A TaqMan real-time RT-PCR assay was developed for detection of RNA transcripts produced by replicating CPV-2. A pair of primers and a TaqMan probe targeting the spliced NS2 mRNA were designed. A synthetic DNA fragment was constructed to mimic the spliced NS2 mRNA by PCR-based gene assembly and was used for generation of standard RNAs. The detection limit of the assay was 1x10(2) RNA copies and standard curve displayed a linear range from 1x10(2) to 1x10(9) copies and a good reproducibility. The assay was then applied to determine the mRNA loads in the tissues of dogs naturally infected by CPV-2. mRNA was detected in a variety of tissues, including the central nervous system.