Table 1 PCR conditions and primers
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 |