Sensitive detection of canine parvovirus DNA by the nested polymerase chain reaction

A polymerase chain reaction (PCR) for the detection of canine parvovirus (CPV) was developed. To increase the sensitivity and specificity of the reaction, the nested PCR with a double-nested primer pair (inner primer pair) was designed. The sequences of the PCR primer pairs were selected from the conserved region in the CPV VP1/VP2 gene. The PCR with the outer or inner primer pair alone (single PCR) could detect 10 fg of viral replicative form (RF) DNA on agarose gel electrophoresis; whereas as little as 100 ag of the RF DNA was detected by the nested PCR, which was shown to be 100 times more sensitive than the single PCR. Samples prepared from feline panleukopenia virus and mink enteritis virus, both having a very close antigenic relationship to CPV, were also amplified by the nested PCR. The specificity of the reaction was confirmed by restriction enzyme analysis and Southern hybridization. Next, fecal samples were examined by the nested PCR. All 10 samples suspected of CPV infection were positive, and two restriction sites (HaeIII and HindIII sites) in the PCR product were conserved among them. On the other hand, specific amplification was not observed in the samples derived from normal dogs. The number of the genome copy in positive samples was estimated about 10(9)-10(11)/g by the single PCR and 10(11)-10(13)/g by the nested PCR. The assay can be completed in 1-1.5 days, and does not need radioisotopes. Thus, the nested PCR seems to be a sensitive, specific and practical method for the detection of CPV in fecal samples.