Ergün Şakalar

Determination of irradiation dose and distinguishing between irradiated and non irradiated fish meat by real-time PCR.

In this study, the effects of gamma irradiation on the DNA of fish (Oncorhynchus mykiss) by real-time PCR were studied. Fish (O. mykiss) were exposed to radiation doses of 0.250, 0.500, 1, 3, 5, 7, and 9 kGy in a gamma cell. Primers were designed for regions with different lengths of both nuclear and mitochondrial DNA, and each primer was used to amplify the DNA from irradiated samples. The amplicon curves for mitochondrial and nuclear DNA, and the correlations among the curves, were obtained. The Ct values for a 519 bp region of the 18S RNA gene on nuclear DNA correlated appropriately. Radiation doses applied to the fillets were estimated using the standard curve data obtained from the correlation values, and the DNA damage caused by each dose was calculated. As a consequence, a molecular methodology to analyze irradiated fish meat qualitatively and also for the estimation of administered dose was developed. This method allowed analysis of irradiated fish, which had been stored for up to 3 months with a dose limit of approximately 0.5 kGy.

Practical Molecular Detection Method of Beef and Pork in Meat and Meat Products by Intercalating Dye Based Duplex Real-Time Polimerase Chain Reaction

In this study, a rapid, specific, and low-cost duplex-detection technique of pork and beef was developed by a real-time polimerase chain reaction assay based on fluorescence. Deoxyribonucleic acid was extracted from variable mixtures of pork and beef in sausage and industrial products to develop the duplex assay using the GIDAGEN® Multi-Fast DNA Isolation Kit. Identification of genomes was accomplished in the same tube by their distinctive melting peak, which was 87.5°C for pork and 80.5°C for beef, respectively. The detection limit of the method was 0.01 ng/µL deoxyribonucleic acidor 0.001% target pork and beef in sausage. The results showed that the intercalating dye based duplex real-time polimerase chain reaction is a potentially sensitive, reliable, and practical assay for the detection of meat species adulterated with beef and pork.

A new RAPD-PCR based analytical assay for detection of sea bass and sea bream treated with ionizing radiation

Random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) was used to evaluate the effects of irradiation on the DNA of sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata). Meats of sea bass and sea bream were exposed to 0.25, 0.5, 1, 3, 5, and 7 kGy of irradiation (dosage rate 1.31 kGy/h) in a gamma cell at room temperature of approximately 10°C. RAPD-PCR was carried out using DNA samples of both control and irradiated groups. Gel electrophoresis showed loss of some amplicons, especially in the upper long bands of irradiated samples. Due to DNA damage caused by irradiation, the visibility of bands decreased as the radiation dosage increased. This study can be used as a basis for screening of irradiated samples based on loss of specific bands for a specific amount of irradiation.

A molecular assay for quantification and simultaneous detection of soybean and poultry DNA in sausages following multi-extraction

A duplex PCR assay was designed for simultaneous identification of soybean and poultry in sausages. A Multi-Fast DNA Isolation Kit was used for simultaneous extraction of amplifiable soybean and poultry DNA. Primers generated specific DNA fragments of 173 and 183 bp in length for soybean and poultry, respectively. The duplex assay sensitivity was 0.35% for specific detection of poultry mixed with soybeans and other additive materials and was 0.05% for specific detection of soybeans mixed with poultry and other additive materials. The optimized duplex PCR assay was applied to 15 commercial sausages. Unintentional and intentional contaminants were analyzed using real-time PCR based Sakalar Quantification Table of DNA (SQT-DNA). Techniques developed here are suitable tools for qualitative and quantitative comparison of sausage contents.

Development of a traceable molecular hygiene control method (TMHCM) for human DNA content in foods.

The aim of this study was to develop a molecular technique to determine the level of human originated DNA contamination in unhygienic food products. In the study, four model foods were prepared under both hygienic (H) and non-hygienic (NH) conditions and the human originated microbial loads of these products were determined. DNA was extracted from the model foods and human buccal samples by GIDAGEN Multi-fast DNA isolation kit. A primer specific region of human mitochondrial D-Loop was designed. The level of human DNA contamination in the model foods was determined by real-time PCR. The sensitivity of the technique developed here was 0.00001ng DNA/PCR. In addition, the applicability of the traceable molecular hygiene control method (TMHCM) was tested in 60 food samples from the market. The results of this study demonstrate that DNA based TMHCM can be used to predict to what extent foods meet the human oriented hygienic conditions.