The viral RNA from samples collected in the in vivo assays was quantified through quantitative reverse transcription polymerase chain reaction (RT-PCR). Total RNA was extracted using QIAamp Viral RNA (Qiagen, Germantown, MD, USA), according to manufacturer’s instructions. Quantitative RT-PCR was performed using Quanti Tect Probe RT-PCR Kit (Qiagen) in a StepOne Plus™ Real-Time PCR System (Thermo Fisher Scientific). Amplifications were carried out in 15 µL reaction mixtures containing 2× reaction mix buffer, 50 µM of each primer, 10 µM of the probe and 5 µL of RNA template. Primers, probes and cycling conditions recommended by the Centers for Disease Control and Prevention (CDC) protocol were used to detect the SARS-CoV-2 (CDC 2020). Amplification of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a reference for the number of cells. The cycle threshold (CT) values for this target were compared to those obtained with different cell quantities (107 to 102), for calibration.
Qiaamp viral rna
Manufactured by Qiagen
Sourced in Germany, United States
The QIAamp Viral RNA Kit is a laboratory equipment product designed for the purification of viral RNA from various sample types. It employs a silica-based membrane technology to efficiently capture and purify viral RNA, which can then be used for downstream analysis and applications.
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Lab products found in correlation
Stepone real time pcr system, by Thermo Fisher Scientific (9 mentions)
Rneasy mini kit, by Qiagen (4 mentions)
Gotaq probe qpcr, by Promega (4 mentions)
Rt qpcr system, by Promega (4 mentions)
Quantitect probe rt pcr kit, by Qiagen (4 mentions)
Eurobiogreen lo rox qpcr mix, by Eurobio Scientific (2 mentions)
Abi prism 7300 sequence detection system, by Thermo Fisher Scientific (2 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Gdna eraser, by Takara Bio (1 mentions)
Quantinova probe rt pcr kit, by Qiagen (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Ptc 100 programmable thermal controller, by Bio-Rad (1 mentions)
Pd n 6, by Cytiva (1 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Realstar sars cov 2, by Altona Diagnostics (1 mentions)
Geneamp pcr system 2400, by Thermo Fisher Scientific (1 mentions)
Rnase inhibitor, by Promega (1 mentions)
Onestep rt pcr kit, by Qiagen (1 mentions)
30 protocols using qiaamp viral rna
1
SARS-CoV-2 Viral RNA Quantification via RT-PCR
2
RNA Extraction from Allantoic Fluid
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IBV RNA and negative control RNA (NDV [183.6 ng/µl], IBDV [67.5 ng/µl], FLUAV [92.1 ng/µl]) were extracted from 140 µl of supernatant from allantoic fluid using the QIAamp Viral RNA or RNeasy Mini Kits (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. Each RNA fraction was eluted in 50 µl of RNase-free water.
3
Longitudinal Surveillance of Pediatric Norovirus
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Routine stool collection was conducted monthly from age 1 to 12 months, and at 15, 18, 21, and 24 months of age. Homes were visited twice weekly for active surveillance of diarrhea and other symptoms, including fever, vomiting, cough, and appetite, to generate a continuous illness history. Diarrhea was defined as ≥3 loose stools in a 24-hour period and separated by at least 2 diarrhea-free days. Children experiencing moderate to severe diarrhea were referred to local health services.
Nucleic acid extraction was performed with the QIAamp viral RNA (Qiagen, Valencia, California), and real-time reverse transcriptase polymerase chain reaction was used to detect genogroups [11 (link)]. The presence of other enteropathogens was assessed per previously reported microbiologic methods [11 (link)].
Norovirus testing was performed on all diarrheal stools collected in the study. Additionally, 10% of children at each site were randomly selected by identification number to have their routine stool samples tested for norovirus. Longitudinal analyses were performed using this subsample, whose symptomatic and asymptomatic history of infection was captured. Additional analyses were performed on diarrheal stools from the full study population to describe coinfection and disease severity.
Nucleic acid extraction was performed with the QIAamp viral RNA (Qiagen, Valencia, California), and real-time reverse transcriptase polymerase chain reaction was used to detect genogroups [11 (link)]. The presence of other enteropathogens was assessed per previously reported microbiologic methods [11 (link)].
Norovirus testing was performed on all diarrheal stools collected in the study. Additionally, 10% of children at each site were randomly selected by identification number to have their routine stool samples tested for norovirus. Longitudinal analyses were performed using this subsample, whose symptomatic and asymptomatic history of infection was captured. Additional analyses were performed on diarrheal stools from the full study population to describe coinfection and disease severity.
4
Viral RNA Extraction and Quantification
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Viral RNAs were extracted using QIAamp viral RNA (Qiagen) and reverse transcriptions were performed using a PrimeScript RT reagent kit with a gDNA Eraser (Takara Bio, Inc., Kusatsu, Shiga Prefecture, Japan). Viral RNA in animal samples were quantified by QuantiNova Probe RT-PCR kit (QIAGEN, Inc., Hilden, Germany) using primers targeting RNA-dependent RNA polymerase (RdRp). Taqman probe and qPCR primers were adopted from previous studies [10 (link)].
5
Nucleic Acid Extraction and cDNA Synthesis
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Nucleic acids were extracted from 140 μL of the eluate to obtain a final volume of 60 μL, using the QIAamp Viral RNA (Qiagen, Inc., Valencia, CA) according to the manufacturer’s instructions.
cDNA synthesis was carried out by RT using a random primer (PdN6; 50A260 units; Amersham Biosciences, Chalfont St Giles, Buckinghamshire, UK) for RV, NoV, HAV 2 μL of dimethyl sulfoxide (Sigma, St. Louis, MO) and 10 μL of RNA were mixed briefly, heated at 97 °C for 7 min, and chilled in ice for 4 min. The components of the mixture and their final concentrations for a 50-μL RT reaction were carried out as follows: 2.5 mM each deoxynucleoside triphosphate (GIBCO BRL, Life Technologies, Inc., Grand Island, NY), 1.5 mM MgCl2, 200U of Superscript III reverse transcriptase (Invitrogen), and 1 μL of PdN6. The RT reaction mixture was incubated in a thermal cycler (PTC-100 Programmable Thermal Controller; MJ Research, Inc., Watertown, MA) at 25 °C for 5 min, 50 °C for 60 min and 70 °C for 20 min.
cDNA synthesis was carried out by RT using a random primer (PdN6; 50A260 units; Amersham Biosciences, Chalfont St Giles, Buckinghamshire, UK) for RV, NoV, HAV 2 μL of dimethyl sulfoxide (Sigma, St. Louis, MO) and 10 μL of RNA were mixed briefly, heated at 97 °C for 7 min, and chilled in ice for 4 min. The components of the mixture and their final concentrations for a 50-μL RT reaction were carried out as follows: 2.5 mM each deoxynucleoside triphosphate (GIBCO BRL, Life Technologies, Inc., Grand Island, NY), 1.5 mM MgCl2, 200U of Superscript III reverse transcriptase (Invitrogen), and 1 μL of PdN6. The RT reaction mixture was incubated in a thermal cycler (PTC-100 Programmable Thermal Controller; MJ Research, Inc., Watertown, MA) at 25 °C for 5 min, 50 °C for 60 min and 70 °C for 20 min.
6
COVID-19 Imaging and Molecular Monitoring
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The local ethical committee approved this study and written informed consent for participation in the study was obtained from all enrolled patients. A cohort of 52 patients was prospectively enrolled in the study. Inclusion criteria included prior COVID-19 confirmed by RT-PCR testing, prior CT scan during the active phase of the disease documenting imaging signs compatible with those known to be related to COVID-19, remission of clinical symptoms, negative PCR outcomes of two separate tests performed 24 h apart and an indication to undergo a follow-up CT scan to document the resolution of prior imaging findings. Patients with absolute contraindications to MRI were excluded.
RT-PCR (RealStar® SARS-CoV-2; Altona Diagnostics, Hamburg, Germany) was performed following the RNA extraction (QIAamp® Viral RNA; Qiagen, Hilden, Germany) of samples collected using upper nasopharyngeal as well as oropharyngeal swabs, both to confirm the diagnosis and to document recovery.
RT-PCR (RealStar® SARS-CoV-2; Altona Diagnostics, Hamburg, Germany) was performed following the RNA extraction (QIAamp® Viral RNA; Qiagen, Hilden, Germany) of samples collected using upper nasopharyngeal as well as oropharyngeal swabs, both to confirm the diagnosis and to document recovery.
7
Viral RNA Extraction and Sequencing
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Total RNAs were extracted from the virus suspensions using the QIAamp Viral RNA (QIAGEN, Venlo, Netherlands). The extracted RNAs were used as targets for amplification of VP1 to VP4 regions by reverse transcription-polymerase chain reaction (RT-PCR) using the One Step RT-PCR kit (QIAGEN). Briefly, a total 50 μl volume for RT-PCR consisted of 10 μl of RNA template in master mix containing 1X RT-PCR buffer, 2 μl of enzyme mix, 2 μl of dNTP mix, 5 units of RNase inhibitor (Promega Corporation, Madison, WI), and 400 nM of primers. The reaction was carried out in a thermal cycler (GeneAmp PCR system 2400, Applied Biosystems, Foster City, CA). The PCR cycling program consisted of one cycle at 50 °C for 30 min and one cycle at 95 °C for 15 min, followed by 35 cycles at 94 °C for 30 s, 55 °C for 30 s, 72 °C for 60 s and a final extension step cycle at 72 °C for 10 min. The amplified product was electrophoresed in 1.5% agarose gel in TAE buffer and purified using a QIAEX II gel extraction kit (QIAGEN). The primer sets, shown in an Additional file 1 (Table S1), were designed by our group and used for both RT-PCR and nucleotide sequencing. The purified PCR products were sequenced at Macrogen Korea. The nucleotide sequences were edited and aligned using Bioedit version 7.2.6.1.
8
Quantitative PCR for Viral RNA
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Viral RNA was extracted from culture supernatants and cells using the QIAamp Viral RNA and RNeasy Minikits, respectively (all from Qiagen, Hilden, Germany), according to the manufacturer’s instructions. Quantitative PCR for viral RNA was performed with the EuroBioGreen Lo-ROX qPCR mix (Eurobio, Les Ulis, France), using primers 5′-CTTTCCCCTGGCGTGTCA-3′ and 5′-GAATTTTGAAGGCTGCCTTGA-3′ for MOPV and 5′-CTCTCACCCGGAGTATCT-3′ and 5′-CCTCAATCAATGGATGGC-3′ for LASV.
9
Quantitative RT-PCR for SARS-CoV-2 Viral Detection
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Total viral RNA from a culture supernatant and/or monolayers was extracted using QIAamp Viral RNA (Qiagen®) according to the manufacturer’s instructions. Quantitative RT‒PCR was performed using GoTaq® Probe qPCR and RT-qPCR Systems (Promega) in a StepOne™ Real-Time PCR System (Thermo Fisher Scientific) ABI PRISM 7500 Sequence Detection System (Applied Biosystems). Amplifications were carried out in 25 µL reaction mixtures containing 2× reaction mix buffer, 50 µM of each primer, 10 µM of probe, and 5 µL of RNA template. Primers, probes, and cycling conditions recommended by the Centers for Disease Control and Prevention (CDC) protocol were used to detect SARS-CoV-252 . The standard curve method was employed for virus quantification. For reference to the cell amounts used, the housekeeping gene RNAse P (F-5′-AGATTTGGACCTGCGAGCG-3′, R-5′GAGCGGCTGTCTCCACAAGT-3′, P-5′-FAM-TTCTGACCTGAAGGCTCTGCGCG-BHQ-1-3′) was amplified. The Ct values for this target were compared to those obtained for different cell amounts, 107 to 102, for calibration. Alternatively, genomic (ORF1; F-5′-ATGAGCTTAGTCCTGTTG-3′, R-5′-CTCCCTTTGTTGTGTTGT-3′, P–5′-FAM-AGATGTCTTGTGCTGCCGGTA-BHQ-1-3′) and subgenomic (ORFE; F-5′ACAGGTACGTTAATAGTTAATAGCGT-3′, R-5′-ATATTGCAGCAGTACGCACACA-3′, P-5′-FAM-ACACTAGCCATCCTTACTGCGCTTCG-BHQ-1-3′) were detected, as described elsewhere53 (link).
10
Viral RNA Extraction and qPCR for MOPV and LASV
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Viral RNA was extracted from culture supernatants and cells using the QIAamp Viral RNA and RNeasy Minikits, respectively (all from Qiagen), according to the manufacturer’s instructions. Quantitative PCR for viral RNA was performed with the EuroBioGreen Lo-ROX qPCR mix (Eurobio, Les Ulis, France), using primers 5’-CTTTCCCCTGGCGTGTCA-3’ and 5’-GAATTTTGAAGGCTGCCTTGA-3’ for MOPV and 5’-CTCTCACCCGGAGTATCT-3’ and 5’-CCTCAATCAATGGATGGC-3’ for LASV.
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