Application Guide - QIAxcel Advanced

QIAxcel Advanced Application Guide 10/2016 121 2. RFLP-PCR allows the identification of various mammal, bird, and fish species (5). Wolf et al. developed a method that allows recognition of 25 animal species. The DNA fragment obtained by amplifying a specific region of the mitochondrial genome (tRNAGlu/cytochrome b) is treated with 11 different restriction endonucleases. 3. Real-time PCR allows quantitative contamination assessment, including the identification of meats of different, even closely related, animal species. It is very efficient in detecting traces of specific animal DNA, even if the DNA has been degraded during a meat preparation process. As such, it serves an excellent screening tool for high- throughput routine testing where the target is known. RFLP-PCR satisfies crucial aspects such as specificity, sensitivity, flexibility, and efficiency. Previous studies demonstrated that RFLP-PCR (5, 6, 7, 8) is successful in identifying meat species. Sample analysis using slab gel electrophoresis is unsuitable for routine work because the method is time-consuming, requires more manual handling, and uses hazardous products such as ethidium bromide. The results are difficult to interpret and may require specific software. Native capillary electrophoresis with the QIAxcel Advanced system overcomes these issues. The automated procedure is fast and inexpensive. The QIAxcel ScreenGel® Software analyzes the electrophoresis data and provides the sizes and concentrations, so no other software is needed. The purpose of this study was to optimize a procedure using RFLP-PCR in conjunction with the QIAxcel Advanced system for use in rapid (results in less than 8 h) and accurate routine analyses. The method involves amplification of a 359-bp product that is common to all vertebrates, followed by one or more enzymatic digestions. The proportion of individual meats could range from 1 to 99%. By applying four enzymes, it was possible to distinguish meat from 15 different animals. In addition, 6 animals served as contamination markers. Criteria for optimization We defined four criteria for optimizing the meat authentication method: sensitivity, flexibility, speed, and simplicity. The limit of detection (LOD) should range between < 0.5% and 1%. Several different pure animal samples were tested and a sensitivity of 0.01% was validated for beef, pork and chicken. The meat was tested either as a water dilution or in a maize mixture. The method should be suitable for a broad range of sample material to give maximum flexibility of application. It should take less than one working day to receive results as longer delays lead to higher costs for food producers. Finally, since the method is intended for routine analyses, it should be easy to implement and perform. If RFLP-PCR is intended for species identification, the reference samples must be analyzed using the same procedure and QIAxcel DNA kit as the unknown samples. The disadvantage of this technique is the possibility of incomplete digestion. In such cases, other methods, such as real-time PCR and/or sequencing, should be used for confirmation.

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