Optimization of polymerase chain reaction to detect channel catfish virus in channel catfish, Ictalurus punctatus

Optimal polymerase chain reaction conditions were determined using nested primers targeted to the DNA polymerase gene of channel catfish virus (CCV). Serial dilutions of CCV DNA in the presence or absence of catfish DNA were used as templates throughout the optimization procedure. The final PCR product was detected by visualization in ethidium bromide stained acrylamide gels. Fewer than ten copies of CCV DNA (about 1 fg) could be detected in the presence of 10{dollar}sp8{dollar} times as much catfish DNA. The optimized two-step PCR assay was evaluated for detection of latent CCV in blood samples from healthy brood fish. Latent CCV DNA was readily detected. The PCR assay developed in this study was both cost-effective and time-economic because of the reduced amount of sample (10 {dollar}mu{dollar}l) and short running time ({dollar}<{dollar}3.5 hours). This makes possible the development of a routine testing procedure for catfish brood stock to eliminate CCV in young catfish.