The base composition of PCR products derived from sodium bisulfite-modified templates is methylation dependent. Hence, methylated and unmethylated, PCR products show different melting profiles when subjected to thermal denaturation. The methylation-sensitive high-resolution melting (MS-HRM) protocol is based on the comparison of the melting profiles of PCR products from unknown samples with profiles specific for PCR products derived from methylated and unmethylated control DNAs. The protocol consists of PCR amplification of bisulfite-modified DNA with primers designed to proportionally amplify both methylated and unmethylated templates and subsequent high-resolution melting analysis of the PCR product. The MS-HRM protocol allows in-tube determination of the methylation status of the locus of interest following sodium bisulfite modification of template DNA in less than 3 h. Here, we provide a protocol for MS-HRM, which enables highly sensitive, labor- and cost-efficient single-locus methylation studies on the basis of DNA high-resolution melting technology.

The possibility of the adulteration of meat products with seagull meat disturbs people living in coastal cities. In order to eliminate the suspicions of consumers a sensitive and reliable method is needed for the detection of seagull meat. In order to identify and quantify seagull meat in meat mixtures a real-time polymerase chain reaction (PCR) assay, using species-specific primers and a TaqMan probe was designed on the mitochondrial NADH dehydrogenase subunit 2 gene. In addition, it was possible to detect the template DNA of seagull at the level of 100 pg without any cross-reactivity with non-target species (bovine, ovine, donkey, pork, horse, chicken, turkey, goose, duck). Also, the method was capable of detecting seagull meat at the level of 0.1% in raw and heat-treated test mixtures, prepared by mixing seagull meat with beef and chicken at different levels (0.01-10%). In conclusion, it can be suggested that the Real-time PCR assay used in this research could be a rapid and sensitive method for the routine identification of seagull meat in raw or cooked meat products.

Brucellosis is a worldwide zoonotic disease transmitted to humans by consumption of contaminated milk and milk products. Brucellosis is endemic in Turkey, and Edirne has a high Brucella prevalence. Brucellosis is prevented by live-attenuated vaccines for animals and the vaccination program has been in place since 1984 in Turkey. Thrace is the pilot region for this vaccination program. The gold standard diagnostic technique for brucellosis is still the isolation of suspicious bacterial colonies followed by bacteriological identification, but it is very time consuming and laborious. In many studies, Brucella has been investigated by PCR techniques. However, PCR-based methods cannot differentiate between the vaccine strain and the virulent strain; thus, the vaccine strain may interfere with the virulent strain and causes false-positive reactions. To monitor brucellosis control programs effectively, it is important to distinguish vaccine and field strains of Brucella spp. In this study, raw milk samples were collected from 99 cows at 12 different barns in 5 villages of Edirne (Turkey). Bacteriological analyses and real-time quantitative (q)PCR experiments were applied to all samples. The DNA was isolated using Biospeedy DNA-Tricky Purification Kit (Bioeksen, Istanbul, Turkey). For all reactions, Roche Light Cycler Nano (Roche Diagnostics, Mannheim, Germany) instrument and Biospeedy EvaGreen qPCR Pre-Mix (Bioeksen) were used. The data were analyzed using Roche LightCycler® NanoSoftware 1.0. For samples that were negative by bacteriological analyses and positive by qPCR, we developed a novel qPCR-based method to differentiate the virulent B. abortus strains and B. abortus S19 vaccine strain. We designed qPCR primers targeting the outer membrane protein of B. abortus. The qPCR products were sequenced using the ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction Kit on an ABI Prism 377 DNA sequencer (Applied Biosystems, Foster City, CA). In total, 2.02% of the samples were Brucella positive, by both bacteriological method and the novel qPCR method. We concluded that, to obtain true-positive results in Brucella spp. screening studies for milk, differentiating the virulent and vaccine strain should not be disregarded.

Nanosilver has well-known antibacterial properties, and is widely used in daily life as various medical and general products. In comparison with silver ion, there is serious lacking of information concerning the biological effects of nanoAg. In this study, we observed the cytotoxic effect of nanoAg in HeLa cells. The nanoAg-induced cytotoxicity was lower than that of AgNO₃, used as a silver ion source. Apoptosis evaluated by flowcytometric analysis was associated with this cell death. Further, the expressions of ho-1 and mt-2A, well-known oxidative stress-related genes, were up-regulated by nanoAg treatment. Our results showed that nanoAg possesses the potential for cytotoxicity, therefore, in the case of exposure at high concentrations, we should consider to protect from nanoAg-induced toxicity.

Use of the real-time polymerase chain reaction (PCR) to amplify cDNA products reverse transcribed from mRNA is on the way to becoming a routine tool in molecular biology to study low abundance gene expression. Real-time PCR is easy to perform, provides the necessary accuracy and produces reliable as well as rapid quantification results. But accurate quantification of nucleic acids requires a reproducible methodology and an adequate mathematical model for data analysis. This study enters into the particular topics of the relative quantification in real-time RT-PCR of a target gene transcript in comparison to a reference gene transcript. Therefore, a new mathematical model is presented. The relative expression ratio is calculated only from the real-time PCR efficiencies and the crossing point deviation of an unknown sample versus a control. This model needs no calibration curve. Control levels were included in the model to standardise each reaction run with respect to RNA integrity, sample loading and inter-PCR variations. High accuracy and reproducibility (<2.5% variation) were reached in LightCycler PCR using the established mathematical model.