Nucleotides sequence alignment of the RPO30 gene of CaPVs highlighting the snapback tail binding site.

<p>The RPO30 gene sequences of 7 CaPVs representing GTPVs, LSDVs and SPPVs were aligned. A sequence of sixteen nucleotides complementary to the snapback tail in GTPV (100% match), as well as the corresponding positions in SPPV and LSDV are shown in the box. Note the targeted single nucleotide mismatches inside the box: T:A between GTPV and SPPV, and T:G between GTPV and LSDV. Conserved nucleotides are shown as dots.</p

Secondary structure of the expected GTPV PCR amplicon.

<p>The Snapback hairpin with a 18-nucleotide stem and a loop of 55 bases is shown. The predictions were done on the Mfold web server <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075971#pone.0075971-Zuker1" target="_blank">[28]</a> using the default parameters of the DNA folding form except for the temperature which was set at 45°C and the salt concentration set at 50 mM.</p

Snapback primer genotyping of CaPVs.

<p>The fluorescence melting curve analysis of the PCR products shows two melting peaks for each of the CaPV three genotypes (GTPV, SPPV and LSDV) corresponding to the snapback stem melting peak at lower temperature and the full-length PCR amplicon melting peak at higher temperature (see arrows).</p

Sequences of the snapback and reverse primers.

<p>The snapback tail of 16 bases is shown as underlined; 2 nucleotides mismatch at the 5′end are indicated as lowercase; and mismatch nucleotide as underlined bold (A).</p

High-Resolution melting curve analysis of CaPVs using the Precision Melt Analysis™ software (BioRad).

<p>A: the normalized melt curve of the full-length amplicon; B: the difference curve of full-length amplicon; C: the normalized melt curve of snapback stem; D: The difference curve of the snapback stem. The species are indicated by the arrows: G = GTPV, S = SPPV and L = LSDV.</p