SENSITIVE DETECTION OF CANINE PARVOVIRUS DNA BY THE NESTED POLYMERASECHAIN-REACTION

A polymerase chain raction (PCR) for the detection of canine parvoviru s (CPV) was developed. To increase the sensitivity and specificity of the reaction, the nested PCR with a double-nested primer pair (inner p rimer pair) was designed. The sequences of the PCR primer pairs were s elected from the conserved region in the CPV VP1/VP2 gene. The PCR wit h the outer or inner primer pair alone (single PCR) could detect 10 fg of viral replicative form (RF) DNA on agarose gel electrophoresis; wh ereas 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 vi rus, both having a very close antigenic relationship to CPV, were also amplified by the nested PCR. The specificity of the reaction was conf irmed by restriction enzyme analysis and Southern hybridization. Next, fecal samples were examined by the nested PCR. All 10 samples suspect ed of CPV infection were positive, and two restriction sites (HaeIII a nd HindII sites) in the PCR product were conserved among them. On the other hand, specific amplification was not observed in the samples der ived from normal dogs. The number of the genome copy in positive sampl es 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 sensi tive, specific and practical method for the detection of CPV in fecal samples.