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Table 1 PCR conditions and primers

From: Molecular identification of zoonotic and livestock-specific Giardia-species in faecal samples of calves in Southern Germany

Target gene Number of reaction Length of amplification (bp) Primer Cycle condition Reaction volume Reference
18S rRNA Primary reaction 292 Forward primer: RH11 a Total volume 25 μl [18]
5’-CATCCGGTCGATCCTGCC-3’
Reverse primer: RH4 96°C, 45 s d
5’-AGTCGAACCCTGATTCTCCGCCAGG-3’ 50°C, 30 s 0.15 μl Taq-Ti hot start DNA polymerasee
  72°C, 45 s
→ 35 cycles 5% dimethyl sulfoxide (DMSO)f
b
Secondary reaction 130 Forward primer: GiarF a 2 μl from the 1st-round PCR reaction [19]
5’-GACGCTCTCCCCAAGGAC-3’
Reverse primer: GiarR 96°C, 45 s
5’-CTGCGTCACGCTGCTCG-3’ 55°C, 30 s
  72°C, 45 s
→ 35 cycles
b
β-giardin Primary reaction 753 Forward primer: G7 a Total volume 25 μl [20]
5’-AAGCCCGACGACCTCACCCGCAGTGC-3’
Reverse primer: G759 95°C, 30 s d
5’-GAGGCCGCCCTGGATCTTCGAGACGAC-3’ 50°C, 30 s 0.15 μl Tth Plus DNA polymerasee
  72°C, 60 s
  → 40 cycles
b
Secondary reaction 511 Forward primer: B-F a 2 μl from the 1st-round PCR reaction [21]
5’-GAACGAACGAGATCGAGGTCCG-3’
Reverse primer: B-R 96°C, 45 s
5’-CTCGACGAGCTTCGTGTT-3’ 55°C, 30 s
  72°C, 45 s
  → 35cycles
  b
GDH Primary reaction not given Forward primer: GDHeF c Total volume 25μl [19]
5’-TCAACGTYAAYCGYGGYTTCCGT-3’
Reverse primer: GDHiR 94°C, 30 s d
5’-GTTRTCCTTGCACATCTCC-3’ 50°C, 30 s 0.2 μl Tth Plus DNA polymerasee
  72°C, 60 s
  → 40 cycles
  b
Secondary reaction 432 Forward primer: GDHiF c 2 μl from the 1st-round PCR reaction [19]
5’-CAGTACAACTCYGCTCTCGG-3’
Reverse primer: GDHiR 94°C, 30 s
5’-GTTRTCCTTGCACATCTCC-3’ 60°C, 30 s
  72°C, 60 s
  → 40 cycles
  b
  1. a: Initial activation step: 96°C, 5 min.
  2. b: Final extension: 72°C, 7 min.
  3. c: Initial activation step: 94°C, 5 min.
  4. d: used substances: 2 μl diluted DNA template, 2.5 μl 10x Reaction Buffer , 2.5 μl MgCl2 (25 mM), 1 μl dNTPs (5 mM) (Promega), 1 μl of each primer (10 μM), Water-ultra pure grade (Fisher Biotech Perth, Australia).
  5. e: Fisher Biotech Perth, Australia.
  6. f: Sigma–Aldrich St. Louis, Missouri.