Eurotium Cristatum Postfermentation of Fireweed and Apple Tree Leaf Herbal Teas
Table 1
Studied DNA regions of the fungus and primers and PCR annealing temperature.
DNA regions/genes
Symbols
Thermocycling programs
Primers (5→3)
Symbols
Sequences
Entire ITS rDNA and fragment of 28S rDNA
ITS-D1/D2
(1) 94°C–5 min (2) 33 cycles with temperature interval 94°C–1 min, 51°C–1 min, 72°C–1 min (3) 72°C–7 min
ITS1f
TCCGTAGGTGAACTTGCG
NL4
GGTCCGTGTTTCAAGG
Large subunit 28S of ribosomal DNA
LSU
(1) 94°C–5 min (2) 33 cycles with temperature interval 94°C–1 min, 49°C–1 min, 72°C–2 min (3) 72°C–7 min
LR0R
ACCCGCTGAACTTAAGC
LR5
TCCTGAGGGAAACTTCG
RNA polymerase II gene
RPB2
(1) 94°C–5 min (2) 9 cycles 94°C–1 min, at 60°C to 50°C–1 min (with 1 degree decrement each cycle), 72°C–1.5 min (3) 32 cycles 94°C–1 min, 50°C–1.5 min, 72°C–1.5 min (4) 72°C–7 min
fRPB2-5F
GAYGAYMGWGATCAYTTYGG
fRPB2-7cR
CCAT(AG)GCTTG(CT)TT(AG)CCCAT
Translation elongation factor 1-α gene
TEF1-α
(1) 94°C–5 min (2) 9 cycles 94°C–1 min, at 66°C to 56°C–1 min (with 1 degree decrement each cycle), 72°C–1.5 min (3) 32 cycles 94°C–1 min, 56°C–1.5 min, 72°C–1.5 min (4) 72°C–7 min
EF-595F
CGTGACTTCATCAAGAACATG
EF-1567R
ACHGTRCCRATACCACCRATCTT
β-tubulin gene
β-tub
(1) 94°C–5 min (2) 9 cycles 94°C–1 min, at 64°C to 54°C–1 min (with 1 degree decrement each cycle), 72°C–1.5 min (3) 32 cycles 94°C–1 min, 54°C–1.5 min, 72°C–1.5 min (4) 72°C–7 min
βt2a
GGTAACCAAATCGGTGCTGCTTTC
βt2b
ACCCTCAGTGTAGTGACCCTTGGC
Glycerol 3-phosphate dehydrogenase gene
GAPDH
(1) 94°C–5 min (2) 9 cycles 94°C–1 min, at 65°C to 55°C–1 min (with 1 degree decrement each cycle), 72°C–1.5 min (3) 32 cycles 94°C–1 min, 55°C–1.5 min, 72°C–1.5 min (4) 72°C–7 min