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http://dx.doi.org/10.4014/mbl.1508.08001

Development of TaqMan Probe Real-Time RT-PCR for Quantitative Detection of Porcine Transmissible Gastroenteritis Virus During the Manufacture of Biopharmaceuticals  

Lee, Jae Il (Center for Biopharmaceuticals Safety Validation, BioPS Co., Ltd. Daedeuk Valley Campus, Hannam University)
Han, Sang Eun (Department of Biological Sciences and Biotechnology, Hannam University)
Kim, In Seop (Department of Biological Sciences and Biotechnology, Hannam University)
Publication Information
Microbiology and Biotechnology Letters / v.43, no.3, 2015 , pp. 267-274 More about this Journal
Abstract
Biopharmaceuticals and the cell substrates used for their manufacture are currently tested for porcine adventitious viruses due to the widespread use of porcine trypsin in cell culture. Porcine transmissible gastroenteritis virus (PTGV) is one of the major adventitious porcine viruses causing contaminated during the manufacture of biopharmaceuticals. Therefore, rapid and sensitive detection of PTGV is essential in ensuring the safety of biopharmaceuticals. A TaqMan probe real-time RT-PCR method was developed for the quantitative detection of PTGV contamination in cell substrates, raw materials, manufacturing processes, and final products, as well as PTGV clearance validation. Specific primers for the amplification of PTGV RNA were selected, and PTGV RNA was quantified by use of a specific TaqMan probe. Specificity, limit of detection (LOD), and robustness of the method was validated according to international guidelines on the validation of nucleic acid amplification tests. The sensitivity of the assay was calculated to be 1.10 × 100 TCID50/ml. The real-time RT-PCR method was validated to be reproducible, very specific to PTGV, and robust. The established real-time RT-PCR assay was successfully applied to the validation of Chinese Hamster Ovary (CHO)-K1 cells artificially infected with PTGV.
Keywords
Porcine transmissible gastroenteritis virus; contamination; TaqMan probe real-time RT-PCR; biopharmaceuticals; cell culture; validation;
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