DNA was extracted from each stool pool using PureLink Genomic DNA Mini Kit (Invitrogen, Cat.No.K1820-02,USA) according to the manufacturer's instructions. A real-time PCR test was performed in a CFX Connect real-time PCR machine (Biorad, USA) with primers and probe previously described by Decaro et al. [24 (link)], as detailed in Table 1 . The reactions for real-time PCR were conducted on 25 μl final volume containing 5 μl of template DNA, 0.75 μl of dNTP, 2.5 μl of 10X buffer, 500 nM of each primer, 300 nM of the probe, 1 μl of Taq polymerase, 2.5 μl of MgCl2, and 10 μl of RNAse-free water. PCR test conditions were adjusted as follows: 1 min at 95 °C, followed by 40 cycles at 95 °C for 15 s, 52 °C for 30 s, and 72 °C for 60 s. Parvovirus vaccine (Novibac, UK) was a positive control in all PCR assays.
Primers and probe were used for CPV PCR testing and sequencing
| Primers & probe | Sequences (5′ 3′) | Amplicon (bp) | References |
|---|---|---|---|
| CPV-for | AAACAGGAATTAACTATACTAATATATTTA | 93 | [24 (link)] |
| CPV-rev | AAATTTGACCATTTGGATAAACT | ||
| CPV-Pb | FAM–TGGTCCTTTAACTGCATTAAATAATGTACC-TAMRA | ||
| Pabs | GAAGAGTGGTTGTAAATAATT | 681 | [25 (link)] |
| Pabas | CCTATATAACCAAAGTTAGTAC | ||
| Hfor | CAGGTGATGAATTTGCTACA-3 | 630 | [3 (link)] |
| Hrev | CATTTGGATAAACTGGTGGT-3 | ||
| 555for | CAGGAAGATATCCAGAAGGA | 583 | [3 (link)] |
| 555rev | GGTGCTAGTTGATATGTAATAAACA |
for Forward; rev Reverse; Pb Probe; bp Base pair
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