Primetime gene expression master mix

A previously developed in-house primer–probe set was used for the TaqMan-chemistry-based qPCR assay [19 (link)]. The primers and probe (Integrated DNA Technologies, IDT, Skokie, IL, USA) targeted a region from the conserved Salmonella invA gene as shown in Supplementary Table S1. The qPCR reaction mixture was prepared by combining 10 μL of 2X PrimeTime^® Gene Expression Master mix (Integrated DNA Technologies, IDT, Skokie, IL, USA), 2 μL of nuclease-free water (Invitrogen™, Life Technologies, Grand Island, NY, USA), 3 μL of the in-house primer–probe mixture (0.22 μM final concentration of the probe, 0.33 μM final concentration of each of the primers), and 5 μL of the DNA template to contain a final reaction volume of 20 μL. Positive DNA for the invA gene and a no-template control (nuclease-free water) were included in each run. qPCR runs for each experiment were performed in triplicate, and each dilution per treatment was plated in triplicate qPCR wells unless otherwise stated. Amplification conditions consisted of 95 °C for 3 min, followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s on the 7500 FAST real-time PCR system (Applied Biosystems, Foster City, CA, USA).

We extracted the viral RNA using the Viral Nucleic Acid Extraction Kit II (Geneaid, Taipei, Taiwan), following the manufacturer’s protocol. cDNA was generated using the QuantiNova Reverse Transcription Kit (Qiagen, Valencia, CA, USA). For qPCR, the reaction mixtures were prepared using PrimeTime^® Gene Expression Master Mix (Integrated DNA Technologies, Bothell, WA, USA) according to the manufacturer’s instructions with virus-specific primers and the probe [23 (link)]. The qPCR reactions were performed as previously described [24 (link),25 (link)].

qPCR was performed using PrimeTime® Gene Expression Master Mix (Integrated DNA Technologies, Inc.). Each reaction contains, 500 nM each of forward/reverse primers, 200 nM probe (Table S1), and 1 µL sample DNA in a total reaction volume of 10 µL using CFX96 Touch Real-Time PCR Detection System (BIO-RAD). The PCR amplification cycle was as follows: 95°C for 2 min; 40 cycles of 95°C for 15 seconds, 65°C for 30 seconds. The recovered pDNA from each organ was normalized by recovered Mitochondria DNA.