In this study, real-time polymerase chain reaction (PCR) was used for identifying the effects of different temperatures and times of heat treatment on the DNA of meat products. For this purpose, beef, pork, and chicken were baked at 200 °C for 10, 20, 30, 40, 50 min, and for 30 min at 30, 60, 90, 120, 150, 180, 210 °C and also cooked by boiling at 99 °C for 10, 30, 60, 90, 120, 150, 180, 210, and 240 min. The DNA was then extracted from all samples after the heat treatment. Further, a region of 374, 290, and 183-bp of mitochondrial DNA of beef, pork, and chicken, respectively, was amplified by real-time PCR. It was found that baking and boiling of the beef, pork, and chicken resulted in decreases in the detectable copy numbers of specific genes, which varied with the heating time and degree. The results indicated that species determination and quantification using real-time PCR are affected by the temperature, duration of the heat treatment, and size of the DNA fragment to be amplified.

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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 GIDAGEN® Multi-Fast DNA Isolation Kit. Genomes were identified based on their specific melting peaks (Mp) which are 82.5℃ and 78℃ 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 real-time 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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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 77.00 6 0.4C for L. monocytogenes and 85.60 6 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/lL 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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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.