Table 1 PCR protocols used for Leishmania detection in dog samples

Nested SSU rRNA-PCR

1^st PCR:

R221: GGTTCCTTTCCTGATTTACG

R332: GGCCGGTAAAGGCCGAATAG

603

10 μl DNA, 2 mM MgCl₂, 0.2 mM dNTPs, 15 pmol primers, 1.4U Taq, 1× buffer (Promega)

den.: 94 °C (30'); ann.: 60 °C (30'); ext.: 72 °C (30'); 35 cycles

[34]

2^nd PCR:

R223: TCCATCGCAACCTCGGTT

R333: AAAGCGGGCGCGGTGCTG

358

5 μl 1^st PCR prod^a., 2 mM MgCl₂, 0.2 mM dNTPs, 1.5 pmol primers, 0.7U Taq, 1× buffer (Promega)

den.: 94 °C (30'); ann.: 65 °C (30'); ext.: 72 °C (30'); 32 cycles

[10]

ITS1-PCR

LITSR: CTGGATCATTTTCCGATG

L5.8S: TGATACCACTTATCGCACTT

311

2 μl DNA, 1.5 mM MgCl₂, 0.2 mM dNTPs, 25 pmol primers, 1U Taq, 1× buffer (Promega)

den.: 95 °C (20'); ann.: 53 °C (20'); ext.: 72 °C (60'); 32 cycles

[35]

MC-PCR

MC1: GTTAGCCGATGGTGGTCTTG

MC2: CACCCATTTTTCCGATTTTG

447

2 μl DNA, 3 mM MgCl₂, 0.2 mM dNTPs, 15 pmol primers, 1U Taq, 1× buffer (Promega)

den.: 94 °C (30'), ann.: 60 °C (30'); ext.: 72 °C (20'); 30 cycles

[13]

Uni21/Lmj4-PCR

Uni21: GGGGTTGGTGTAAAATAGGCC

Lmj4^b: CTAGTTTCCCGCCTCCGAG

800

2 μl DNA, 1.5 mM MgCl₂, 25 pmol primers, 12,5 μl Biomix (Bioline)

den.: 94 °C (30), ann.: 62 °C (30'); ext.: 72 °C (45'); 35 cycles

[36]