Detection of Canine Parvovirus Type ۲ by Designing Multiple Methods and Genetic Characterization in Iran

Canine parvovirus infection is the most highly infectious in dogs younger than six months. Our study aimed to design and optimize an In-house PCR Assay for Rapid Detection of parvovirus type ۲ and compares it with REAL-TIME PCR and LAMP Assay and phylogenetic analysis. The virulence gene selected for the categories was vp۲ for CPV-۲. PCR products were cloned in pTZ۵۷R/T plasmid for preparation of positive control. Determination of the specificity of primers was done with the negative control virus genomes, and the limit of detection was determined for the Homemade PCR, REAL-TIME PCR, LAMP, and to perform a phylogenetic study using partial vp۲ gene sequences. Added analysis of PCR products using agarose gel electrophoresis for the vp۲ gene showed ۴۸۵bp, and GAPDH ۹۰۰ bp bands, respective amplification using negative control genomes as template was negative. The least detectable copy number for the vp۲ gene in a ۲۵ µl PCR reaction equals ۱۹ copies by homemade PCR, LAMP, and REAL-TIME PCR ۲۵ and ۲۱ copies, respectively. The phylogenetic analysis for the five field sequences formed three distinct clusters. The in-house PCR has advantages such as high specificity, sensitivity, and the ability to detect major CPV-۲ pathogens. This assay may replace the previous laboratory methods and work as an essential supplement to the more time-consuming assays. Phylogenetic analysis is necessary for epidemiological studies to control and prevent disease.

Keywords:

Polymerase Chain Reaction , CPV , Molecular diagnostic , VP۲ , Phylogenetic analysis