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KMID : 0364820080440010014
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Korean Journal of Microbiology
2008 Volume.44 No. 1 p.14 ~ p.21
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Real-Time PCR for Quantitative Detection of Bovine Herpesvirus Type 1
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Lee Dong-Hyuck
Kim In-Seop
Jeong Hyo-Sun
Lee Jung-Hee
Kim Tae-Eun
Lee Jung-Suk
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Abstract
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Bovine blood, cell, tissue, and organ are used as raw materials for manufacturing biopharmaceuticals, tissue
engineered products, and cell therapy. Manufacturing processes for the biologicals using bovine materials have
the risk of viral contamination. Therefore viral validation is essential in ensuring the safety of the products.
Bovine herpesvirus type 1 (BHV-1) is the most common bovine pathogen found in bovine blood, cell, tissue,
and organ. In order to establish the validation system for the BHV-1 safety of the products, a real-time PCR
method was developed for quantitative detection of BHV-1 in raw materials, manufacturing processes, and final
products as well as BHV-1 clearance validation. Specific primers for amplification of BHV-1 DNA was
selected, and BHV-1 DNA was quantified by use of SYBR Green I. The sensitivity of the assay was calculated
to be 2 TCID50/ml. The real-time PCR method was validated to be reproducible and very specific to BHV-1.
The established real-time PCR assay was successfully applied to the validation of Chinese hamster ovary
(CHO) cell artificially infected with BHV-1. BHV-1 DNA could be quantified in CHO cell as well as culture
supernatant. Also the real-time PCR assay could detect 10 TCID50/ml of BHV-1 artificially contaminated in
bovine collagen. The overall results indicated that this rapid, specific, sensitive, and robust assay can be reliably
used for quantitative detection of BHV-1 contamination during the manufacture of biologics.
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KEYWORD
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bovine herpesvirus type 1, Chinese hamster ovary (CHO) cell, collagen, real-time PCR, virus validation
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