Erkan Mozioğlu

Comparison of DNA extraction methods for meat analysis

Preventing adulteration of meat and meat products with less desirable or objectionable meat species is important not only for economical, religious and health reasons, but also, it is important for fair trade practices, therefore, several methods for identification of meat and meat products have been developed. In the present study, ten different DNA extraction methods, including Tris-EDTA Method, a modified Cetyltrimethylammonium Bromide (CTAB) Method, Alkaline Method, Urea Method, Salt Method, Guanidinium Isothiocyanate (GuSCN) Method, Wizard Method, Qiagen Method, Zymogen Method and Genespin Method were examined to determine their relative effectiveness for extracting DNA from meat samples. The results show that the salt method is easy to perform, inexpensive and environmentally friendly. Additionally, it has the highest yield among all the isolation methods tested. We suggest this method as an alternative method for DNA isolation from meat and meat products.

The use of digital PCR to improve the application of quantitative molecular diagnostic methods for tuberculosis

BackgroundReal-time PCR (qPCR) based methods, such as the Xpert MTB/RIF, are increasingly being used to diagnose tuberculosis (TB). While qualitative methods are adequate for diagnosis, the therapeutic monitoring of TB patients requires quantitative methods currently performed using smear microscopy. The potential use of quantitative molecular measurements for therapeutic monitoring has been investigated but findings have been variable and inconclusive. The lack of an adequate reference method and reference materials is a barrier to understanding the source of such disagreement. Digital PCR (dPCR) offers the potential for an accurate method for quantification of specific DNA sequences in reference materials which can be used to evaluate quantitative molecular methods for TB treatment monitoring.MethodsTo assess a novel approach for the development of quality assurance materials we used dPCR to quantify specific DNA sequences in a range of prototype reference materials and evaluated accuracy between different laboratories and instruments. The materials were then also used to evaluate the quantitative performance of qPCR and Xpert MTB/RIF in eight clinical testing laboratories.ResultsdPCR was found to provide results in good agreement with the other methods tested and to be highly reproducible between laboratories without calibration even when using different instruments. When the reference materials were analysed with qPCR and Xpert MTB/RIF by clinical laboratories, all laboratories were able to correctly rank the reference materials according to concentration, however there was a marked difference in the measured magnitude.ConclusionsTB is a disease where the quantification of the pathogen could lead to better patient management and qPCR methods offer the potential to rapidly perform such analysis. However, our findings suggest that when precisely characterised materials are used to evaluate qPCR methods, the measurement result variation is too high to determine whether molecular quantification of Mycobacterium tuberculosis would provide a clinically useful readout. The methods described in this study provide a means by which the technical performance of quantitative molecular methods can be evaluated independently of clinical variability to improve accuracy of measurement results. These will assist in ultimately increasing the likelihood that such approaches could be used to improve patient management of TB.

Efficiency of the TK Culture System in the diagnosis of tuberculosis.

We have evaluated the efficiency of the TK Rapid Mycobacterial Culture System in isolating mycobacteria from clinical samples and in susceptibility testing. The TK Medium indicates mycobacterial growth by changing its color from red to yellow. During a 1-year period, 16,303 clinical samples were inoculated to TK selective (TK SLC) and Löwenstein-Jensen media (LJ). Mycobacteria were isolated in 2150 (13.04%) samples in at least 1 type of medium. While LJ isolated mycobacteria from 1920 (11.69%) of all samples, TK SLC isolated 2070 (12.63%). Among all positives, the isolation rates for LJ and TK SLC were 89.30% and 96.27%, respectively. Contamination of cultures by other organisms was observed in 878 (5.33%) LJ tubes and in 90 (0.55%) TK SLC tubes. On average, time-to-growth detection was 15.57 days in TK SLC and 25.14 days in LJ. The modes of time-to-growth detection were 12 and 25 days for TK SLC and LJ, respectively. The reliability of antimycobacterial susceptibility testing was checked by 36 Mycobacterium tuberculosis strains with known susceptibility patterns which were obtained from the World Health Organization collection and by participating in an external quality control program. All susceptibility results, except for a few borderline-resistant strains, were consistent with the expected susceptibility patterns. The TK Rapid Mycobacterial Culture System is a practical and reliable automated system that shortens the time required for both culture and susceptibility results. All types of TK Media are ready to use, saving time and effort as well as drastically reducing contamination during testing.

Fatty acid composition and chemotaxonomic evaluation of species of Stachys

The fatty acid composition of the seed oil of 23 Stachys taxa was analysed by GC/MS. The main compounds were found to be linoleic (27.1–64.3%), oleic (20.25–48.1%), palmitic (4.3–9.1%), stearic (trace to 5.2%) and 6-octadecynoic (2.2–34.1%) acids. The latter compound could be used as a chemotaxonomic marker of the genus Stachys. A cluster analysis was performed for comparison and characterisation of the seed oil from Stachys species.

Inter-laboratory assessment of different digital PCR platforms for quantification of human cytomegalovirus DNA

Quantitative PCR (qPCR) is an important tool in pathogen detection. However, the use of different qPCR components, calibration materials and DNA extraction methods reduces comparability between laboratories, which can result in false diagnosis and discrepancies in patient care. The wider establishment of a metrological framework for nucleic acid tests could improve the degree of standardisation of pathogen detection and the quantification methods applied in the clinical context. To achieve this, accurate methods need to be developed and implemented as reference measurement procedures, and to facilitate characterisation of suitable certified reference materials. Digital PCR (dPCR) has already been used for pathogen quantification by analysing nucleic acids. Although dPCR has the potential to provide robust and accurate quantification of nucleic acids, further assessment of its actual performance characteristics is needed before it can be implemented in a metrological framework, and to allow adequate estimation of measurement uncertainties. Here, four laboratories demonstrated reproducibility (expanded measurement uncertainties below 15%) of dPCR for quantification of DNA from human cytomegalovirus, with no calibration to a common reference material. Using whole-virus material and extracted DNA, an intermediate precision (coefficients of variation below 25%) between three consecutive experiments was noted. Furthermore, discrepancies in estimated mean DNA copy number concentrations between laboratories were less than twofold, with DNA extraction as the main source of variability. These data demonstrate that dPCR offers a repeatable and reproducible method for quantification of viral DNA, and due to its satisfactory performance should be considered as candidate for reference methods for implementation in a metrological framework.

Syntheses and evaluation of multicaulin and miltirone-like compounds as antituberculosis agents

Abstract Four multicaulin and miltirone-like phenanthrene derivatives were synthesised and evaluated as antituberculosis agents. The crucial step of the synthesis was Pschorr coupling of 4-(3-isopropyl-4-methoxyphenyl)-2-(2-aminophenyl)ethane (13) to give 2-isopropyl-3-methoxy-9,10-dihydrophenanthrene (9) and 4-isopropyl-3-methoxy-9,10-dihydrophenanthrene (9a). Compound 9 was converted to multicaulin and miltirone-like phenanthrene derivatives by further reactions. The best antituberculosis activity was exhibited by 2-isopropylphenanthrene-3-ol (11).