For virus RNA extraction, 120 μL of thawed samples were incubated at 56 °C for 10 min with 330 μL of lysis solution and transferred to the Maxwell® RSC Instrument cartridges for RNA extraction according to manual instructions (Maxwell® 16 Viral Total Nucleic Acid Purification Kit, Promega, Wisconsin, USA). Undiluted RNA samples were subjected to a simplex RT-qPCR using the QuantiTect Probe RT-qPCR Kit (QIAGEN, Hilden, Germany) at 50 °C for 30 min (step 1), 95 °C for 15 min (step 2), and 45 cycles at 94 °C for 15 s (step 3) and 55 °C for 1 min (step 4) using the CFX96 Touch Real-Time PCR Detection System. Data collection occurred on step 4 using the FAM channel for all targets (N1, N2, and RP) and it was evaluated using the CFX Maestro Software. Human specimen control (HSC), Positive (2019-nCoV_N_Positive Control, Integrated DNA Technologies Inc, Iowa, USA), and Non-template control reactions were performed in every RT-qPCR run. The interpretation of the results (positive, negative, invalid or inconclusive) was performed according to the CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-qPCR Diagnostic Panel (USA CDC, 2020 ).
Quantitect probe rt qpcr kit
Manufactured by Qiagen
Sourced in Germany, United States
The QuantiTect probe RT-qPCR kit is a real-time reverse transcription-quantitative PCR (RT-qPCR) assay designed for the sensitive and accurate quantification of RNA targets. The kit includes all the necessary components for the reverse transcription and subsequent real-time PCR detection of RNA samples.
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Lab products found in correlation
Cfx96 touch real time pcr detection system, by Qiagen (1 mentions)
Maxwell 16 viral total nucleic acid purification kit, by Promega (1 mentions)
2019 ncov n positive control, by Integrated DNA Technologies (1 mentions)
Stepone v2, by Thermo Fisher Scientific (1 mentions)
E z n a viral rna extraction kit, by Omega Bio-Tek (1 mentions)
E z n a total rna extraction kit, by Omega Bio-Tek (1 mentions)
Stepone software, by Thermo Fisher Scientific (1 mentions)
4 protocols using quantitect probe rt qpcr kit
1
Automated SARS-CoV-2 RNA Extraction and Detection
2
Quantification of Respiratory Viruses by RT-qPCR
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Virus RNA was extracted using E.Z.N.A viral RNA extraction kit (Omega, Norcross, USA, R6874-02) and quantified by real-time RT-qPCR using a specific set of primers and probes targeting RSV-A [20 (link)] and HMPV (primers forward 5′-CATAYAARCATGCTATATTAAAAGAGTCTCA-3′ and reverse 5′-CCTATYTCWGCAGCATATTTGTARTCAG-3′; and probe 5′-Fam-CAACHGCAGTRACACCYTCATCATTRCA-TAMRA-3′) and QuantiTect probe RT-qPCR kit (Qiagen, Amtsgericht Düsseldorf, The Netherlands, 204443) in a StepOne Applied Biosystems thermocycler. Results were analyzed using the StepOneTM V2.0 Software (Applied Biosystems, Waltham, MA, USA).
3
Quantitative Viral RNA Detection Protocol
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Viral RNA was extracted from apical washes using the E.Z.N.A. viral RNA extraction kit (R6874-02; Omega Bio-tek, Inc., Norcross, GA, USA) and from tissue lysate using the E.Z.N.A. total RNA extraction kit (R6834-02; Omega Bio-tek, Inc., Norcross, GA, USA). For RNA quantification, primers and probes specific for each virus were used as previously described20 (link). The RNAseP housekeeping gene was used as an internal control for cell-associated virus quantification (4331182; Life Technology, Zug, Switzerland).
Quantitative real-time reverse transcriptase polymerase chain reaction (RT-qPCR) assays were performed using the QuantiTect probe RT-qPCR kit (no. 204443; Qiagen, Valencia, CA, USA) in a StepOne Applied Biosystems thermocycler (Thermo Fisher Scientific, Waltham, MA, USA). In each run and for each virus, four 10-fold serial dilutions of RNA reference standards were included. Results were analyzed using the StepOne version 2.0 software (Thermo Fisher Scientific, Waltham, MA, USA).
Quantitative real-time reverse transcriptase polymerase chain reaction (RT-qPCR) assays were performed using the QuantiTect probe RT-qPCR kit (no. 204443; Qiagen, Valencia, CA, USA) in a StepOne Applied Biosystems thermocycler (Thermo Fisher Scientific, Waltham, MA, USA). In each run and for each virus, four 10-fold serial dilutions of RNA reference standards were included. Results were analyzed using the StepOne version 2.0 software (Thermo Fisher Scientific, Waltham, MA, USA).
4
Real-time RT-qPCR for SARS-CoV-2 Quantification
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The assay was performed using the QuantiTect probe RT-qPCR kit (Qiagen #204443) in a StepOne Applied Biosystems thermocycler with the following cycling conditions: 50 • C for 30 min, 95 • C for 15 min, and 45 cycles of 94 • C for 15 s and 60 • C for 1 min. The reaction mixture (25 l) contains 900 nM of each primer and 200 nM of probe, two units of reverse transcriptase and 5 l of viral RNA extracts or transcripts. Results were analyzed using the StepOne TM software (Applied Biosystems). Of note the reaction mixture for the commercially available kit of multiplex real-time qPCR (Fast Track Diagnostics ® ; # FTD-2), contained 10 l of extracted RNA and 15 l of master mix, according to the manufacturer's instructions. To establish the standard curve for RNA quantification, four 10-fold serial dilutions of RNA reference standards were included in each batch of test. The threshold cycle (CT) values were used to quantify the RNA copy numbers per reaction using the slope-intercept form. The number of RNA copies per reaction was then corrected for the dilution factor performed between the extraction and the real-time RT-qPCR to obtain a final viral RNA copy number per ml of initial sample.
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