Repository of Research and Investigative Information

Repository of Research and Investigative Information

دانشگاه علوم پزشکی و خدمات بهداشتی درمانی زنجان

Quantitative real-time PCR technique for the identification of E. coli residual DNA in streptokinase recombinant product

(2017) Quantitative real-time PCR technique for the identification of E. coli residual DNA in streptokinase recombinant product. Preparative Biochemistry & Biotechnology. pp. 986-989. ISSN 1532-2297 (Electronic) 1082-6068 (Linking)

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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 (10(1) to 10(7) copies/microL) was tested in triplicate. The sensitivity of the test was determined to be 101 copies/microL or 35 fg/microL. 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.

Item Type: Article
Keywords: Escherichia coli/chemistry/*genetics Genome, Bacterial Genome, Human Humans Limit of Detection RNA, Bacterial/*analysis/genetics Real-Time Polymerase Chain Reaction/*methods Recombinant Proteins/chemistry/*genetics Streptokinase/chemistry/*genetics E. coli real-time PCR recombinant streptokinase residual DNA
Page Range: pp. 986-989
Journal or Publication Title: Preparative Biochemistry & Biotechnology
Abstract and Indexing: ISI, Pubmed, Scopus
Quartile : Q4
Volume: 47
Number: 10
Identification Number: https://doi.org/10.1080/10826068.2017.1365251 http://www.ncbi.nlm.nih.gov/pubmed/28857670
ISSN: 1532-2297 (Electronic) 1082-6068 (Linking)
Depositing User: خانم مریم زرقانی
URI: http://repository.zums.ac.ir/id/eprint/302

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