Itaq universal sybr green one step kit

RNA was extracted from individual dry swabs and kidneys. Briefly, the swabs were eluted in 500 µl of PBS and the viral RNA was extracted using a QIAamp viral RNA minikit (QIAGEN, Hilden, Germany) following the manufacturer’s protocol, whereas 30 mg of each kidney was homogenized using MagNA Lyser Instrument (Roche, Germany) and the RNA was isolated using the RNeasy minikit (QIAGEN, Hilden, Germany), following the manufacturer’s recommendations. In each round of RNA isolation, a QX isolate with known titer (10⁶ EID₅₀/ml) was included as positive control, while PBS was used as negative control. qRT-PCRs were performed using the iTaq universal SYBR Green one-step kit (Bio-Rad Laboratories, Hercules, California, USA). A genotype QX specific SYBR Green qRT-PCR developed and validated in our laboratory [15] based on the S1 gene sequence was used to detect and quantify viral RNA. The limit of detection of the assay was equal to 10^1.31 EID₅₀/100 µl. The qRT-PCR reactions were carried out in a Bio-Rad CFX Connect real-time PCR system. In each qRT-PCR round, three ten-fold dilutions of the positive control were used to create a standard curve to quantify the viral RNA in the samples.