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    A molecular assay for quantification and simultaneous detection of soybean and poultry DNA in sausages following multi-extraction
    (Korean Society Food Science & Technology-Kosfost, 2015) Sakalar, Ergun
    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.
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    A new measurement approach of ionizing radiation in irradiated trout (Oncorhynchus mykiss) by Randomly Polymorphic DNA-Polymerase Chain Reaction
    (Springer India, 2016) Sakalar, Ergun; Mol, Suhendan
    Trout (Oncorhynchus mykiss) were irradiated at doses of 0.250, 0.500, 1, 3, 5, 7 and 9 kGy in gamma cell. DNAs were extracted from the irradiated samples before and after storage. 1ERP primers were designed, and RAPD-PCR (Randomly Polymorphic DNA-Polymerase Chain Reaction) was applied to make randomly amplifications on the DNA of the irradiated samples. Agarose gel profiles of irradiated fish were obtained to determine change of band profiles. In addition, DNA fragmentation occurring in each dose was determined by comet assay for the verification of methodology developed in this study. The molecular methodology was developed to estimate ionizing radiation (IR) level in irradiated fish. This methodology allows the analysis of the trout irradiated up to the dose limit of around 0.5 kGy and stored for a period of three months.
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    A new RAPD-PCR based analytical assay for detection of sea bass and sea bream treated with ionizing radiation
    (Korean Society Food Science & Technology-Kosfost, 2015) Sakalar, Ergun; Arman, Kaifee
    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 10A degrees 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.
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    A Rapid and Cost-Efficient Technique for Simultaneous/Duplex Detection of Listeria Monocytogenes and Escherichia Coli O157:H7 Using Real Time PCR
    (Wiley, 2016) Kaynak, Ahmet; Sakalar, Ergun
    The increasing industrial interest in easy, economical and reliable methods has led to the development and application of DNA-based methods for the detection of microbial pathogens in food. In the present article, we describe the development of a cost-efficient EvaGreen-based real-time Polymerase chain reaction (PCR) technique for simultaneous and practical detection of Listeria monocytogenes (L. monocytogenes) and Escherichia coli O157:H7 (E. coli O157:H7) in the food matrix. EvaGreen-based simplex and duplex real-time PCR showed that specific PCR products were identified by melting curve analysis, and had a reproducible T-m of 77.000.4C for L. monocytogenes and 85.60 +/- 0.2 for E. coli O157:H7.The absolute detection limit of Eva Green-based simplex and duplex real-time PCR was 0.0001 ng/L for DNA of both pathogens. The relative detection limit of the simplex and duplex technique was less than 10 cells/mL for the two pathogens. Practical ApplicationsThis study illustrates a rapid, effective and cheap identification method to simultaneously monitor two foodborne pathogens in food samples. The specificity and sensitivity of this method can be used to clearly identify these two foodborne pathogens in practical food samples based on the specific genes of the species. Hence, this detection method is applicable to surveillance measures for these two foodborne pathogens in the food production chain. Moreover, this technique is used by food control laboratories in a secure way.
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    A Simultaneous Analytical Method for Duplex Identification of Porcine and Horse in the Meat Products by EvaGreen based Real-time PCR
    (Korean Soc Food Science Animal Resources, 2015) Sakalar, Ergun; Ergun, Seyma Ozcirak; Akar, Emine
    A duplex real-time polymerase chain reaction (PCR) based assay for the detection of porcine and horse meat in sausages was designed by using EvaGreen fluorescent.dye. Primers were selected from mitochondrial 12S rRNA and 16S rRNA genes which are powerful regions for identification of horse and porcine meat. DNA from reference samples and industrial products was successfully extracted using the GI-DAGEN Multi-Fast DNA Isolation Kit. Genomes were identified based on their specific melting peaks (Mp) which are 82.5 degrees C and 78 degrees C for horse and porcine, respectively. The assay used in this study allowed the detection of as little as 0.0001% level of horse meat and 0.001% level of porcine meat in the experimental admixtures. These findings indicate that EvaGreen based duplex realtime PCR is a potentially sensitive, reliable, rapid and accurate assay for the detection of meat species adulterated with porcine and horse meats.
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    Determination of irradiation dose and distinguishing between irradiated and non irradiated fish meat by real-time PCR
    (Elsevier Sci Ltd, 2015) Sakalar, Ergun; Mol, Suhendan
    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. (C) 2015 Elsevier Ltd. All rights reserved.
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    Development of a traceable molecular hygiene control method (TMHCM) for human DNA content in foods
    (Elsevier Sci Ltd, 2017) Sakalar, Ergun; Ergun, Seyma Ozcirak; Pala, Cigdem; Akar, Emine; Atasoglu, Cengiz
    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 0.00001 ng 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. (C) 2016 Published by Elsevier Ltd.
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    Molecular DNA-based detection of ionising radiation in meat
    (Wiley, 2017) Sakalar, Ergun
    BACKGROUNDIonising radiation induces molecular alterations, such as formation of ions, free radicals, and new stable molecules, and cleavage of the chemical bonds of the molecules present in food. Irradiation-treated meat should be labelled to control the process and to ensure free consumer choice. Therefore, sensitive analytical methods are required to detect the irradiation dose. RESULTSMeat samples were exposed to radiation doses of 0, 0.272, 0.497, 1.063, 3.64, 8.82 and 17.42 kGy in an industrial Co-60 gamma cell. Primers were designed to amplify 998, 498 and 250-base pair (bp) regions of the 18S rRNA gene of nuclear DNA from the irradiated samples. A new DNA-based method was developed to quantify the radiation exposed to the unstored meat and the meat stored at -20 degrees C for 3 and 6 months. The method was able to detect meat samples stored and unstored with dose limits of 1.063 and 3.64 kGy, respectively. CONCLUSIONThe level of irradiation can be detected using primer pairs that target particularly different-sized sequences for DNA amplification by PCR. This method can be widely used for the analysis of not only meat samples, but also all biological materials containing DNA. (c) 2016 Society of Chemical Industry
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    Practical Molecular Detection Method of Beef and Pork in Meat and Meat Products by Intercalating Dye Based Duplex Real-Time Polimerase Chain Reaction
    (Taylor & Francis Inc, 2016) Sakalar, Ergun; Kaynak, Ahmet
    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((R)) Multi-Fast DNA Isolation Kit. Identification of genomes was accomplished in the same tube by their distinctive melting peak, which was 87.5 degrees C for pork and 80.5 degrees C for beef, respectively. The detection limit of the method was 0.01 ng/mu L 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.