To assess if our disinfection process could effectively clean any possible SARS-CoV-2 contamination from a tablet computer surface, we performed an ex vivo study by spraying 20 microliters of a positive SARS-CoV-2 virus solution onto a computer in a P3 laboratory at NCKUH. After the computer surface was air-dried for 30 minutes, it was swabbed by technicians to obtain the first sample (V0), which indicated the surface virus load after initial exposure to the virus. After V0 sample collection, the technicians used 75% alcohol to mimic the disinfection procedure that was recommended in the current Taiwan CDC guideline. Samples V1 and V2 were collected after disinfecting the surface once and twice using 75% alcohol, respectively. The crossing point (CP), which is the maximum second derivative of the amplification curve of the real time–polymerase chain reaction (RT-PCR), was measured to obtain the viral load on the tablet computer surface using the COVID-19 Genesig RT-PCR assay (Primerdesign Ltd) using a LightCycler 480 II PCR platform (Roche Holding AG). A positive viral load was defined by the CP of amplification curve <45 of the RT-PCR result. Two specific genes, RNA-dependent RNA polymerase (RdRp) and Envelope (E) genes, were simultaneously assayed to determine the results.
Lightcycler 480 2 pcr platform
Manufactured by Roche
Sourced in Belgium, United Kingdom
The LightCycler 480 II is a real-time PCR (Polymerase Chain Reaction) platform manufactured by Roche. It is designed for fast and accurate detection and quantification of nucleic acid targets. The system employs a thermal cycler unit and optical detection module to perform real-time PCR experiments. It supports multiple detection chemistries and can accommodate a variety of sample types and reaction volumes.
Automatically generated - may contain errors
Lab products found in correlation
Sensifast sybr no rox kit, by Meridian Bioscience (4 mentions)
Iscript cdna synthesis kit, by Bio-Rad (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Covid 19 genesig rt pcr assay, by Primerdesign (1 mentions)
Sybr green supermix kit, by Takara Bio (1 mentions)
Reverse transcriptase, by Takara Bio (1 mentions)
Tri reagent, by Merck Group (1 mentions)
6 protocols using lightcycler 480 2 pcr platform
1
Evaluating Disinfection Efficacy Against SARS-CoV-2
2
qRT-PCR Analysis of Cas9 Expression
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Total RNA was extracted from cells using Trizol (ThermoFisher). For each sample, 1 μg total RNA was used for reverse transcription with reverse transcriptase (TaKaRa, Kyoto, Japan). qRT-PCR analysis was performed on the LightCycler 480 II PCR platform (Roche, Basel, Switzerland) using the SYBR Green Supermix kit (Takara). The PCR condition was as follows: 1 min of hot start at 95 °C, followed by 45 cycles of 95 °C for 10 s, 60 °C for 10 s, and 72 °C for 10 s. Blank controls with no cDNA templates were used to rule out contamination. The ampicillin resistance gene on the plasmid was used as the internal control. The specificity of the PCR product was confirmed by melting curve analysis and gel electrophoresis. Gene expression levels were normalized to the GAPDH housekeeping gene. Relative expression levels were calculated by the ΔΔCt method. The primers used were as follows:
AmpR-F: TTCTCAGAATGACTTGGTTG.
AmpR-R: GATCAAGGCGAGTTACATGA.
Cas9-F: GACATCGGCACCAACTCTG.
Cas9-R: TCCGGTTCTTCCGTCTGGT.
AmpR-F: TTCTCAGAATGACTTGGTTG.
AmpR-R: GATCAAGGCGAGTTACATGA.
Cas9-F: GACATCGGCACCAACTCTG.
Cas9-R: TCCGGTTCTTCCGTCTGGT.
3
Aorta Aneurysm Transcriptome Analysis
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MFS (n = 6) and non-MFS (n = 6; 3× Loeys-Dietz syndrome, 2× TAA, and 1× BAV) patients’ ascending aorta aneurysm tissues were collected during prophylactic aortic surgery. Permission was granted for the use of the anonymized samples by our local Ethical Board (reference: W16_037#16.052).
Human and murine aortic tissues were crushed in liquid nitrogen and dissolved in Trizol to isolate RNA according to the manufacturer’s protocol. Copy-DNA (cDNA) was synthesized using iScript cDNA synthesis kit (BioRad). Quantitative PCR (qPCR) was performed using SensiFAST SYBR No-ROX Kit (Bioline, Antwerp, Belgium) on a LightCycler 480II PCR platform (Roche). Cycle quantification and primer set amplification efficiency were calculated using the LinRegPCR software package (32 (link)). Primers were designed to quantify mRNA by qPCR (primer list provided in Supplemental Table 1, see section onsupplementary materials given at the end of this article). Target gene expression was normalized for housekeeping gene Rplp0 (P0). High CD45 (inflammation marker) mRNA expression was considered as aorta pathology and screened for complement factor expression.
Human and murine aortic tissues were crushed in liquid nitrogen and dissolved in Trizol to isolate RNA according to the manufacturer’s protocol. Copy-DNA (cDNA) was synthesized using iScript cDNA synthesis kit (BioRad). Quantitative PCR (qPCR) was performed using SensiFAST SYBR No-ROX Kit (Bioline, Antwerp, Belgium) on a LightCycler 480II PCR platform (Roche). Cycle quantification and primer set amplification efficiency were calculated using the LinRegPCR software package (32 (link)). Primers were designed to quantify mRNA by qPCR (primer list provided in Supplemental Table 1, see section on
4
Quantitative Real-Time PCR Procedure
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For quantitative real-time PCR (qPCR), total RNA was isolated using Trizol reagent (Invitrogen, USA) and gene expression was determined by qPCR using SensiFAST SYBR No-ROX Kit (Bioline, UK) on the LightCycler 480 II PCR platform (Roche, Switzerland). Primer sequences are shown in ESM Table 1 .
5
RNA Isolation and qPCR Analysis
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Total RNA was isolated using Trizol reagent (Invitrogen) according to the manufacturer's protocol. cDNA was synthesized using iScript cDNA synthesis kit (BioRad). Quantitative PCR was carried out using SensiFAST SYBR No-ROX Kit (Bioline) on a LightCycler 480 II PCR platform (Roche). Cycle quantification and primer set amplification efficiency were calculated using the LinRegPCR software package (Ruijter et al., 2009) . Target gene expression was normalized by dividing the geometric mean of the gene expression of Rplp0 and β-actin. Primer sequences are listed in Table S1.
6
Quantitative gene expression analysis
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Total RNA from cultured cells was isolated using TRIreagent (Sigma). Reverse transcription (RT-)PCR was performed on 1 μg RNA using iScript cDNA Synthesis Kit (BioRad). qPCR was carried out using SensiFAST SYBR No-ROX Kit (Bioline) on a LightCycler 480 II PCR platform (Roche). Quantification cycles (Cq), primer set amplification efficiencies, and transcript starting concentrations (N0) were calculated using LinRegPCR. Target gene expression was normalized by dividing by the geometric mean of Actb and Rplp0 housekeeping gene expression. Sequences of the primers used for RT-qPCR can be found in Table S3 .
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