2-3- RT-PCR and sequencing
The RNA was extracted from the clinical samples using the Extraction of viral DNA/RNA kit (FAVORGEN Biotech Corporation, Taiwan) based on the manufactures’ instructions. All the extracted RNAs were evaluated for the quality of the extraction via spectrophotometry method using NanoDrop ND-1000® (Thermo Fisher Scientific Inc., Waltham, MA, USA). PrimeScript One Step RT-PCR Kit (Takara Bio, Japan) was used for the synthesis of complementary DNA (cDNA) and PCR according to the manufactures’ instructions.
We used the specific primers for amplifying the RdRp (RNA dependent RNA polymerase), the S protein, and nsp-2 (non-structural protein 2)21,22. Conventional PCR method was used for amplification of 344 bp region of RNA-dependent RNA polymerase (RdRp) gene via forward 5ʹ-CAA GTG GGG TAA GGC TAG ACT TT-3ʹ and reverse 5ʹ-ACT TAG GAT AAT CCC AAC CCA T-3ʹ primers; and for the 158 bp region of Spike (S) protein forward 5ʹ-CCT ACT AAA TTA AAT GAT CTC TGC TTT ACT-3ʹ and reverse 5ʹ- CAA GCT ATA ACG CAG CCT GTA-3ʹ primers; and a nested-PCR for amplification of 346 and 322 bp region of nsp-2 gene by first round forward 5ʹ-CTC GAA CTG CAC CTC ATG G-3ʹ and reverse 5ʹ- CAG AAG TTG TTA TCG ACA TAG C-3ʹ primers and second round 5ʹ-ACC TCA TGG TCA TGT TAT GG-3ʹ and 5ʹ-GAC ATA GCG AGT GTA TGC C-3ʹ primers.
A total volume of 50 ul Master Mix was performed using 25ul 2x EnzymeMix (Takara Bio, Japan) for one-step RT PCR of RdRp, S and the first round of nsp-2 PCR, and 2x Super PCR Mastermix (Yekta Tajhiz Azma Co., Iran) for the second round of PCR for nsp-2 gene amplification, 1ul (10 pMol concentration) of each primers, 4 μl (0.2–0.5 μM concentration) of extracted specimens, and distilled RNase/DNase free water added to reach the rest of total volume.
A Bio-Rad (T100™ Thermal Cycler) was performed for heating program. Heating protocol, for RdRp, S, and the first round of nsp-2 PCR, was 1 step at 50ºC 40min, 94ºC 5min, 40 cycles at 94ºC 30s, 58ºC 30s, 72ºC 30s, and 1 step at 72ºC 5 min. The second round of PCR for nsp-2 gene included 94ºC 3 min, 94ºC 20s, 58ºC 20s, 72ºC 20s, and 72ºC 3 min. Visualization of PCR products was performed using 1.5% concentration of agarose gel into an electrophoresis system and stained using safe stain (YTA, Yekta Tajhiz Azma Co., Iran) behind UV translaminator.