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

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.