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Evaluation of Limit of Detection and Range of Quantitation for RT-PCR, Real-Time RT-PCR and RT-PCR-ELISA Detection of Bovine Viral Diarrhoea Virus Contamination in Biologics Derived from Cell Cultures
Journal of Bacteriology and Virology ; : 161-168, 2003.
Artigo em Coreano | WPRIM | ID: wpr-20920
ABSTRACT
Risk of viral contamination is one of major concerns common to all biologics derived from cultivated cells. Bovine viral diarrhoea virus (BVDV) has widely been known as a contaminant of cell culture-derived vaccines. The objective of the study was to assess the limit of detection and range of quantitation of the detection methods for BVDV using a reverse transcription-polymerase chain reaction (RT-PCR) assay, real-time RT-PCR assay, and RT-PCR-ELISA. One milliliter of cell culture supernatant containing 106.5+/-0.2 median tissue culture infectious dose (TCID50)/ml of BVDV NADL strain was subjected to RNA isolation. The isolated RNA was 10-fold serially diluted and each diluted sample (10-1 to 10-6) was subjected to RT-PCR on a GeneAmpR PCR System 9700 and/or LightCycler(TM). The amplified products were analyzedly (1) agarose gel electrophoresis for RT-PCR assay, (2) melting curve analysis for real-time RT-PCR assay (in this case a program is automatically linked to amplification step), and (3) ELISA using capture and detection probes for RT-PCR-ELISA. The limit of detection of the 3 assay methods was equally estimated to be 316 TCID50/ml of starting virus culture supernatant subjected to the assay. The quantitation range of real-time RT-PCR assay and RT-PCR-ELISA was estimated to be from 3.16x105 to 3.16x102 TCID50/ml of starting virus culture supernatant. The overall results suggested that the 3 assay methods for BVDV detection can be reliably applied to evaluate BVDV contamination in biologics derived from cell cultures.
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Texto completo: DisponíveL Índice: WPRIM (Pacífico Ocidental) Assunto principal: Produtos Biológicos / RNA / Ensaio de Imunoadsorção Enzimática / Vacinas / Reação em Cadeia da Polimerase / Técnicas de Cultura de Células / Transcrição Reversa / Eletroforese em Gel de Ágar / Limite de Detecção / Congelamento Tipo de estudo: Estudo diagnóstico Idioma: Coreano Revista: Journal of Bacteriology and Virology Ano de publicação: 2003 Tipo de documento: Artigo

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Texto completo: DisponíveL Índice: WPRIM (Pacífico Ocidental) Assunto principal: Produtos Biológicos / RNA / Ensaio de Imunoadsorção Enzimática / Vacinas / Reação em Cadeia da Polimerase / Técnicas de Cultura de Células / Transcrição Reversa / Eletroforese em Gel de Ágar / Limite de Detecção / Congelamento Tipo de estudo: Estudo diagnóstico Idioma: Coreano Revista: Journal of Bacteriology and Virology Ano de publicação: 2003 Tipo de documento: Artigo