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    Complete genome sequences and phylogenetic analysis of hepatitis delta viruses isolated from nine Turkish patients
    (Springer Wien, 2011) Celik, Inci; Karatayli, Ersin; Cevik, Emrah; Kabakci, S. Gokce; Karatayli, Senem Ceren; Dinc, Bedia; Cmar, Kubilay
    Hepatitis delta virus (HDV) is a subviral agent of hepatitis B virus (HBV), and its life cycle is dependent on HBV. It is commonly accepted that HDV has eight distinct genotypes. In this study, the complete nucleotide sequences of HDV genomes isolated from nine Turkish patients were obtained by RT-PCR using two pairs of primers that cover the entire HDV genome. PCR products were sequenced directly. The results showed that these 9 isolates were approximately 1680 base pairs in length and clustered in the genotype HDV-1 branch when phylogenetic analysis was done with the sequences together with the complete sequences of HDV genomes representing each genotype retrieved from GenBank. Analysis of a portion of the large hepatitis D antigen (L-HDAg) gene showed that sequence similarity among these Turkish isolates is between 87.4 and 97.1%, and the Turkish isolates have the most sequence similarity to HDV-1 (90.5%), while they have the least sequence similarity to HDV-3 (64.1%). Full-genome analysis indicates that the sequence similarity is between 80.7 and 95.4%, and the highest sequence similarity is 84.8% (between the Turkish isolates and HDV-1). The lowest sequence similarity is 56.4% (between the Turkish isolates and HDV-3). In conclusion, phylogenetic analysis shows that the Turkish HDV isolates belong to HDV-1.
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    Evaluation of 2015-2016 MOTAKK HBV DNA and HCV RNA External Quality Assessment National Program Results
    (Ankara Microbiology Soc, 2018) Karatayli, Ersin; Soydemir, Ege; Aksoy, Zeynep Busra; Kizilpinar, Mehtap; Altay Kocak, Aylin; Karatayli, Senem Ceren; Yurdcu, Esra
    MOTAKK, as a national external quality control program has been launched to evaluate the molecular detection of viral infections including HBV DNA and HCV RNA in molecular microbiology diagnostic laboratories in Turkey. This program is prepared in compliance with ISO 17043:2010 (Conformity assessment general requirements for proficiency testing) standards, and aims to take the place of external quality control programs from abroad, contributing to standardization and accuracy of molecular diagnostic tests in our country. The aim of this study was to evaluate 2015 and 2016 results of the MOTAKK External Quality Control Program for HBV DNA and HCV RNA viral load. The calls were announced on the web page of MOTAKK (www.motakk.org). The quality control samples were sent to participating laboratories in 2015 and 2016. Main stocks were prepared from patients with chronic hepatitis B and C who had viral load detection with reference methods according to WHO reference materials for viral load studies to improve quality control sera. From these main stocks, samples with different viral loads were prepared from dilutions of plasma with HBV, HCV, HAV, HIV, Parvovirus B19 and CMV negative serologic markers. Quality control samples were sent to the participating laboratories along with the negative samples in the cold chain. The laboratories accomplished the related tests within 2-3 weeks and entered their results on the MOTAKK web page. These results were analysed according to ISO 13528 (Statistical methods for use in proficiency testing by interlaboratory comparison) and scoring reports were created by a software developed by MOTAKK and sent to participating labs. Each laboratory evaluated their own results in comparison with the other laboratory results, reassessed the tests via observing the distance from the mean result and the reference values. The number of laboratories participating in the HBV DNA and HCV RNA external quality control program was 70-73 in 2015-2016. Participants were able to comply with the program tools, registering, entering results and receiving the results reports problem. In HBV panel, 72.6-89.1% and 84.7-90.3% of the participant laboratories were in 1 standard deviation (SD) in 2015-2016, respectively. In HCV panel, 70.8-89.1% and 84.7-90.3% of the participant laboratories were in 1 SD in 2015-2016, respectively. A national external quality control program for HBV DNA and HCV RNA in Turkey has been prepared for the first time with this project and implemented successfully. All the data provided in the MOTAKK external quality control program final report, compensate all the data provided by the quality control program final reports from abroad; additionally, the report allows comparison of used technologies and commercial products.
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    A one step real time PCR method for the quantification of hepatitis delta virus RNA using an external armored RNA standard and intrinsic internal control
    (Elsevier Science Bv, 2014) Karatayli, Ersin; Altunoglu, Yasemin Celik; Karatayli, Senem Ceren; Alagoz, S. Gokce K.; Cinar, Kubilay; Yalcin, Kendal; Idilman, Ramazan
    Background: Hepatitis delta virus (HDV) RNA viral load measurement is critical in diagnosis and monitoring the response to antiviral treatment. Objectives: Our aim is to design a real time PCR method for accurate quantitation of HDV RNA in clinical specimens using an armored RNA as external standard, and an intrinsic internal control. Study design: A plasmid bearing delta antigen region of genotype I HDV genome was used to develop an armored RNA. Serial dilutions of the armored HDV RNA standard with 10(12) copy/mL were used as standards for quantitation. A primer-probe set derived from HDAg region was used in one step EZ RT PCR kit chemistry which uses rTth enzyme allowing reverse transcription and polymerization in the same tube. The kit also uses the advantage of uracil-N-glycosylase (UNG) enzyme treatment to prevent PCR contamination. Results: The established assay has a dynamic range of 10(2)-10(11) copy/mL with a PCR efficiency of 96.9%. Detection limit was 858 +/- 32 copy/mL with 95% confidence interval. Intra-and inter-assay variabilities were low for high, medium and low levels of viremia. Incorporation of freely circulating GAPDH in serum into the assay as an intrinsic internal control prevented false negative results and failures in PCR amplifications due to inhibitors, inefficient extraction procedures or enzymatic reactions. Conclusion: In conclusion, this study defines a novel assay for sensitive and reliable quantification of HDV RNA using an armored HDV RNA as a standard and GAPDH in plasma or serum as an intrinsic internal control in a single tube. (c) 2014 Elsevier B.V. All rights reserved.