Kapa sybr fast universal 2 qpcr master mix

According to previously described method^{31 (link)}, total RNA was extracted from the kidneys using TRIzol (Life Technologies, Inc., Carlsbad, CA) and Direct-zol™ RNA MiniPrep (Zymo Research Corporation., Irvine, CA). Two hundred nanograms of total RNA was reverse transcribed to synthesize cDNA using a PrimeScript RT reagent kit with gDNA Eraser (Takara Bio Inc., Shiga, Japan). The real-time detection of PCR products was performed using KAPA SYBR FAST qPCR Master Mix (2 ×) Universal (Kapa Biosystems, Wilmington, MA) and a Thermal Cycler Dice Real Time System (Takara Bio Inc.). All reactions were performed in duplicate. The primers for targets are listed in Supplementary Table S2.

Viral genome copy number was quantified on a PikoReal 96 machine (Thermo Scientific) using a standard protocol (95 °C 2 min; 40 cycles of 95 °C 10 s, 60 °C 20 s, 72 °C 20 s). Each quantitative PCR analysis was performed in triplicate. Nontemplate controls (water) were included in triplicates in each assay. The KAPA SYBR FAST qPCR Master Mix (2×) Universal (Kapa Bio-systems) was used, in a 10 μL final volume. For each analysis 2 μL of the diluted cDNA was used (dilution factor of 4) and specific primers VOV-1-qRT-F1 and VOV-1-qRT-R1 at a concentration of 0.25 μM each (Table S1). The specificity of the amplicons synthesized during the PCR run was ascertained by performing a dissociation curve protocol from 60 °C to 95 °C. Specific primers used for quantification are provided in Table S1 in Supplementary material.

qPCR was performed on an ABI ViiA 7 quantitative thermocycler (Applied Biosystems, USA). Primer set amoA-1F/amoA-2R (Chen et al. 2008 (link); Rotthauwe et al. 1997 (link); Zhang et al. 2015 (link)) was used to amplify bacterial amoA gene. The thermal program for qPCR of bacterial amoA gene was: 3 min at 94 °C, 40 cycles of 30 s at 94 °C, 55 s at 60 °C, and 45 s at 72 °C (Chen et al. 2008 (link)). Primer set 341F/518R was used to quantify bacterial 16S rRNA gene, with thermal program: 3 min at 95 °C, 40 cycles of 30 s at 95 °C, 30 s at 60 °C, and 40 s at 72 °C (He et al. 2007 (link)). The 20-μL reaction mixture consisted of 10 μL 2 × KAPA SYBR FAST qPCR Master Mix² Universal (KAPA Biosystems; Beijing), 0.4 μL Forward Primer (10 µM), 0.4 μL reverse primer (10 µM), 0.4 μL 50 × ROX/Low, 2 μL diluted DNA template (<20 ng), and 6.8 μL double-distilled H₂O. A standard curve was constructed using recombined plasmid with bacterial 16S rRNA gene as template, and AOB amoA gene (Bai et al. 2012 (link)). All reactions, including standards and the 19 SHARON samples, were performed in triplicate.