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7 protocols using primedirect probe rt qpcr mix

1

Characterizing Cellular Response to Infection

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MTT reagent Thiazolyl Blue Tetrazolium Bromide (Sigma Aldrich), All cell culture dishes (Nunc), TRIzol (Ambion), DEPC, Diethyl pyrocarbonate (Ambion), High-Capacity Reverse Transcription Kit (Applied Biosciences, Inc. Foster, CA), DyNAmo ColorFlash SYBR Green qPCR kit (Thermo Scientific), EDTA-free Protease-cocktail inhibitor (Roche Mannheim Germany), Agarose (Invitrogen by Life Technologies), Crystal violet (Sigma-Aldrich), Gelatin (Merck), PFA, Paraformaldehyde (Merck), Mouse monoclonal Anti-N, Nucleocapsid protein of MHV-JHM (monoclonal clone 1-16-1, kindly provided by Julian Leibowitz, Texas A&M, College Station, TX), Anti-CD11b (Abcam; catalog no. ab133357), Anti-Iba1 (Wako, Richmond, VA, USA, Cat no. 019-19741, RRID:AB_839504) antibody, Avidin-biotin immunoperoxidase technique (Vector Laboratories), Refrax mounting medium (Anatech Ltd., MI, USA), Direct-Zol RNA MiniPrep (Zymo Research), Turbo DNA-Free Kit (Life Technology), High Fidelity cDNA Synthesis Kit (Roche), Fast Start Universal Probe Master (Rox) (Roche), Viral-ToxGlo assay (Promega), Prime-direct probe RT-qPCR mix (Takara).
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2

SARS-CoV-2 RNA Detection by RT-qPCR

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Positive and negative serum were diluted with a proportional concentration gradient. All details pertaining to the incubation, extension, and washing processes are described in our previous reports (Yan et al., 2021 (link)). Prior to performing RT-PCR, the SARS-CoV-2 RNA fragments, PrimeDirect™ Probe RT-qPCR Mix (Takara, Japan), RNase-free H2O, and four sets of probe primers were simultaneously added to the reaction tubes. The reaction system is shown in Table S4. All PCR reactions were performed in a 25 μL reaction volume on a LightCycler 96 (Roche, Switzerland) under the manufacturer's recommended fast cycling conditions for 40 cycles. The instrument was adjusted to simultaneously record signals from all four channels.
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3

SARS-CoV-2 Viral Load Quantification

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The SARS-CoV-2 viral load in the cells was determined as described (Acharya et al., 2021 (link)). Briefly, RT-qPCR was performed on a set of primers targeting the E gene of SARS-CoV-2 using PrimeDirect Probe RT-qPCR Mix (TaKaRa Bio USA, Inc) and Applied Biosystems QuantStudio3 real-time PCR system (Applied Biosystems, Waltham, MA, USA). The SARS-CoV-2 genome equivalent copies were calculated by quantitative PCR control RNA from heat-inactivated SARS-CoV-2, isolate USA-WA1/2020 (BEI, Catalog# NR-52347). The percent inhibition of SARS-CoV-2 replication by JQ1 and OTX015 treatment was measured based on viral concentration in positive control wells (considered 0% inhibition) and negative control wells (uninfected cells). IC50 values were calculated using four-parameter variable slope sigmoidal dose-response models using Graph Pad Prism 9.0 software.
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4

SARS-CoV-2 RNA Detection from Nasal Swabs

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Each residual sample of nasal swab from four patients was used for this assay. Briefly, 0.5% Tween 20 (45 μl) and monobody clone 4 (5 μl of 200 nM) were added into 400 μl of the swab sample in PBS. The mixture was incubated at 25°C for 10 min, followed by addition of Dynabeads M-280 streptavidin (2.5 mg/ml, 10 μl, prewashed by HBST buffer). After 10 min of incubation, the beads were washed with 1 ml of HBST three times and resuspended with 2.5 μl of HBST containing carrier RNA. The bead suspension or the original swab solution (2.5 μl) was mixed with an RT-qPCR reaction mixture (47.5 μl; PrimeDirect Probe RT-qPCR Mix, Takara Bio) with a primer/probe set (data file S1). The RNA copy numbers were determined using the Thermal Cycler Dice Real Time System III (Takara Bio), as aforementioned. The detection level of positive control RNA using the PrimeDirect Probe RT-qPCR Mix was ≳25 copies per well.
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5

Quantifying SARS-CoV-2 viral load inhibition

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The SARS-CoV-2 viral load in the cells was determined as described (Acharya et al., 2021 (link)). Briefly, RT-qPCR was performed on a set of primers targeting the E gene of SARS-CoV-2 using PrimeDirect Probe RT-qPCR Mix (TaKaRa Bio USA, Inc) and Applied Biosystems QuantStudio3 real-time PCR system (Applied Biosystems, Waltham, MA, USA). The SARS-CoV-2 genome equivalent copies were calculated by quantitative PCR control RNA from heat-inactivated SARS-CoV-2, isolate USA-WA1/2020 (BEI, Catalog# NR-52347). The percent inhibition of SARS-CoV-2 replication by JQ1 and OTX015 treatment was measured based on viral concentration in positive control wells (considered 0% inhibition) and negative control wells (uninfected cells). IC50 values were calculated using four-parameter variable slope sigmoidal dose-response models using Graph Pad Prism 9.0 software.
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6

Examining BVDV in Mouse Tissues

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The mice were observed daily for clinical symptoms and were killed when they exhibited listlessness, rough coat, decreased diet, shapeless feces, or obvious diarrhea. Subsequently, blood was collected from the eyes, and the kidney, spleen, liver, duodenum, and colon tissues were collected.
The fresh feces (500 mg) was obtained from the mouse rectum and soaked in 200 μL pre-cooled diethylpyrocarbonate (DEPC) water for 15 min to dissolve the virus contained in the feces. The DEPC water was centrifuged at 4°C at 10,000 × g for 5 min and the supernatant was stored for subsequent use.
The tissue samples (100 mg) was ground into powder with liquid nitrogen, and 1 mL pre-cooled saline was added. After repeated freeze-thawing, the sample was centrifuged at 10,000 × g at 4°C for 5 min, and the supernatant was obtained.
Total RNA was extracted from the fecal and tissues samples with an RNA extraction kit (Takara Bio, Shiga, Japan). Complementary DNA (cDNA) was synthesized by reverse transcription and the BVDV content was analyzed by qRT-PCR using a Prime Direct™ Probe RT-qPCR Mix (Takara Bio). The relative BVDV content was calculated in relation to that of the normal control. Table 3 shows the primers used in this study.
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7

Quantification of SARS-CoV-2 RNA Levels

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The relative amount of SARS-CoV-2 RNA present in the tissue-extracted RNA was determined by qRT-PCR targeting the E-gene of the virus using the Takara PrimeDirect probe, RT-qPCR mix (Takara Bio Inc, Japan). qRT-PCR was performed in a 25 uL reaction volume, with 175 ng input RNA, 400 nM each primers (Eurofins, USA; Forward: 5’-ACAGGTACGTTAATAGTTAATAGCGT-3’ and  reverse: 5’-ATATTGCAGCAGTACGCACACA-3’),  200 nM probes (Sigma-Aldrich, UK; 5’- [FAM]-ACACTAGCCATCCTTACTGCGCTTCG-[BBQ650]) and cycling conditions of  initial denaturation 90℃ for 3 min, reverse transcription 60℃ for 5 min, followed by 45 cycles of 95°C for 5 sec, 58°C for 30 sec (PMID: 34006825). The human RNase P gene was used as an internal control in the PCR to validate the quality of the extracted RNA. The relative quantification of viral copies per ng of input RNA was performed by comparing the results to the known viral copies/reaction (Lowest detectable: 750 copies/reaction) of the All-WHO-CDC-Genes-nCoV-Control-Plasmid (Eurofins Genomics, #5004ALL001).
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