Meat Species Identification Using PCR-RFLP and Native Capillary Electrophoresis System

Introduction: For consumers protection it is necessary to develop effective methods able to verify species composition of different food products. These methods mostly rely on DNA analysis using PCR because it allows unequivocal species identification from raw as well as processed foods. The DNA fragment obtained by amplification of a specific gene is digested with restriction enzymes (PCR-RFLP) to discriminate the species. Usually, a region of the mitochondrial genome, such as tRNAGlu/cytochrome b, is amplified, followed by digestion with a number of restriction endonucleases sufficient for species identification.

Purpose: The purpose of this study was to optimize and validate an efficient PCR-RFLP protocol for meat species identification using automated detection and data analysis of the QIAxcel Advanced system.

Methods: After meat homogenization and lysis, DNA purification was performed using QIAsymphony and Qiasymphony DSP DNA mini kit. Following PCR, the resulting product of 359 bp was digested with four restriction enzymes: AluI, HaeIII, HinfI and RsaI. The digests were analyzed by native capillary electrophoresis and the results were interpreted utilizing a programmed Excel sheet.

Results: Raw meat was used, as well as the meat processed by cooking, freezing, cooking and smoking, dehydration and sterilization. The method was tested on 108 reference samples containing either only one meat (80) or mixed meat species (28). For validation, the samples were analyzed by real-time PCR and the results were compared to the data obtained by PCR- RFLP following the analysis by QIAxcel ScreenGel software.  The two methods gave 100% congruent results.

Significance: Using the native and automated capillary electrophoresis significantly reduces the analysis time and minimizes the potential procedural errors, thus providing reliable and fast method for meat species identification, especially for large-scale analyses encountered in the routine food control of meat and meat products.