Comparative Analysis of Verification Parameters of Event-Specific Methods in GMO Maize

The second most dominant genetically modified (GM) crop is maize. Increasing number of GM maize events puts significant pressure on GMO testing laboratories to achieve the level of competence necessary to fulfill legal requirements. In the European Union, Polymerase Chain Reaction (PCR) is the method of choice for identification and quantification of GMOs. We performed verification of validated methods for identification of four GM maize events. Additionaly we aimed to explore the option of designing a method for simultaneous detection of these events in a multiplex PCR reaction. DNA was extracted from certified reference materials (CRM) using validated CTAB extraction protocol. Concentration of DNA was measured using Qubit dsDNA Broad Range Assay. Amplification of taxon specific marker for maize and all event-specific methods was performed according to the JRC Compendium of Reference Methods for GMO Analysis. Absolute limit of detection (LODabs) was determined for taxon specific and four event specific RealTime PCR based methods. DNA extracted from CRMs showed sufficient concentration for downstream analyses and preparation of dilutions for determination of LODabs. Determined LODabs for all tested methods meet acceptance criteria. As expected, the methods performance with respect to the repeatability and precision decline with the decrease in concentration of the target. Event-specific GA21 and NK603 methods show high Ct values at the determined LODabs. However, by adjusting the concentrations of primers and probes sensitivity of these two methods should be improved. Considering that the amplicons for all five methods are quite short (<120 bp) optimization of multiplex reaction conditions for simultaneous amplification should be feasibl

SCREENING FOR GMO IN FERMENTED SOY SAUCE

Soy sauce is worldwide popular condiment of Asian origin. With the advent of GM soybean production, soy sauce drew the interest of food safety control. Samples collected for inspection are generally of industrial grade soy sauce type, which is produced from hydrolyzed soybean and grain. Following the failure to perform RealTime PCR based GMO screening on a number of submitted samples we tested our screening system on soy sauce produced following traditional method based on fermentation. Four batches of soy sauce were produced and DNA extracted. DNA concentration ranged from 32,68 to 65,36 ng/μl. Amplification of taxon specific target was successful with rather high Ct ( > 30). Promoter P-35S sequence was not detected, but T-NOS was detected in three samples with values reaching or exceeding LOD of the method. The results show that it is possible to detect transgenic elements in traditionally produced soy sauce while DNA extraction from industrial grade soy sauce is not possible

Overview of Human Population-Genetic Studies in Bosnia and Herzegovina during the Last Three Centuries: History and Prospective

Modern Bosnia and Herzegovina is a multi-ethnic and multi-religion country, with a very stormy history. Certain archaeological findings indicate continuous population of its territory since the Paleolithic. In time, vast number of different factors jointly influenced fascinating diversity of local human populations. A great number of small, more or less isolated, indigenous populations, make this area quite attractive for population-genetic surveys of different levels and approaches. Austro-Hungarian military physicians conducted the very first known bio-anthropological analyses of Bosnia-Herzegovina population at the end of the 19th century. Thus, the first step towards resolving the genetic structures of local B&H human populations was made. The studies that followed (conducted throughout most of the 20th century) were primarily based on the observation of various phenotypic traits. This stage was followed by the examination of various cytogenetic and fundamental DNA based molecular markers. The efforts undertaken over the last three centuries revealed »human genetic treasure« in Bosnia and Herzegovina. However, even now, after all the studies that were conducted, many interesting features remain to be discovered and described within the existing local human populations

Incidence of horse meat in processed food on B&H market

Following the “horse meat scandal” in 2013, European Union countries have conducted official control of EU market and unraveled food fraud which implicated a number of processed food products and food businesses. Five years after the breakout of the scandal, no official information on market surveillance in Bosnia and Herzegovina is available. Therefore, 73 randomly selected meat products from retail were collected and analyzed for the presence of horse DNA. Horse DNA was detected in 21 products (28.77%). Particularly disturbing for B&H consumers is high proportion of sujuk samples positive for horse DNA (46.15%) with lower incidence among the products of small manufactures. Also disturbing is the finding that 71.43% of the products that contain horse DNA were produced in B&H. According to our data there is a requirement for stricter surveillance of both import and internal market

Effect of War and Postwar Genotoxins on Micronuclei Frequency in Sarajevo Study Group

During the 1992-1995 siege, as well as after the war activities, citizens of Sarajevo were most probably exposed to various potential genotoxic agents. The effects of those potential genotoxins were evaluated by micronucleus-cytokinesis blocked assay. The study included 30 individuals who resided in the area of Sarajevo during the war and the postwar period. Point bi-serial coefficient analysis did not reveal any relationship between the frequencies of binuclear cells with micronuclei as well as total number of micronuclei and smoking habits or gender. Simple linear regression revealed statistically significant positive correlation between the age and micronuclei formation. Due to the war related environmental contamination more extensive study is recommended

Effect of War and Postwar Genotoxins on Micronuclei Frequency in Sarajevo Study Group

During the 1992-1995 siege, as well as after the war activities, citizens of Sarajevo were most probably exposed to various potential genotoxic agents. The effects of those potential genotoxins were evaluated by micronucleus-cytokinesis blocked assay. The study included 30 individuals who resided in the area of Sarajevo during the war and the postwar period. Point bi-serial coefficient analysis did not reveal any relationship between the frequencies of binuclear cells with micronuclei as well as total number of micronuclei and smoking habits or gender. Simple linear regression revealed statistically significant positive correlation between the age and micronuclei formation. Due to the war related environmental contamination more extensive study is recommended

Genetic diversity of Thoroughbred horse population from Bosnia and Herzegovina based on 17 microsatellite markers

The focus of this study was on genetic diversity of TB horse population raised in B&H. Genomic DNA was genotyped by using 17 microsatellite markers. A total of 103 alleles were detected. The average number of alleles per locus was 6.059 and effective number of alleles was 3.293. Means of observed and expected heterozygosity were calculated 0.645 and 0.696, respectively. The average PIC values was 0.649 and inbreeding coefficient was 0.090. Based on all observed parameters, ASB2 locus showed the highest genetic diversity while locus HMS2 was the least diverse. These results suggest that the population of TB horses from B&H is not affected by substantial loss of genetic diversity, indicating the presence of reasonably high level of genetic variability

Optimisation of Forensic Genetics Procedures Used in Disputed Paternity Testing: Adjustment of the PCR Reaction Volume

Standard molecular techniques, with only a slight modification, are very useful in obtaining and interpreting the final results in the field of forensic genetic. Data obtained through such analysis are highly reliable and can be used as a very powerful tool that produces valuable results. However, success and swiftness of DNA typing of biological evidence either that found at a crime scene or used in disputed paternity testing, depends on the optimization of numerous factors. One of the most important and critical phases that ensures reliability of the whole procedure is the choice of the most suitable volume for the amplification protocol. Buccal swabs were collected from volunteers. DNA was extracted by Qiagen Dnaeasy Tissue Kit. PowerPlex 16 kit was used to simultaneously amplify 15 STR loci by PCR. Amplification was carried out as described previously. The tested total working reaction volumes were 5, 10 and 25 microl. The PCR amplification was carried out in PE Gene Amp PCR System Thermal Cycler (ABI, Foster City, CA). Amplification products were analyzed on an ABI PRISM 377 instrument (ABI, Foster City, CA) in 5% bis-acrilamide gel. Amplification was generally successful for all the tested reaction volumes. Lower partial to complete DNA profiles ratio, the quality of obtained STR profiles, significantly reduced amount of reaction's components give advantage to 5 microl reaction volume over other two tested volumes in this case