Bigdye terminator cycle sequencing kit 1

For initial genotyping of IBV viruses, sequencing for IBV HVR 1, 2, and 3 of the S1 gene was done using primer sets and reaction conditions as described by (Adzhar et al., 1997 (link), Naguib et al., 2017a (link)) by direct amplification of the RNA extracted from the sample homogenate. Primer sensitivity was tested using RNA stock from IBV isolate NR725/16 with an initial Ct value of 13 that could be diluted × 10^–5. For sequencing of gene 3ab gene a specific primer set was generated to amplify gene 3ab from diagnostic samples consisting of two forward primers (IBV/3ab/FW1: CAA TAC AGA CCT AAA AAG TCT G, IBV/3ab/FW2: TAT TAA GTG GCC TTG GTA TGT) and one reverse primer (IBV/3ab/RV: CCA CTA CCC ATG TRT ACC A). The SuperScript®III One-Step RT-PCR kit (Invitrogen, Carlsbad, CA, USA) was used for amplification with the following cycling conditions: RT step 45 °C for 10 min, followed by an initial denaturation step at 95 °C for 10 min. PCR amplification was done for 35 cycles each with 95 °C:15 s, 55 °C:15 s, and 68 °C:30 s with a final extension step of 68 °C for 5 min. Agarose gel size separated PCR products were excised and purified using the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany). The product was analyzed by nucleotide sequencing with the BigDye Terminator Cycle Sequencing Kit 1.1 (PE Applied Biosystems, Weiterstadt, Germany).