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.
- Author:
Seung Rel RYU
1
;
Jin Ho SHIN
;
Sun Young BAEK
;
Jae Ok KIM
;
Kyung Il MIN
;
Bok Soon MIN
;
Byoung Guk KIM
;
Do Keun KIM
;
Mi Kyung PARK
;
Mi Jin AHN
;
Kyung Sook CHAE
;
Hye Sung JEONG
;
Seok Ho LEE
;
Sue Nie PARK
Author Information
1. Division of Viral Products, Department of Biologics Evaluation, Korea Food and Drug Administration, Seoul, Korea. suenie@kfda.go.kr
- Publication Type:Original Article
- Keywords:
Bovine viral diarrhoea virus;
Biologics;
Reverse transcription;
Polymerase chain reaction
- MeSH:
Biological Products*;
Cell Culture Techniques*;
Electrophoresis, Agar Gel;
Enzyme-Linked Immunosorbent Assay;
Freezing;
Limit of Detection*;
Polymerase Chain Reaction;
Reverse Transcription;
RNA;
Vaccines
- From:Journal of Bacteriology and Virology
2003;33(2):161-168
- CountryRepublic of Korea
- Language:Korean
-
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.
