세포배양 유래 생물의약품 중 Bovine Viral Diarrhoea Virus 검출을 위한 RT-PCR, Real-Time RT-PCR 및 RT-PCR-ELISA 기법의 검출한계와 정량범위 평가

• Published in: Journal of bacteriology and virology Vol. 33; no. 2; pp. 161 - 168
• Main Authors: 류승렬, Seung Rel Ryu, 신진호, Jin Ho Shin, 백선영, Sun Young Baek, 김재옥, Jae Ok Kim, 민경일, Kyung Il Min, 민복순, Bok Soon Min, 김병국, Byoung Guk Kim, 김도근, Do Keun Kim, 박미경, Mi Kyung Park, 안미진, Mi Jin Ahn, 채경숙, Kyung Sook Chae
• Format: Journal Article
• Language: Korean
• Published: 대한바이러스학회 2003; 대한미생물학회
• Subjects: Biologics; Bovine viral diarrhoea virus; Polymerase chain reaction; Reverse transcription; 생물학; Polymerase chain reaction; Reverse transcription; Bovine viral diarrhoea virus; Biologics
• ISSN: 1598-2467; 2093-0429

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 $10^{6.5{\pm}0.2}$ median tissue culture infectious dose ($TCID_{50}$)/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 $GeneAmp^{(R)}$ 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 $TCID_{50}/ml$ 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 $3.16{\times}10^5$ to $3.16{\times}10^2$ $TCID_{50}/ml$ 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.