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

Hoang, Minh; Wu, Hung-Yi; Lien, Ying-Xiu; Chiou, Ming-Tang; Lin, Chao-Nan

diagnosis. Because a point mutation at the amino acid position 426 in the VP2 protein has been identified to be associated with the different types of CPV-2, sequencing analysis by conventional PCR can be utilized to provide details in terms of CPV typing. 4 In addition, this amino acid substitution is caused by mutating AAT (2a) to GAT (2b) at nucleotide position 1276 or to GAA (2c) at nucleotide positions 1276 and 1278 in the VP2 gene. 4 Traditionally, identifying the CPV-2 variants has been performed by hemagglutination inhibition (HI) testing using monoclonal antibodies (MAbs), 8, 18 PCR-RFLP using enzyme MboII, 4 PCR-based methods, 19 and sequence analyses. HI testing using MAbs helps to predict CPV-2 antigen specificity, which distinguishes the CPV-2 variants. Although types 2a and 2b differ in their lack of MAb B4A2 reactivity to CPV-2b, this MAb cannot recognize type CPV-2c. Thus, a MAb was developed to differentiate the new variant, 2c, from type 2b. 8 However, only samples with high HA titers (≥1:64) can be characterized using MAbs, and several samples containing high viral DNA titers that tested negative or poorly positive by HA have been calculated by real-time PCR. 20, 21 In addition, nonhemagglutinating strains have been described. 22 The PCR-RFLP assay with enzyme MboII 4 can only identify CPV-2c. Both types 2a and 2b are unrecognized by the enzyme and consequently are indistinguishable by utilizing this method; thus, this sequence analysis is often required to definitively characterize the strain, which is more expensive and time-consuming. 21 PCR-based methods have been developed to identify type 2, 2a and 2b CPVs 23 by utilizing nucleotide differences between the primers restricted to one base on their 3′ end. However, these mismatches at the 3′ end of the primers would be insufficient to prevent other CPV-2 types from amplifying. 4 In addition, among the type-specific PCR assays, CPV-2c is undetectable, as the Asp426Glu substitution is due to a change (T to A) at the third codon position at nucleotide 1278 of the VP gene, this mutant is recognized erroneously as type 2b using this PCR strategy. 4 The minor groove binder (MGB) probe real-time PCR assay was developed to rapidly and unambiguously characterize CPV-2 variants. 5 The MGB probe assays with two probe designs can recognize single nucleotide polymorphisms (SNPs) that exist between types 2b/2b (A1276G) and (T1278A), which determine the presence of amino acids, Asn, Asp and Glu, in types 2a, 2b and 2c, respectively, at residue 426 of the capsid protein. 4 Both type 2a/2b and type 2b/2c assays are highly sensitive and specific despite the type 2a-specific probe not discriminating type 2a CPVs from the original type 2.