Quantitative real-time PCR technique for the identification of E. coli residual DNA in streptokinase recombinant product |
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Authors: | Mansoureh Fazelahi Vahid Kia |
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Affiliation: | 1. Department of Genetics Biology, Ashkezar Branch, Islamic Azad University, Yazd, Iran;2. Department of Medical Biotechnology, Faculty of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran |
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Abstract: | Recombinant streptokinase is a biopharmaceutical which is usually produced in E. coli. Residual DNA as a contamination and risk factor may remain in the product. It is necessary to control the production procedure to exclude any possible contamination. The aim of the present study was to develop a highly specific and sensitive quantitative real-time PCR-based method to determine the amount of E. coli DNA in recombinant streptokinase. A specific primers and a probe was designed to detect all strains of E. coli. To determine the specificity, in addition to using NCBI BLASTn, 28 samples including human, bacterial, and viral genomes were used. The results confirmed that the assay detects no genomic DNA but E. coli’s and the specificity was determined to be 100%. To determine the sensitivity and limit of detection of the assay, a 10-fold serial dilution (101 to 107 copies/µL) was tested in triplicate. The sensitivity of the test was determined to be 101 copies/µL or 35 fg/µL. Inter-assay and intra-assay were determined to be 0.86 and 1.69%, respectively. Based on the results, this assay can be used as an accurate method to evaluate the contamination of recombinant streptokinase in E. coli. |
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Keywords: | E. coli real-time PCR recombinant streptokinase residual DNA |
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