Abi veriti 96

Total RNA was isolated using Trizol reagent (Takara, Tokyo, Japan) following the manufacturer’s protocol. Reverse transcription of total RNA was performed using PrimeScript™II1st strand cDNA Synthesis Kit (Takara,Tokyo, Japan). PCR analysis was performed using the ABI Veriti96 (Applied Biosystems, Foster City, USA). The primers were shown in Table 1. The cycling parameters for PCR were as follows: denaturation at 95 °C 4 min, 94 °C 50 s, annealing at 54 °C, 56 °C, and 58 °C (for EPOR, GAPHD and EPO, respectively) for 30 s, and extension at 72 °C for 50 s for a total 30 cycles, which were followed by an extension at 72 °C for 10 min.Table 1

Sequences of primers

Target gene	Conventional RT-PCR	Real-time PCR
GAPDH
Forward	5′-CTCTGATTTGGTCGTATTGGG-3′	5′-ATTGCCCTCAACGACCACTT-3′
Reverse	5′-CTGGAAGATGGTGATGGGAT-3′	5′-CCCTGTTGGTCAACTCTTCC-3′
EPO
Forward	5′-ATCACGACGGGCTGTGCTGAACAC-3′	5′-CCCTGTTGGTCAACTCTTCC-3′
Reverse	5′-ATCACGACGGGCTGTGCTGAACAC-3′	5′-GTGTACAGCTTCAGCTTTCC-3′
EPOR
Forward	5′- GGGAGATGGCTTCCTTCTGGGCTC-3′	5′-GCACCGAGTGTGTGCTGAGCAA-3′
Reverse	5′-CGGGGACAGATGATGAGG-3′	5′-GGTCAGCAGCACCAGGATGAC-3′

Each PCR reaction was performed in a 25-μL reaction volume with 1× HotStar Taq^® Plus Master Mix (QIAGEN GmbH, Hilden, Germany), various concentrations of forward and reverse primers and 50 ng genomic DNA as the template. The tested primer concentrations were 0.16, 0.24, 0.32, 0.40 and 0.8 μM. Two primer pairs (CITS-F10′/CITS- R′-2 and COI-F/COI-R) were simultaneously added into the PCR reaction system to develop a duplex PCR assay for Chinese cordyceps identification. Primer combinations at different concentrations were tested to determine the optimal proportions that influenced the amplification efficiency. A gradient PCR was performed with annealing temperatures ranging from 50 °C to 60 °C in an ABI Veriti 96 thermal cycler (Applied Biosystems). Six temperatures in a gradient, 50 °C, 52 °C, 54 °C, 56 °C, 58 °C and 60 °C, were tested to determine the optimal annealing temperature. The program was as follows: one step of 5 min at 95 °C, 35 cycles of 30 s at 95 °C, 30 s at 50 °C–60 °C and 30 s at 72 °C; followed by one step of 7 min at 72 °C. The PCR products were size separated using electrophoresis on 2.5% agarose gel in 1 × Tris-acetate-EDTA buffer and stained with GelRed Nucleic Acid Stain (Cat. 41003, Biotium, Inc. Fremont, CA, USA) for visualisation. The amplicons were further confirmed by cloning and sequencing (Sangon Biotech (Shanghai) Co. Ltd.).