A synthetic RNA template, including the envelope gene region targeted by the Gibb system [16 (link)], was produced using the MEGAshortscript T7 Transcription Kit (Thermo Fisher Scientific) and quantified by spectrophotometry. EBOV genomic RNA was detected in NHP plasma samples by real-time reverse transcription PCR using the Gibb system and the GoTaq Probe 1-Step RT-qPCR Kit (Promega) following manufacturer’s instructions. Quantification was performed with reference to the standard curve obtained from serial dilutions of the standardized synthetic RNA template.
Gotaq probe 1 step rt qpcr kit
Manufactured by Promega
Sourced in United States, France
The GoTaq Probe 1-Step RT-qPCR kit is a reagent system for the reverse transcription and real-time quantitative PCR (RT-qPCR) detection of RNA targets. The kit includes all necessary components, including a thermostable reverse transcriptase and DNA polymerase, to perform a one-step RT-qPCR reaction.
Automatically generated - may contain errors
Lab products found in correlation
Qiaamp viral rna mini kit, by Qiagen (3 mentions)
Quantstudio12k flex real time pcr machine, by Thermo Fisher Scientific (2 mentions)
Qiaamp viral rna kit, by Qiagen (2 mentions)
Qiacube, by Qiagen (2 mentions)
Express one step superscript rt qpcr, by Thermo Fisher Scientific (2 mentions)
Rnase free dnase set, by Qiagen (2 mentions)
Megashortscript t7 transcription kit, by Thermo Fisher Scientific (1 mentions)
Cfx96 touch real time pcr detection system, by Bio-Rad (1 mentions)
Purelink viral rna dna mini kit, by Thermo Fisher Scientific (1 mentions)
Evagreen dye, by Biotium (1 mentions)
Bst 2.0 warmstart dna polymerase, by New England Biolabs (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Betaine, by Merck Group (1 mentions)
Calcein, by Merck Group (1 mentions)
Warmstart rtx reverse transcriptase, by New England Biolabs (1 mentions)
Synthetic sars cov 2 rna control, by Twist Bioscience (1 mentions)
Mgso4, by New England Biolabs (1 mentions)
Quantstudio 5 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Steponeplus real time pcr machine, by Thermo Fisher Scientific (1 mentions)
Stepone software, by Thermo Fisher Scientific (1 mentions)
8 protocols using gotaq probe 1 step rt qpcr kit
1
Quantitative EBOV RNA Detection in NHP Plasma
2
Extraction and Detection of HIV RNA
3
Multiplex RT-qPCR for Respiratory Viruses
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Nucleic acids were extracted from respiratory samples using the PureLink™ Viral RNA/DNA Mini Kit (Invitrogen®, Thermo Fisher Scientific©), according to the manufacturer’s protocol. All samples were initially tested for Influenza A and B in a TaqMan® one-step real-time RT-PCR (RT-qPCR) assay using specific primers and probes for influenza (CDC, USA), according to the manufacturer’s recommendations. Additionally, an RT-qPCR assay was performed to identify positive RSV cases using a GoTaq® Probe 1-Step RT-qPCR Kit (Promega, Madison, WI, EUA). RSV positive samples (i.e. those with cycle threshold [CT] ≤ 40) were subtyped using specific primers and probes for RSV-A and RSV-B N genes. In parallel, Ribonuclease P RNA (RNase P) was used as an internal control for each sample and, in all batches, RNA extraction negative control (MOCK) and a PCR negative control (NTC) were used. All primers and probes are described in the S1 Table .
4
SARS-CoV-2 Detection Protocol
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Betaine (5.0 M), calcein (5 G), manganese (II) chloride tetrahydrate (100 G), and dimethyl sulfoxide (DMSO) were purchased from MilliporeSigma (Burlington, MA). EvaGreen dye (20 ×) was purchased from Biotium (Fremont, CA). Microseal 'B' PCR Plate Sealing Film, adhesive, optical #MSB1001, and mineral oil #1632129 were purchased from Bio-Rad Laboratories (Hercules, CA). The QIAamp Viral RNA Mini Kit (50) was purchased from Qiagen (Hilden, Germany). Nuclease-free water, dNTP mix (10 mM of each), 10 × isothermal amplification buffer, MgSO4 (100 mM), extreme thermostable single-stranded DNA binding protein (ET SSB; 500 µg mL-1), Bst 2.0 WarmStart DNA polymerase (Bst 2.0 WS; 8000 U mL-1), and WarmStart RTx reverse transcriptase (WS RTx; 15,000 U mL-1) were purchased from New England BioLabs (Ipswich, MA). The GoTaq Probe 1-Step RT-qPCR kit was purchased from Promega (Madison, WI). The synthetic SARS-CoV-2 RNA control was purchased from Twist Bioscience (South San Francisco, CA). POP7 plasmid, SARS-CoV N plasmid control, MERS-CoV N plasmid control, and all of the primers were purchased from Integrated DNA Technologies (Coralville, IA). A total of 20 de-identified, clinical NP swab samples were handled in compliance with ethical regulations and the approval of the Institutional Review Board of the University of Connecticut Health Center (protocol #P61067).
5
SARS-CoV-2 RNA Detection by RT-PCR
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Extraction of the total RNA from samples was performed using the QIAamp Viral RNA Mini Kit (Qiagen, USA), according to protocols provided by the manufacturer. From the 1.0 mL of DS on the collection tube, 140 μL were used for RNA extraction and the remaining volume was frozen at −80 °C for eventual repetitions or further testing. The real-time polymerase chain reaction (RT-PCR) was performed using primers and probes described in two different protocols: the United States Center for Disease Control and Prevention (CDC) protocol [20 ] and the Berlin (Charité/Berlin) protocol [21 (link)]. The viral gene coding for the N protein was targeted for the detection of SARS-CoV-2 RNA using the CDC protocol, and the viral gene E was used in the Charité/Berlin protocol. Probes and primers for the human RNAse P mRNA were used in both protocols as an endogenous reaction control. Reactions were carried out with the Promega GoTaq® Probe 1-Step RT-qPCR Kit (Promega, France) according to the manufacturer’s recommendations, using the QuantStudio™ 5 real-time PCR system (Thermo Fisher, USA).
6
RT-qPCR Assay for SARS-CoV-2 Detection
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RT-qPCR diagnostic assay for SARS-CoV-2 was performed using the CDC 2019-Novel Coronavirus Real-Time RT-PCR Diagnostic Panel for N1 and N2 assays (Research Use only kits, IDT technologies), following the in-vitro diagnostic procedures specified by the US Centers for Disease Control and Prevention (CDC-006-00,019, Revision 6). The GoTaq Probe 1-step RT-qPCR kit (Promega) was used, with the Twist Synthetic SARS-CoV-2 RNA control (Twist Bioscience) as template. Briefly, a 20 μl reaction was set up consisting of 5ul RNA template, 3.1 μl nuclease-free water, 1.5 μl primer mix, 10 μl qPCR master mix, 0.4 μl Go Script RT Mix. RT-qPCR was performed using the StepOne Plus Real Time PCR machine (Thermo Fisher), using the thermal profile: 45 °C for 15 min; 95 °C for 2 min; 45 cycles of 95 °C for 3 s, 55 °C for 30 s. The ΔRn values and cycle numbers were extracted for analysis using the Step One software (Thermo Fisher).
7
SARS-CoV-2 RNA Quantification from Samples
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Viral RNA was isolated from 100μL of cell supernatant medium using a QIAamp Viral RNA kit and RNase-Free DNase Set on the automated QIAcube (Qiagen) facility, following the manufacturer's instructions. Relative quantification of viral RNA was performed using the express One-Step SuperScript ® RT-qPCR (Invitrogen). To isolate viral RNA from tissues, 100µL of organ clarified homogenates, spiked with 10µL of internal control (bacteriophage MS2) 27 , were transferred into an S-block containing the recommended volumes of VXL, proteinase K and RNA carrier. The RT-qPCR reaction mixture (for SARS-CoV-2 and MS2 viral genome detection) was processed using the GoTaq Probe 1-Step RT-qPCR kit (Promega) and contained 5μL of Master Mix 2X, 0.25μL of each primer (500 nM), 0.07μL of probe (75 nM), 0.2μL of GoScript RT mix, 0.4µL of H2O and 3.8μL of extracted nucleic acids. Assays were performed using the QuantStudio 12K Flex Real-Time PCR machine (ThermoFisher Scientific) with the following conditions: 50°C for 15 min and 95°C for 2 min, followed by 45 cycles of 95°C for 3 s and 60°C for 30 s. Data collection occurred during the 60°C step. The amount of viral RNA was calculated from standard curves using synthetic RNA. The primers and probes used are described in supplemental table 4.
8
SARS-CoV-2 RNA Quantification from Samples
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Viral RNA was isolated from 100μL of cell supernatant medium using a QIAamp Viral RNA kit and RNase-Free DNase Set on the automated QIAcube (Qiagen) facility, following the manufacturer's instructions. Relative quantification of viral RNA was performed using the express One-Step SuperScript ® RT-qPCR (Invitrogen). To isolate viral RNA from tissues, 100µL of organ clarified homogenates, spiked with 10µL of internal control (bacteriophage MS2) 27 , were transferred into an S-block containing the recommended volumes of VXL, proteinase K and RNA carrier. The RT-qPCR reaction mixture (for SARS-CoV-2 and MS2 viral genome detection) was processed using the GoTaq Probe 1-Step RT-qPCR kit (Promega) and contained 5μL of Master Mix 2X, 0.25μL of each primer (500 nM), 0.07μL of probe (75 nM), 0.2μL of GoScript RT mix, 0.4µL of H2O and 3.8μL of extracted nucleic acids. Assays were performed using the QuantStudio 12K Flex Real-Time PCR machine (ThermoFisher Scientific) with the following conditions: 50°C for 15 min and 95°C for 2 min, followed by 45 cycles of 95°C for 3 s and 60°C for 30 s. Data collection occurred during the 60°C step. The amount of viral RNA was calculated from standard curves using synthetic RNA. The primers and probes used are described in supplemental table 4.
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