Discriminating the eight genotypes of the porcine circovirus type 2 with TaqMan-based real-time PCR

Abstract

Background: The porcine circovirus type 2 (PCV2) is divided into eight genotypes including the previously described genotypes PCV2a to PCV2f and the two new genotypes PCV2g and PCV2h. PCV2 genotyping has become an important task in molecular epidemiology and to advance research on the prophylaxis and pathogenesis of PCV2 associated diseases. Standard genotyping of PCV2 is based on the sequencing of the viral genome or at least of the open reading frame 2. Although, the circovirus genome is small, classical sequencing is time consuming, expensive, less sensitive and less compatible with mass testing compared with modern real-time PCR assays. Here we report about a new PCV2 genotyping method using qPCR.

Methods: Based on the analysis of several hundred PCV2 full genome sequences, we identified PCV2 genotype specific sequences or single-nucleotide polymorphisms. We designed six TaqMan PCR assays that are specific for single genotypes PCV2a to PCV2f and two qPCRs targeting two genotypes simultaneously (PCV2g/PCV2d and PCV2h/PCV2c). To improve specific binding of oligonucleotide primers and TaqMan probes, we used locked nucleic acid technology. We evaluated amplification efficiency, diagnostic sensitivity and tested assay specificity for the respective genotypes.

Results: All eight PCV2 genotype specific qPCRs demonstrated appropriate amplification efficiencies between 91 and 97%. Testing samples from an epidemiological field study demonstrated a diagnostic sensitivity of the respective genotype specific qPCR that was comparable to a highly sensitive pan-PCV2 qPCR system. Genotype specificity of most qPCRs was excellent. Limited unspecific signals were obtained when a high viral load of PCV2b was tested with qPCRs targeting PCV2d or PCV2g. The same was true for the PCV2a specific qPCR when high copy numbers of PCV2d were tested. The qPCR targeting PCV2h/PCV2c showed some minor cross-reaction with PCV2d, PCV2f and PCV2g.

Conclusion: Genotyping of PCV2 is important for routine diagnosis as well as for epidemiological studies. The introduced genotyping qPCR system is ideal for mass testing and should be a valuable complement to PCV2 sequencing, especially in the case of simultaneous infections with multiple PCV2 genotypes, subclinically infected animals or research studies that require large sample numbers.

Keywords: Genotype; Locked nucleic acid; PCV2; QPCR; Real-time PCR; TaqMan PCR.

Fig. 1

Phylogenetic analysis of PCV2 based on the complete genome of selected PCV2 strains representing the heterogeneity of the eight PCV2 genotypes. The tree was constructed using the neighbour joining method (P-distance model; 1000 bootstraps). The scale bar indicates nucleotide substitutions per site. Underlined reference sequences were used for the evaluation of the PCV2 genotype specific qPCR assays

Fig. 2

Position of the oligonucleotide primers and TaqMan probes of the eight genotype specific qPCR assays (A-H), relative to the PCV2a genome (upper scale) and ORF2. Primers of PCR A were used in PCR C, G and H, too. For PCR C, G and H the identical TaqMan probe was used

Fig. 3

Binding sites of oligonucleotide primers and TaqMan probes. Consensus sequences of the PCV2 genotypes (with indicated position in the full genome) show the binding position of the used oligonucleotide primer or probes (bold and underlined). Letters in red demonstrate motifs discriminating the PCV2 genotypes

Fig. 4

Comparison of PCV2a (left panel) and PCV2d (right panel) genotype specific qPCR with our standard pan-PCV2 qPCR. Shown are the Cq values of diagnostic samples (oral fluids) from pigs infected with PCV2a or PCV2d, respectively

Fig. 5

Standard curves of PCV2 genotype specific PCR assays (A-H) based on serial log10 dilutions of genotype specific DNA template. Mean Cq values of two independent experiments