Quanta qscript xlt one step rt pcr kit

Processed samples were initially tested for the presence of coronavirus using a pancoronavirus nested PCR targeting the RdRp gene as previously published (Table S2) (16 (link)). Briefly, 100 μL of each sample was used to extract RNA using the MagMAX core nucleic acid purification kit (Applied Biosystems, Carlsbad, CA, USA) and KingFisher duo prime (Thermo Scientific, Carlsbad, CA, USA), according to the manufacturers’ instructions. Purified RNA was used to perform the first round of RT-PCR using the Quanta qScript XLT one-step RT-PCR kit (Quantabio, Beverly, MA, USA). The first-round product was used for a second PCR using Dream Taq green master mix (Thermo Scientific, Carlsbad, CA, USA). The final product of 440 bp was gel purified using a MEGAquick-spin plus kit (iNtRON, Seoul, South Korea) and analyzed by Sanger sequencing. In addition, purified RNA was tested for the presence of BCoV RNA by targeting the BCoV nucleocapsid gene as previously described (Table S2) (15 (link), 40 (link)). Briefly, purified RNA was used to perform RT-qPCR using the Quanta qScript XLT one-step RT-qPCR ToughMix kit (Quantabio, Beverly, MA, USA) and analyzed using the Bio-Rad CFX 96 real-time detection system (Bio-Rad, Hercules, CA, USA).

Total RNA was extracted from the ISR1127 source sample as described above and used to amplify the S1 region of the spike glycoprotein, using the Quanta qScript XLT one-step RT-PCR kit (Quantabio, Beverly, MA, USA). For S1 amplification, we used a six-primer set previously described, with slight modification to allow the amplification of nucleotides 1 to 2731 of the S glycoprotein of ISR1127 (Table S2) (23 (link)). RT-PCR products were loaded on a 1.5% agarose gel, and corresponding bands were gel extracted and purified using a MEGAquick-spin plus kit (iNtRON, Seoul, South Korea). Purified PCR products were verified by Sanger sequencing (Hylabs, Rehovot, Israel). The sequenced S1 region of ISR1127 was further used in BLAST and phylogenetic analysis.