Detection and Typing of CPV with Real-Time PCR and MiniSequencing

Canine parvovirus causes an acute enteric disease in dogs. All the infections currently reported, worldwide, are caused by either of the genetic variants of CPV2 viz., CPV2a, CPv2b and CPV2c. This study describes a novel, combinatorial approach for rapid and direct detection of CPV2 from canine fecal samples followed by typing to determine the variants (CPV2a, CPV2b and CPV2c) involved in the infection. The combined approach adapts a SYBR green based real-time PCR assay for the detection followed by the mini-sequencing assay of the resultant PCR amplicons for the variant typing. The real-time PCR assay reported here was standardized and validated with plasmid constructs bearing target DNA of CPV2a, 2b, 2c; and CPV-2b genome as source of templates. The assay could detect all three variants of CPV2 with a lower limit of detection (LOD) of one copy per reaction. Subsequently, the real-time PCR with viral genomic DNA extracted from tissue culture fluid (TCF) post viral culture showed a LOD of 5fg of genomic DNA per reaction. Melt-cure analysis of the reactions showed specific amplification of a ~145bp length fragment (Tm = ~73.3°C) matching the size of the target DNA. Further, reaction set-up with genomic DNA from unrelated viruses which causes infection in canines showed no amplified product. Together, it indicates the specificity of the assay. Downstream analysis of the PCR amplicons with the mini-sequencing method could adequately determine the variants at an initial concentration of one copy of plasmid construct or 5.0fg of genomic DNA. In a field study, of the fecal samples (n=60) collected from dogs showing enteric symptoms, 5, 2, 14 and 22 numbers of samples were determined positive by the virus isolation, hemagglutination (HA), PCR and real-time PCR assays, respectively. Samples determined positive by real-time PCR (n=22) were further genotyped by mini-sequencing. and all were found to be of CPV type 2a. The combined approach of real-time PCR followed by mini-sequencing for detection and typing respectively is specific and rapid. We reckon, this approach has the potential for extensive on-field applications in routine diagnosis and epidemiology.