IDENTIFICATION OF THE COMMON CARP (CYPRINUS CARPIO) SPECIES USING REAL-TIME PCR METHODS

Before being put out onto the market many fish species sold around the world need to be processed, which may result in the subsequent removal of characteristics used for their classification (head, fins, internal organs). The biochemical characterization of fish species could be achieved using proteins or DNA sequences as species-specific markers. However, since different fish products undergo different processes, the method of analysis has to be chosen according to the modifications undergone by fish constituents during processing. As DNA molecules are more resistant than proteins to various processes (including thermal treatment), DNA analysis appears to be a promising method for fish species identification. For the species identification of the Common Carp (Cyprinus carpio) among 15 different freshwater fish species a specific pre-designed molecular - genetic marker of Common Carp (Cyprinus carpio) was used, which comes from the mtDNA control D - loop area. Next we analyzed the presence of mtDNA in DNA isolates of the 15 kinds of freshwater fish and compared them with the Common Carp markers by using the following two PCR identification methods. The isolates were diluted to 10 % concentration, using the TaqMan Real-Time PCR method and the SYBR (R) Green Real-Time PCR method. The results of using the optimized the SYBR (R) Green Real-Time PCR method for species identification of the Common Carp (C. carpio) pointed to its suitability. We were able to create an analysis of the monitored standard curve which represented the PCR positive control (C. carpio), containing the characteristic melting peak (up to the melting point 80.72 degrees C). A single peak indicated a single product (C. carpio) which can be verified upon characterization of the PCR product by agarose gel electrophoresis. The TaqMan Real-Time PCR method with a TaqMan probe is a very sensitive and reliable method of authentication used on food of animal origin. The suitability of this method, which we used for species identification of the Common Carp (C. carpio), was confirmed. Thanks to using this method, already in the 17th cycle of the PCR amplification procedure, the presence of the Common Carp gene (C. carpio) was detected in the positive control and not detected in the rest of the fish samples.