Applicability of a duplex and four singleplex real-time PCR assays for the qualitative and quantitative determination of wild boar and domestic pig meat in processed food products.

Appropriate analytical methods are needed for the detection of food authentication. We investigated the applicability of a duplex real-time PCR assay targeting chromosome 1 and two singleplex real-time PCR assays targeting chromosome 9, both published recently, for the qualitative and quantitative determination of wild boar and domestic pig in processed food products. In addition, two singleplex real-time PCR assays targeting chromosome 7 were tested for their suitability to differentiate the two subspecies. Even by targeting the three genome loci, the probability of misclassification was not completely eliminated. Application of the real-time PCR assays to a total of 35 commercial meat products, including 22 goulash products, revealed that domestic pig DNA was frequently present, even in 14 out of 15 products declared to consist of 100% wild boar. Quantitative results obtained with the real-time PCR assays for wild boar (p < 0.001) and those for domestic pig (p < 0.001) were significantly different. However, the results obtained with the real-time PCR assays for wild boar (r = 0.673; p < 0.001) and those for domestic pig (r = 0.505; p = 0.002) were found to be significantly correlated. If the rules given in the paper are followed, the real-time PCR assays are applicable for routine analysis.

Differentiation between wild boar and domestic pig in food by targeting two gene loci by real-time PCR.

Studies indicate that many meat products are not authentic, most frequently because the meat species differ from those given on the food labels. At present, DNA based methods play the most important role in meat species authentication. Discrimination of wild boar and domestic pig meat in food is challenging because it is differentiation on the subspecies level. We developed and validated two singleplex real-time PCR assays targeting SNP rs81416363 on chromosome 9 and a duplex real-time PCR assay targeting SNP g.299084751 C > T in the NR6A1 gene located on chromosome 1. The singleplex real-time PCR assays led to some ambiguous results for Mangalica and Krskopolje pig breeds and wild boar individuals from Germany, the duplex real-time PCR assay particularly for the Turopolje pig breed. We demonstrate that the probability of misclassification can be substantially reduced if the results of both the singleplex real-time PCR assays and the duplex real-time PCR assay are taken into consideration. 86 (91.5%) of a total of 94 individuals, comprising 64 domestic pigs (14 different breeds and 6 cross-breeds) and 30 wild boars (from Austria, Germany, Romania, USA and Estonia), were classified correctly.

Comparison of R5 and G12 Antibody-Based ELISA Used for the Determination of the Gluten Content in Official Food Samples.

Celiac Disease (CD) is one of the most common food intolerances. It comes along with serious damage of the mucosa in the small intestine and is caused by the storage proteins-termed "gluten"-of wheat, rye, barley and possibly oats. Sensitive individuals need to stick to a strict gluten-free diet. The gluten level in food products labeled as "gluten-free", must not exceed 20 mg/kg. It is obvious that effective test methods are needed to accurately determine the gluten concentration in foods. The determination of the presence of gluten in foodstuffs is mainly done by means of an immunochemical method called ELISA (enzyme-linked immunosorbent assay). To check the suitability of a G12 antibody-based gluten detection kit for its use in official control systems a number of routine samples were tested in parallel with two different test kits, as would be done in a routine lab. The determination of the gluten content was performed on samples entering the official laboratory including samples from official control plans, commercially available and private samples to request gluten-free labels. The results obtained with the G12 antibody ELISA assay were comparable to the official R5 method. A validation of the two different methods was not part of this study.

Authenticity control of game meat products--a single method to detect and quantify adulteration of fallow deer (Dama dama), red deer (Cervus elaphus) and sika deer (Cervus nippon) by real-time PCR.

This contribution presents a single real-time PCR assay allowing the determination of the deer content (the sum of fallow deer (Dama dama), red deer (Cervus elaphus) and sika deer (Cervus nippon)) in meat products to detect food adulteration. The PCR assay does not show cross-reactivity with 20 animal species and 43 botanical species potentially contained in game meat products. The limit of quantification is 0.5% for fallow deer and red deer and 0.1% for sika deer. The deer content in meat products is determined by relating the concentration obtained with the deer PCR assay to that obtained with a reference system which amplifies mammals and poultry DNA. The analysis of binary meat mixtures with pork, a meat mixture containing equal amounts of fallow deer, red deer and sika deer in pork and a model game sausage showed that the quantification approach is very accurate (systematic error generally <25%).

Development and validation of a TaqMan real-time PCR assay for the identification and quantification of roe deer (Capreolus capreolus) in food to detect food adulteration.

In order to protect the consumer from meat adulteration it is necessary to identify and quantify the meat content in foodstuffs. Game meat is particularly susceptible for fraudulent labeling since it is more valuable than meat from domestic animals. The paper presents a TaqMan real-time PCR assay for the quantitative determination of roe deer in meat products. The assay developed does not show cross-reactivity with 23 animal and 43 plant species tested and is therefore specific for roe deer. The amplification efficiency determined by analyzing serially diluted roe deer DNA extracts was found to be 93.9%. For quantifying the roe deer content in % (w/w), a reference system based on the myostatin gene was used. The quantification strategy was validated by determining the roe deer content in model meat mixtures and a model sausage. In addition, the real-time PCR assay was applied to the analysis of commercially available meat products.