Evaluation of Unpurified Cell Culture Supernatant as Template for the Polymerase Chain Reaction (PCR) with Largemouth Bass Virus

Abstract Polymerase chain reaction (PCR) has been used to identify largemouth bass virus (LMBV) in cell culture, and this method has included extraction of the sample DNA. To simplify this procedure, we centrifuged the cell culture fluid to remove cellular debris and then used the supernatant directly in the PCR without DNA extraction. For supernatants from cell cultures inoculated with 1:100 dilutions of homogenized swim bladder or pooled spleen and trunk kidney, only 70.0% of the LMBV-positive samples (based on all available evidence) tested positive with this new PCR method. After a 1:500 dilution of these cell culture supernatants, 85% of the failed samples became PCR positive. In contrast, 97.2% of LMBV-positive samples were PCR-positive when undiluted supernatants from subcultivations (cells inoculated with supernatant from cell cultures showing cytopathic effect [CPE]) were used directly in the PCR. Unpurified cell culture fluid supernatant from blind passage samples (cells inoculated with supernatant from CPE-negative cultures) was PCR-positive for 93.8% of the samples that were eventually shown to be LMBV-positive. The use of cell culture supernatant directly in the PCR reduced the cost and time required to identify LMBV isolated in cell culture. Extraction of DNA from cell cultures to obtain template for PCR identification of LMBV was required for only 2% of the LMBV-positive samples.