Itaq universal probe one step kit

Total RNA was isolated using TRIzol Reagent (Invitrogen, Mississauga, Canada). cDNA was synthesized from 500 ng RNA using SuperScript III (Invitrogen), or RNA was directly used for RT–PCR analysis using the iTaq Universal Probe One‐Step Kit (Bio‐Rad, Mississauga, Canada), according to the manufacturer's instructions. Quantitative reverse transcription–PCR (qRT–PCR) was performed using TaqMan probes (Applied Biosystems, Mississauga, Canada) for the following genes: HMGCR (Hs00168352), HMGCS1 (Hs00266810), INSIG1 (Hs01650979), and GAPDH (Hs99999905).

To extract SARS-CoV-2 RNA, 200 µL of food rinsing fluid from each food sample was mixed with an equal volume of TRI Reagent LS (ThermoFisher, Waltham, MA, USA). Viral RNA was extracted and purified using Direct-zol^TM RNA microPrep kit (Zymo Research, Irvine, CA, USA). A NanoDrop 2000 spectrophotometer (ThermoFisher, Waltham, MA, USA) was used to measure concentrations and purity of the extracted viral RNA using reading absorbances at 260 nm and 280 nm. To determine RNA genome copy number, 10 µL qRT-PCR reactions were prepared using the iTaq Universal Probe One-Step Kit (BioRad, Hercules, CA, USA), mixed with primers and probes specific for SARS-CoV-2 N protein, and then amplified on a ViiA 7 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) [33 ]. The standard setting used for qRT-PCR is as follows: 1 cycle of 10 min at 50 °C followed by 2 min at 95 °C; and 45 cycles of 3 s at 95 °C and 30 s at 55 °C. Final results were reported as genome copy number per mL of food rinsing fluid, to allow for direct comparison to infectious viral titer measured in PFU/mL.

ZIKV-, UV-inactivated ZIKV or mock-infected mice were euthanized 6, 12, 24, 48 or 72 h post-infection and spleens were collected and weighed. Total RNA was harvested by Trizol extraction according to the manufacturer’s instructions and RNA concentration was determined using a NanoDrop 2000 spectrophotometer. Quantification of ZIKV RNA in the mouse spleen was determined by TaqMan one-step quantitative real time PCR (qRT-PCR) on a Bio-Rad CFX96 Touch Real-Time System using an iTaQ Universal Probe One-Step Kit (Bio-Rad) under standard cycling conditions. The primer set used to detect ZIKV RNA included the following primers: forward, 5’-CCG CTG CCC AAC ACA AG-3’; reverse, 5’-CCA CTA ACG TTC TTT TGC AGA CAT-3’; probe, 5’-/56-FAM/AGC CTA CCT/ZEN/TGA CAA GCA ATC AGA CAC TCA A/3IABkFQ/-3’ (Integrated DNA Technologies) [11 (link), 41 (link)]. A standard curve was generated of C_t value versus log₁₀ PFU using serial 10-fold dilutions of ZIKV RNA extracted from previously titrated viral stocks. Viral burden is expressed as PFU equivalents per gram of tissue after comparison with the standard curve (S3 Fig). The limit of detection was set as the average PFU equivalent per gram of tissue from the mock-infected samples.