The virus specific real time PCR mix for all viruses except rabies virus (RV) was composed of 1× QuantiTect Virus + ROX Vial Kit (Qiagen, Manchester, UK), forward and reverse primers at a final concentration of 0.4 μM and virus specific TaqMan probe at a final concentration of 0.2 μM, 1× ROX, 3 μl of template DNA and water to total a volume of 20 μl (Mcgoldrick et al., 1998 (link), Lanciotti et al., 2000 (link), Marriott et al., 2006 (link), Bilk et al., 2012 ) The thermal profile used was 95 °C for 5 min and 45 cycles of 95 °C for 15 s, 60 °C for 45 s. The 18S rRNA real time PCR was performed using 0.6 μl 18S rRNA primers/probe mix (Life Technologies, Paisley, UK), the QuantiTect Virus + ROX Vial Kit as described above and 2 μl template DNA. For RV, 10 μl Brilliant® II SYBR® Green QPCR with low ROX master mix (Agilent Technologies, Cheshire, UK) was used with JW12 and N165-146 primers, each totalling a final concentration of 1 μM, 3 μl template DNA and water to a final volume of 20 μl (Wakeley et al., 2005 (link)). The thermal profile used was 94 °C for 2 min, 45 cycles of 95 °C for 1 min, 55 °C for 30 s and 72 °C for 20 s. Each sample was tested in duplicate and a no-template control (NTC) was also included in each run to check for cross contamination and background noise.
Quantitect virus rox vial kit
Manufactured by Qiagen
Sourced in Canada
The QuantiTect Virus + ROX Vial Kit is a laboratory equipment product designed for the quantitative detection of viral nucleic acids. It includes reagents and consumables necessary for the real-time reverse transcription-polymerase chain reaction (RT-qPCR) analysis of viral samples.
Automatically generated - may contain errors
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8 protocols using quantitect virus rox vial kit
1
Real-Time PCR for Virus Detection
2
SARS-CoV-2, Influenza A, and RSV Detection
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A volume of 100 μL of apical washes was used to extract viral RNA (MagNA Pure LC, Total nucleic acid isolation kit, Roche Molecular System, Laval, QC, Canada). The viral RNA loads were determined by one-step reverse-transcription quantitative PCR (RT-qPCR) assays by using primers and probes to target the E gene of SARS-CoV-2 [24 (link)], the M gene of influenza A/H1N1 (available upon request) and the N gene of RSV-A2 [25 (link)] with the QuantiTect Virus + ROX Vial Kit (Qiagen, Toronto, ON, Canada) on a LightCycler® 480 system (Roche Molecular System).
3
Quantifying Influenza Virus Infection in Human Airway Epithelial Cells
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The apical layers of HAE were gently washed with warm opti-MEM before infection. The epithelium was infected at an MOI of 0.002 with MCV19 or rg-PKT13 and their corresponding PA I38T variants. Viruses were adsorbed at 33 °C under a 5% CO2 atmosphere for 30 min. Then, the inoculum was harvested and the HAE was cultured at the air–liquid interface. Apical washes were collected at 0, 12, 24, 48, 96, and 120 h p.i. RNA extraction was performed from 90 µL of HAE apical washes using the MagNA Pure LC (Total nucleic acid isolation kit, Roche Molecular System, Laval, QC, Canada). Reverse transcription-quantitative PCR (qRT-PCR) assay was performed using primers targeting the influenza NS1 gene of IBV (available upon request). This assay was performed with the QuantiTect Virus + ROX Vial Kit (Qiagen, Toronto, ON, Canada) on a LightCycler® 480 system (Roche Molecular System).
4
Quantitative RT-qPCR for EAV Genome Detection
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RNA extraction was performed from 150 µl of ED cell supernatant using the QIAamp viral RNA kit (Qiagen). EAV genome copy number was determined by one step RT-qPCR using the QuantiTect Virus + ROX Vial Kit (Qiagen). Each RNA sample was tested in triplicate. The primers used to quantify the EAV genome copy number are EAV ORF7F 5′-GGCGACAGCCTACAAGCTACA-3′, EAV ORF7R 5′-CGGCATCTGCAGTGAGTGA-3′ and probe is EAV ORF7P [6FAM]-TTGCGGACCCGCATCTGACCAA-[TAMRA]. The RT-qPCR program started with a retro-transcription step of 20 min at 50 °C, an initial incubation step of 5 min at 95 °C, and 40 cycles of a 2-step program combining 15 sec at 95 °C followed by 45 sec at 60 °C and fluorescence measurement. The RT-qPCR reactions were performed on a CFX Connect Real-Time PCR System (Biorad).
5
SARS-CoV-2 RT-PCR Optimization and Validation
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Choice criteria for SARS-CoV-2 real-time RT-PCR protocol were mainly WHO recommendations (WHO, 2020) with reliable published validation protocols (Corman, 2020; Konrad, 2020) and rapid availability of reagents.
The targeted viral genes were E gene for Sarbecovirus assay as first-line screening and RdRp gene for SARS-CoV-2 specific assay as SARS-CoV-2 confirmatory screening. Primer-probe sets were chosen and ordered from Tib-MolBiol as described by Corman et al. (2020), with the use of SARS-CoV-2 specific probe RdRP-SARSr-P2 -Table 1 .
Real time RT-PCR was initially performed with the QuantiTect Virus + ROX Vial kit (QIAGEN) on the Rotor-Gene Q Real-Time PCR Detection System (QIAGEN), using Green channel for reading. Since we had a supply problem for RT-PCR Master Mixes, we had to adapt PCR protocol for four different reagents during the first month of establishment (Takyon™ two-step RT-PCR mix (Eurogentec), Takyon™ one-step RT-PCR mix (Eurogentec), LightCycler Multiplex RNA Virus Master (Roche) and SuperScript™ III Platinum™ RT-PCR mix (Invitrogen)); cycling conditions are presented inTable 2 . Moreover, each time we had to pass the standards dilution ranges, two negative eluates and up to three SARS-CoV-2 positive eluates. Primers and probes were however the same for all RT-PCR mixes.
The targeted viral genes were E gene for Sarbecovirus assay as first-line screening and RdRp gene for SARS-CoV-2 specific assay as SARS-CoV-2 confirmatory screening. Primer-probe sets were chosen and ordered from Tib-MolBiol as described by Corman et al. (2020), with the use of SARS-CoV-2 specific probe RdRP-SARSr-P2 -
Real time RT-PCR was initially performed with the QuantiTect Virus + ROX Vial kit (QIAGEN) on the Rotor-Gene Q Real-Time PCR Detection System (QIAGEN), using Green channel for reading. Since we had a supply problem for RT-PCR Master Mixes, we had to adapt PCR protocol for four different reagents during the first month of establishment (Takyon™ two-step RT-PCR mix (Eurogentec), Takyon™ one-step RT-PCR mix (Eurogentec), LightCycler Multiplex RNA Virus Master (Roche) and SuperScript™ III Platinum™ RT-PCR mix (Invitrogen)); cycling conditions are presented in
6
Quantitative RT-PCR for Clinical Isolates
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The reaction was performed using a QuantiTect® Virus + ROX Vial Kit (QIAGEN) according to the manufacturer's instructions and as described previously (Nakauchi et al., 2011a (link)). For testing the clinical isolates using cultured medium, the 20 μL assay contained 4 μL of 5× QuantiTect Virus NR Master Mix, 0.2 μL QuantiTect Virus RT Mix, 1.2 μL each of two 10 μM forward primers, 1.2 μL each of two 10 μM reverse primers, 0.4 μL each of two 5 μM probes, 7.6 or 8 μL distilled water, and 2 μL culture medium. For testing the clinical specimens using extracted RNA, 5 μL of RNA template were used. Cycling was performed as follows: 20 min at 50 °C to activate RT, followed by an initial denaturation step for 5 min at 95 °C and 45 cycles of amplification (denaturation at 95 °C for 15 s and annealing as well as extension at 56 °C for 45 s) using a LightCycler® 480 (Roche Molecular Biochemicals). Fluorescent signals were collected during the annealing and extension steps, and the amplification and endpoint data were analyzed using Light Cycler® 480 SW1.5 software according to the manufacturer's instructions.
7
SARS-CoV-2 Detection and Genomic Surveillance
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Laboratory testing involved two swabs (nasopharyngeal and oropharyngeal, pooled) that were stored in viral transport medium and cooled. RNA was extracted using the QiAamp Bio Robot Kit (Qiagen; Hilden, Germany) on a Hamilton Microlab Star as recommended by the manufacturer. Real-time RT-PCR was done with the QuantiTect Virus +Rox Vial Kit (Qiagen, Hilden, Germany) on the Bio-Rad CFX96 Touch Real-Time PCR Detection System. Primer and probes were used as described by Corman and colleagues5 and provided by Tib-Molbiol (Berlin, Germany). The reference laboratory worked exactly as described in Corman and colleagues.5 Whole genome sequencing involved Roche KAPA HyperPlus library preparation and sequencing on Illumina NextSeq and MiSeq instruments as well as RT-PCR product sequencing on Oxford Nanopore MinION using the primers described in Corman and colleagues.6 Patient 1 was sequenced on all three platforms; patients 2–7 were sequenced on Illumina NextSeq, both with and without RT-PCR product sequencing with primers as in Corman and colleagues;6 and patients 8–11, 14, and 16 were sequenced on Oxford Nanopore MinION. Sequencing of patient 15 was not successful. Sequence gaps were filled by Sanger sequencing.
8
Influenza Virus RNA Extraction and Detection
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Viral RNA extraction was performed from 90 μL of supernatants from A549 and Calu3 cells or of HAE apical washes using the MagNA Pure LC system (Total nucleic acid isolation kit, Roche Molecular System, Laval, QC, Canada). Reverse transcription quantitative PCR (qRT-PCR) assay was performed using primers targeting the influenza NS1 gene of influenza B viruses (available upon request). This assay was performed with the QuantiTect Virus + ROX Vial Kit (Qiagen, Toronto, ON, Canada) on a LightCycler® 480 system (Roche Molecular System).
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