A SimpleProbe ® real-time PCR assay for differentiating the canine parvovirus type 2 genotype

Abstract

Background: Canine parvovirus type 2 (CPV-2) causes an important canine viral disease worldwide. CPV-2 belongs to the Protoparvovirus genus in the family Parvoviridae. An amino acid change at position 426 of the VP2 protein differentiate types of CPV-2, designated as CPV-2a (Asn), CPV-2b (Asp), and CPV-2c (Glu). In this study, we compared CPV-2 genotyping results obtained by SimpleProbe ® real-time PCR and DNA sequencing analysis to identify the accuracy and sensitivity of these methods. Methods: One hundred rectal swabs were collected from CPV-2 naturally infected dogs from 2015 to 2017 at the Animal Disease Diagnostic Center, National Pingtung University of Science and Technology. CPV-2 genotyping was performed by SimpleProbe ® real-time PCR and DNA sequencing to compare results. Results: CPV-2a (n = 23), 2b (n = 6) and 2c (n = 71) genotyping results obtained by both techniques were identical with specificity of 100% for SimpleProbe ® assay. In the SimpleProbe ® assay, amplifying the DNAs prepared from the clinical specimens showed three distinct melting curve peaks. CPV-2b had the highest melting peak of 57.8°C (CI 95%: 57.7-58.5°C) followed by CPV-2c with a slightly lower melting peak of 52.3°C (CI 95%: 52.2-53.2°C) and CPV-2a with the lowest peak of 50.2°C (CI 95%: 50.1-50.5°C). Conclusion: This study developed a novel method for genotyping CPV-2 strains using the SimpleProbe ® real-time PCR assay. This assay is a reliable and sensitive tool for differentiating between the CPV-2a, 2b and 2c and this technique can be used for molecular CPV-2 epidemiology studies.