e., several times below the limit of detection (LOD = 0.01%) and limit of quantification (LOQ = 0.1%). The standard curves obtained by consecutive dilutions were linear (R 2 > 0.99), the slopes ranged from 3.2 to − 3.6, and the retardation factors were 23-26. The amplitude threshold analysis demonstrated that significant differences between successive matrices make it impossible to select one reference material to produce a standard curve effective for analyzing each type of matrix. A comparison between the reference material and the test material reveals that the accuracy of the developed method (the match between the actual and the measured value) is above 78%. Validation methods indicate that this method can be used to identify duck and goose DNA in the analyzed matrices.

Proste, skuteczne i czułe metody identyfikacji gatunkowej komponentów mięsnych w produktach spożywczych są ważnym aspektem produkcji żywności. Są one istotne zarówno dla producentów, jak i konsumentów, którzy są coraz bardziej zainteresowani rzeczywistym składem żywności. W identyfikacji komponentów pochodzenia zwierzęcego w żywności największe znaczenie mają obecnie metody polegające na analizie mtDNA, co wynika ze znacznej trwałości oraz specyficzności gatunkowej tej cząsteczki. Celem pracy było opracowanie prostych i skutecznych testów identyfikacji gatunkowej komponentów bydlęcych, wieprzowych i owczych, przydatnych do analizy żywności. Badaniom poddano próbki mięsa o zawartości [%]: 6, 35, 60, 80 i 100 wołowiny w wieprzowinie, wieprzowiny w wołowinie i baraniny w wieprzowinie. Metoda polega na identyfikacji gatunkowo specyficznych dla bydła, świń i owiec fragmentów genu kodującego COX1 o długościach 68 pz oraz 53 pz. W opracowanych testach granica wykrywalności komponentu wieprzowego wynosiła 0,07 %, bydlęcego-0,4 % i owczego-0,5 %. Uzyskane wyniki wskazują na specyficzność gatunkową stosowanych testów. Reaktywność krzyżowa albo nie zachodzi, albo można ją zaobserwować w późnych cyklach reakcji. Metoda ma zastosowanie zarówno do mięsa surowego, jak i jego przetworów, takich jak kiełbasa, mielonka, szynka. Metoda nie jest czasochłonna (4 h), natomiast jest powtarzalna (względne odchylenie standardowe w przypadku mięsa surowego wynosi ≤ 1,57 %) oraz dokładna (błąd pomiaru ≤ 2,33). W analizach mięsa przetworzonego dokładność pomiaru jest mniejsza, a rozrzut wyników nieznacznie większy, co może wskazywać na konieczność dostosowania krzywej standardowej do próbek badanych pod względem przetworzenia.

Chicken is a common protein source in pet foods and is concurrently listed among food allergens. Commercial over-the-counter (OTC) diets with an alternative animal protein source are considered suitable for dietary elimination trials by pet owners. The potential presence of undeclared chicken-derived ingredients in these diets can compromise the outcome of the trial during the diagnosis of adverse food reactions. The aim of this study was to selectively verify the absence or presence of chicken DNA in 10 OTC dry canine foods, using qualitative and quantitative approaches. The method of identification of chicken-derived protein was elaborated with the polymerase chain reaction (PCR) technology, whereas quantitative real-time PCR was used for the quantitative assessment. In most of the analysed samples, the chicken DNA was detectable; however, the quantified amounts were predominantly low, although differences between batches were observed.

Background These days the number of potential food allergens is very large, but chicken is one of the most common allergens in dogs. Elimination diet is one of the clinical tools for the diagnosis of allergies and allergy tests are not very reliable. The restriction diet is most commonly carried out by feeding pet foods, relying on the ingredients on the label to select an elimination diet not containing previously eaten foods. Unfortunately, mislabeling of pet food is quite common. The purpose of this study was to determine the absence or presence of chicken DNA using both qualitative and quantitative polymerase chain reaction (PCR) analysis methods in dry and wet maintenance complete pet foods for adult dogs. Results were used to verify the declared composition on the labels. Results Eleven out of fifteen (73%) dog foods were produced as declared by the manufacturer, two of which showed the presence of chicken protein as stated on the label. The remaining nine foods contained amounts of chicken DNA below 1%, consistent with declarations that no chicken was added in the composition. Four of tested dog foods (27%) were not produced consistently with the declaration on the packaging. Two dog foods (one dry and one wet) did not contain the claimed chicken protein. In two foods the addition of chicken DNA was detected at the level of over 2% and almost 6%, respectively. Conclusions In this study, we focused on one of the most commonly undeclared animal species on the label—chicken protein—and performed DNA analyzes to investigate possible contamination and mislabeling. The results showed some inaccuracies. However, most of them are trace amounts below 1%, which proves compliance with the label. Our results showed that undeclared animal species can be as common as missing an animal protein declared on the label. The conducted research indicates that both dry and wet analyzed foods should not be recommended as a diagnostic tool in elimination tests, because it may result in false negative results. Over-the-counter maintenance foods for dogs should not be recommended for the diagnosis and treatment of food hypersensitivity.