Detection of pestivirus contamination in cellcultures by nested-PCR

ABSTRACT

In this study a nested-PCR assay was optimized for detection of two BVDVbiotype 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 DNAfragment was amplified in nested-PCR. The 4 sensitivity of RT-PCR and nested-PCR for detection of virus in cell culture were found to be 10 2 TCID50 and 10 TCID50, respectively. Seven cell cultures were tested for BVDVcontamination 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