Real-Time RT-PCR for Quantitative Detection of Bovine Viral Diarrhoea Virus during Manufacture of Biologics

생물의약품 제조공정에서 Bovine Viral Diarrhoea Virus 정량 검출을 위한 Real-Time RT-PCR

  • Cho, Hang-Mee (Department of Biological Sciences, Hannam University) ;
  • Lee, Dong-Hyuck (Department of Biological Sciences, Hannam University) ;
  • Kim, Hyun-Mi (Department of Biological Sciences, Hannam University) ;
  • Kim, In-Seop (Department of Biological Sciences, Hannam University)
  • 조항미 (한남대학교 생명.나노과학대학 생명과학과) ;
  • 이동혁 (한남대학교 생명.나노과학대학 생명과학과) ;
  • 김현미 (한남대학교 생명.나노과학대학 생명과학과) ;
  • 김인섭 (한남대학교 생명.나노과학대학 생명과학과)
  • Published : 2008.03.28

Abstract

Bovine blood, cell, tissue, and organ are used as raw materials for manufacturing biologics such as biopharmaceuticals, tissue engineered products, and cell therapy. Manufacturing processes for the biologics using bovine materials have the risk of viral contamination. Therefore viral validation is essential in ensuring the safety of the products. Bovine viral diarrhoea virus (BVDV) is the most common bovine pathogen and has widely been known as a contaminant of biologics. In order to establish the validation system for the BVDV safety of biologics, a real-time RT-PCR method was developed for quantitative detection of BVDV contamination in raw materials, manufacturing processes, and final products. Specific primers for amplification of BVDV RNA was selected, and BVDV RNA was quantified by use of SYBR Green I. The sensitivity of the assay was calculated to be 1 $TCID_{50}/mL$. The rent-time RT-PCR method was validated to be reproducible and very specific to BVDV. The established real-time RT-PCR assay was successfully applied to the validation of Chinese hamster ovary (CHO) cell artificially infected with BVDV. BVDV RNA could be quantified in CHO cell as well as culture supernatant. Also the real-time RT-PCR assay could detect $10TCID_{50}/mL$ of BVDV artificially contaminated in bovine collagen.

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