Detection of pestivirus contamination in cellcultures bynested-PCR

In this study a nested-PCR assay was optimized for detection of two BVDV biotype of NADL strain. Apart of 5' non-coding region of virus, 249 bp in size, was amplified in RT-PCR. PCR product was cloned in a pTZ57R/T vector and sequencing results confirmed the specificity of the test. Internal primers were designed and a 155 bp DNA fragment was amplified in nested-PCR. The sensitivity of RT-PCR and nested-PCR for detection of virus in cell culture were found to be 10 4 TCID50 and 10 2 TCID50, respectively. Seven cell cultures were tested for BVDV contamination using ELISA, RT-PCR and nested-PCR. Results indicate that sensitivity of molecular tests for detection of virus in cell culture samples is higher than ELISA.