The S-gene of PEDV positive samples was amplified using four overlapping fragments with the primers described in Table 1 and the Verso 1-Step RT-PCR ReddyMix kit (Thermo Scientific). The reaction was conducted under the following conditions: 50°C for 30 min, 95°C for 2 min, 45 cycles at 95°C for 20 s, 50°C for 30 s, and 72°C for 2 min, followed by a final extension step at 72°C for 10 min. The RT-PCR products were purified using the GeneMATRIX Basic DNA Purification Kit (EurX). The complete sequences of the S-gene were obtained by using forward and reverse Sanger sequencing. The complete sequence of the S gene of 36 PEDV isolates from different farms can be accessed at the NCBI GenBank with the accession numbers MW251343 -MW251378 (Table 2 ). The remaining five PEDV isolates included in this research were previously sequenced by using a RNA virus-specific tailor-made NGS protocol (15 (link)).
Verso 1 step rt pcr reddymix kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Verso 1-Step RT-PCR ReddyMix kit is a reagent designed for reverse transcription and polymerase chain reaction (RT-PCR) in a single step. It contains all the necessary components, including reverse transcriptase and DNA polymerase, to perform RNA-to-cDNA conversion and subsequent PCR amplification.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (2 mentions)
Genematrix basic dna purification kit, by EURx (1 mentions)
C1000tm thermal cycler, by Bio-Rad (1 mentions)
Gel doc 1000, by Bio-Rad (1 mentions)
Redsafe nucleic acid staining solution, by iNtRON Biotechnology (1 mentions)
Geneamp 9700, by Thermo Fisher Scientific (1 mentions)
Rnase free dnase, by Qiagen (1 mentions)
Genejet rna purification, by Thermo Fisher Scientific (1 mentions)
Paclitaxel taxol, by Bristol-Myers Squibb (1 mentions)
Naringenin, by Merck Group (1 mentions)
Naringin, by Merck Group (1 mentions)
10 protocols using verso 1 step rt pcr reddymix kit
1
PEDV S-gene Amplification and Sequencing
2
RT-PCR Detection of Plant Viruses
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All RNA extracts were tested using primers targeting the coat protein region of the RNA2 of AMVV1, AMPV1 and AMPV2 (Table 2 ). Total RNA (1 μl) was added to a 24 μl reaction containing Verso™ 1-Step RT-PCR ReddyMix™ Kit (Thermo Scientific) and 400 mM of each primer. The assays were performed in a Bio-Rad C1000TM thermal cycler (Bio-Rad laboratories) using the cycling conditions,48 °C for 30 mins for the reverse transcription step, 98 °C for 2 mins, followed by 35 cycles of 98 °C for 10 s, 63 °C for 30 s and 72 °C for 1 min and a final step of 72 °C for 5 min. Amplification products were separated on a 1.2% agarose gel stained with ethidium bromide and visualised on a UV transilluminator.
3
RNA Extraction and RT-PCR Protocol
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Total RNA was isolated using the Qiagen RNeasy Mini Kit (Qiagen). RT–PCR was conducted using the Verso 1-Step RT-PCR ReddyMix Kit (Thermo Scientific, Waltham, MA, USA). For a standard reaction, 50 ng of total RNA was mixed together with 200 nM each of primers in a final volume of 25 μl. All primers and melting temperatures (Tm) used in the study are listed in Additional file 1 : Table S1. The amplification cycles for PCR were 50°C for 15 minutes, 95°C for 2 minutes, followed by 35 cycles of 95°C for 20 seconds, 50 to 60°C for 30 seconds and 72°C for 1 minute, followed by an additional extension for 5 minutes at 72°C. The amplified fragments were separated on 1.5% agarose gel in 1 × Tris-acetate EDTA (TAE) buffer at 100 V for one hour and visualized with ethidium bromide staining on a GelDoc 1000 (BioRad, Hercules, CA, USA).
4
Detection of Swine Coronaviruses by Duplex RT-PCR
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Two duplex RT-PCRs were developed for the detection of SeACoV (ORF1ab), TGEV/SeCoV (N gene), PEDV/SeCoV (S gene) and PDCoV (N gene) (Table 1 ). Two conventional RT-PCRs were also developed to confirm SeCoV by excluding a potential PEDV-TGEV co-infection, by detecting the M gene of PEDV and the S gene of TGEV. The RT-PCR was carried out with Verso 1-Step RT-PCR ReddyMix kit (Thermo Scientific). The reaction was conducted under the following conditions: 50°C for 30 min, 95°C for 2 min, 40 cycles at 95°C for 20 s, 50°C for 30 s, and 72°C for 1 min, followed by a final extension step at 72°C for 10 min.
The RT-PCR products were visualized on a 1.5% agarose gel containing RedSafe Nucleic Acid Staining Solution (iNtRON Biotechnology, Inc.). The length of the PCR fragment generated for each CoV is shown inTable 1 .
The RT-PCR products were visualized on a 1.5% agarose gel containing RedSafe Nucleic Acid Staining Solution (iNtRON Biotechnology, Inc.). The length of the PCR fragment generated for each CoV is shown in
5
Carrot Viral Pathogen Detection
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RNA was extracted from carrot roots by magnetic bead extraction using Invimag Virus DNA/RNA mini-kit (Invitek GMBH). Conventional RT-PCR was carried out for the presence of the four carrot viruses known to be common in the UK namely Parsnip yellow fleck virus[19] and the viruses of the Carrot Motley Dwarf complex, Carrot red leaf virus, Carrot Mottle virus [20] (link) and Carrot red leaf associated viral RNA [21] . All RT-PCR reactions were carried out using Verso 1-Step RT-PCR ReddyMix Kit (Thermo Scientific) on a GeneAmp 9700 (Applied Biosystems) (Annealing Temperature 50°C).
6
U87 Cell Total RNA Extraction and RT-PCR
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Total RNA was extracted from U87 cells with an RNeasy mini kit and depleted of contaminating DNA with RNase-free DNase (QIAGEN). RT-PCR was performed using the Verso 1-step RT-PCR ReddyMix Kit (Thermo) and 100 ng total RNA. The information of primers and procedures are detailed in Supplemental Materials.
7
FMDV Serotype Identification via RT-PCR
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The total RNA was extracted from FMDV isolates according to the manufacturer's instructions using Thermo scientific Gene Jet RNA purification (Thermoscientific, USA). Amplification of the VP1 coding region for each serotype were performed using one step PCR reaction kits with specific oligonucleotide primers according to (Table. 1), using Verso 1-step (RT-PCR) Reddy Mix kit (Thermoscientific, USA). The RT-PCR reaction was done in a final volume of 25 μl consists of consisted of 12.5 μl of 2X 1-step PCR Reddy Mix, 0.5 μl of Verso Enzyme Mix, 1.25 μl RT-Enhancer, 1 μl of specific forward and reverse primer for each serotype and 5 μl of RNA template. The cycling conditions were 50 °C for 30 min and 95 °C for 15 min, then 35 cycles of denaturation at 95 °C for 60 s, annealing at 60 °C for 30 sec and elongation at 72 °C for 1.5 min, followed by a final extension at 72°C for 5 min. The PCR products were analysed by electrophoresis on a 1.5% agarose gel.
8
cDNA Synthesis from Total RNA
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Preparation of C-DNA from total extracted RNA of five tissue samples, was carried out via Verso 1-Step RT-PCR Reddy Mix Kit (Thermo Scientific, AB-1454/LD/A) according to manufacturer protocol with the addition of two specific primers (primer listed in Table 1).
9
Taxol Hepatoprotective Assessment Protocol
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Taxol (paclitaxel) of batch number: 7E05628 was purchased from Bristol-Myers Squibb global biopharmaceutical company. Sigma (MO, USA) provided naringenin and naringin (batch codes: BCBM4171V and BCBJ2179V, respectively). Alkaline phosphatase (ALP) reagent kits, alanine transaminase (ALT), aspartate transaminase (AST), gamma-glutamyltransferase (GGT), and GGT were all bought from Biosystem S.A. (Spain) with catalogue numbers of M11533c-21, M11531c-21, M11584c-11, and M11592-0610, respectively. Lactate dehydrogenase (LDH) reagent kit (catalogue number: MX41214) acquired from Spin React (Spain). Albumin and total bilirubin kits (catalogue numbers 10560 and 10,742 respectively) were attained from HUMAN Gesellschaft für Biochemica und Diagnostica mbH, Wiesbaden, Germany. Verso 1-Step RT-PCR Reddy Mix Kit from Thermo Scientific (Applied Biosystems, Foster City, CA, USA) (catalogue number: AB-1454-LD). All other chemicals were purchased commercially and were of analytical quality.
10
Quantifying Hepatic AFP mRNA Expression
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To determine the mRNA expression of alpha-fetoprotein (AFP), total RNA was isolated from the liver tissue using the methods of Chomzynski and Sacchi (1987 (link)) and Hassan et al. (2021 ) and Thermo Scientific Verso 1-Step RT-PCR Reddy Mix Kit (Applied Biosystems, Foster City, CA, USA) as directed by the manufacturer in the presence of particular primers. The forward and reverse primer sequences for AFP are 5′dAACAGGGCGATGTCCATAA3′ and 5′dAATGGTGGGAGCATACAGG3′, respectively (Tsamandas et al. 2007 (link)). The AFP data was expressed as a fold change from the typical control.
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