Application Guide - QIAxcel Advanced

122 QIAxcel Advanced Application Guide 10/2016 A B Materials and methods Meat homogenization was followed by lysis with chemical (QIAGEN ATL lysis buffer), thermal (up to 65°C), and mechanical (1400 rpm agitation) treatment for up to one hour (internal method). DNA purification was performed on a QIAsymphony® SP using the QIAsymphony DSP DNA mini kit. PCR was performed as previously described (7, 8) using TopTaq Master Mix (QIAGEN) and the primersCYT b1 and CYT b2. This yielded a product of 359 bp that is common to all vertebrates. The PCR product was digested with different enzymes as previously described (7), and the digests were analyzed with the QIAxcel system, which provided information about the size and concentration of the products. The results were further interpreted using a programmed Excel sheet. The sample analysis was performed according to method OM500 for the QIAxcel Advanced system, using the QIAxcel High Resolution Kit, QX Alignment Marker 15 bp/600 bp and QX DNA Size Marker 25 – 500 bp. Results and discussion We used the PCR-RFLP approach where the amplified mitochondrial DNA region encoding cytochrome b (7) undergoes enzyme digestion for species identification. This was achieved using four restriction enzymes: AluI, Hae III, Hinf I, and RsaI. The meat was tested raw and after processing methods, such as cooking, freezing, reheating, smoking, dehydration, and sterilization. The results proved that the PCR-RFLP method can successfully identify and authenticate the species of meat used in commercial products subjected to various processing. The method was first tested on a range of reference samples containing either a single meat or mixed meat species. Initially, the samples were analyzed with real-time PCR and the results were compared to data obtained using RFLP-PCR to validate the procedure and establish whether detection using the QIAxcel system was comparable to detection using real-time PCR. A number of pure and mixed animal products were used as references (Figure 1). The number of enzymes needed for the analyses was species-specific. For example, AluI is sufficient to detect cattle DNA but not that of the other species. In our study, a maximum of four enzymes was needed to identify all of the animals tested. A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 Figure 1. Successful identification of meat species using the QIAxcel Advanced System. The analysis of the 359-bp PCR fragment digested with A. HinfI enzyme and B. HaeIII enzyme. The 359-bp fragment was amplified from nine reference samples. The starting material was fresh meat. Lane 1: Cattle; Lane 2: Pig; Lane 3: Sheep; Lane 4: Deer; Lane 5: Rabbit; Lane 6: Chicken; Lane 7: Duck; Lane 8: Turkey; Lane 9: Goose. Only two mixed samples were prepared in the laboratory: Lane 10: Beef and pork and Lane 11: Chicken and turkey.

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