We assessed the suitability of the Dulbecco’s modified Eagle medium (DMEM) for use in testing specimens by comparing it with the universal transport medium (UTM). First, ten positive NPS specimens were simultaneously spiked in 3 ml DMEM and 3 ml UTM and then analyzed by rRT-PCR using the Roche Cobas 6800 system. Then, to further evaluate the sensitivity of the UTM, a patient positive sample showing a Ct value around 33 was serial diluted in DMEM or UTM transport media and then analyzed by rRT-PCR using the Roche Cobas 6800 system.
Cobas 6800 system
Manufactured by Roche
Sourced in Switzerland, United States, Germany
The Cobas 6800 system is a fully automated, high-throughput molecular testing platform designed for clinical laboratories. It offers efficient sample processing and delivers reliable results for a variety of diagnostic applications.
Automatically generated - may contain errors
Lab products found in correlation
Cobas sars cov 2 test, by Roche (14 mentions)
Abi 7500 fast, by Thermo Fisher Scientific (6 mentions)
Taqpath covid 19 combo kit, by Thermo Fisher Scientific (5 mentions)
Xpert xpress sars cov 2, by Cepheid (5 mentions)
Cobas sars cov 2 kit, by Roche (4 mentions)
Qiasymphony platform, by Qiagen (3 mentions)
Qiaamp viral rna mini kit, by Qiagen (3 mentions)
Genexpert system, by Cepheid (3 mentions)
Kingfisher flex, by Thermo Fisher Scientific (3 mentions)
Exiprep 96 lite, by Bioneer (3 mentions)
Abi 7500 fast, by Roche (2 mentions)
Elecsys anti sars cov 2, by Roche (2 mentions)
Liaison xl murex hiv ab ag kit, by DiaSorin (2 mentions)
Cobas 8000, by Roche (2 mentions)
Chemagic 360, by PerkinElmer (2 mentions)
Chemagic 360 instrument, by PerkinElmer (2 mentions)
Alinity m sars cov 2 assay, by Abbott (2 mentions)
Magna pure compact system, by Roche (2 mentions)
Cobas omni lysis reagent, by Roche (2 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (2 mentions)
103 protocols using cobas 6800 system
1
Evaluating DMEM and UTM for SARS-CoV-2 RT-PCR
2
HCV Detection in Plasma and DBS
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HCV RNA in plasma samples were either determined by the quantitative cobas®HCV assay (Roche Diagnostics GmbH, Mannheim, German) run at the cobas®6800 system (Roche Molecular system) or by the Aptima HCV Quant Dx assay (Hologic GmbH, Vienna, Austria) run at the Panther® system (Hologic, Marlborough, MA, USA). The lower quantification limit were 15 IU/mL and 10 IU/mL, respectively, and all analyses were performed according to the manufacturer’s instructions.
DBS were obtained on Whatman® 903 protein saver cards (Sigma-Aldrich, Copenhagen, Denmark) and were allowed to dry for 1 to 3 days before they were eluted and tested for anti-HCV. HCV antibodies were determined by the Alinity i Anti-HCV assay (Abbott Diagnostics, Delkenheim, Germany). Anti-HCV positive samples were tested for the presence of HCV RNA by nucleic acid amplification testing using the cobas® MPX assay (Roche Diagnostics GmbH, Mannheim, German) run at the cobas® 6800 system (Roche Molecular system). The sensitivity when using DBS samples has previously been reported to be >95% [22 (link)].
DBS were obtained on Whatman® 903 protein saver cards (Sigma-Aldrich, Copenhagen, Denmark) and were allowed to dry for 1 to 3 days before they were eluted and tested for anti-HCV. HCV antibodies were determined by the Alinity i Anti-HCV assay (Abbott Diagnostics, Delkenheim, Germany). Anti-HCV positive samples were tested for the presence of HCV RNA by nucleic acid amplification testing using the cobas® MPX assay (Roche Diagnostics GmbH, Mannheim, German) run at the cobas® 6800 system (Roche Molecular system). The sensitivity when using DBS samples has previously been reported to be >95% [22 (link)].
3
Comparative Analysis of SARS-CoV-2 Detection Tests
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AccuPlex™ SARS-CoV-2 Reference Material Kit (material number: 0505-0126) was used as the SARS-CoV-2 reference standard material. The kit contains 5000 copies/ml RNA materials with a viral protein coat, comprising the nucleotides of SARS-CoV-2 sequence 417-1899, 3094-3360 for ORF1a, 13291-13560, 14700-15950, 18577-19051 for RdRp, 25801-28200 for E, and 27952-29873 for N. To assess the sensitivity of the RT-qPCR assay, the positive RNA materials with a viral protein coat in above kit were serially diluted, extracted, and then performed by the protocols of both SARS-CoV-2 detection tests. The LOD is the lowest concentration of SARS-CoV-2 RNA that can be detected in >95% of samples tested with acceptable precision by our systems. To compare the LOD among two systems, the two-fold dilutions were prepared and performed in triplicates using Cobas SARS-CoV-2 Test on Cobas 6800 System and Cobas SARS-CoV-2 & Flu A/B Test on Cobas Liat System according to the manuals of manufacturers’ instruction. The mean value and standard deviation (SD) were calculated regarding the Ct values of targets either the ORF1 a/b and E genes for singleplex RT-PCR assays (Cobas SARS-CoV-2 Test) or RdRp and N genes for multiplex RT-PCR (Cobas SARS-CoV-2 & Flu A/B Test). The result of both SARS-CoV-2 detection tests was reported as positive and negative according to manufacturers’ instructions.
4
SARS-CoV-2 RT-PCR Testing Protocol
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SARS-CoV-2 testing was performed on nasopharyngeal swabs via RT-PCR on the Cobas 6800 system (Cobas, Roche, Basel, Switzerland). As obliged by the German Infection Protection Act (Infektionsschutzgesetz), patients with suspected or confirmed COVID-19 were reported to the local health department.
5
SARS-CoV-2 Detection by RT-PCR
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Viral RNA genome detection was performed by RT-PCR using the Roche Cobas 6800 system (Cobas SARS-CoV-2 Ref 09175431190; Cobas SARS-CoV-2 Control kit Ref 09175440190; Cobas 6800/8800 Buffer Negative Control kit Ref 07002238190). This technology allows nucleic acid extraction, purification, PCR amplification, and detection of SARS-CoV-2, targeting ORF1a/b and a pan-sarbecovirus conserved region of the E-protein gene.
6
Evaluating Cobas CMV Test Linearity and LOD
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The linearity of the Cobas CMV test, following the simplified sample preparation step, was verified following the Clinical & Laboratory Standards Institute guideline (CLSI) EP06-A. A random breast milk sample with a CMV viral load of approximately 5.41 × 104 IU/mL was utilized. To include seven concentrations within the targeted linearity interval, with equal spacing in log concentration, the sample was serially diluted by a factor of 3, until 38, with distilled water. The CMV viral load was then measured per sample dilution in triplicates using the Cobas 6,800 system. Subsequently, the average log-transformed CMV titers obtained from the serial dilution were examined for linearity. A maximum allowable deviation of 10% from the expected value was applied during the assessment.
The limit of detection (LOD) was verified following the CLSI EP17-A2 guidelines. For this evaluation, two distinct breast milk samples, each harboring a CMV load of 20.6 IU/mL, which was the LOD claimed by the manufacturer, were prepared. These samples were further divided into 10 replicates each. Subsequently, each replicate was measured across 3 days. Given that a total of 20 measurements were conducted, the threshold for observed proportion was set to 85% during the assessment.
The cross-reactivity of the assay was evaluated in our previous study (Roh et al., 2021 (link)).
The limit of detection (LOD) was verified following the CLSI EP17-A2 guidelines. For this evaluation, two distinct breast milk samples, each harboring a CMV load of 20.6 IU/mL, which was the LOD claimed by the manufacturer, were prepared. These samples were further divided into 10 replicates each. Subsequently, each replicate was measured across 3 days. Given that a total of 20 measurements were conducted, the threshold for observed proportion was set to 85% during the assessment.
The cross-reactivity of the assay was evaluated in our previous study (Roh et al., 2021 (link)).
7
SARS-CoV-2 RT-PCR Viral Dynamics
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SARS-CoV-2 RT-PCR was executed using Roche Cobas® 6800 system (Roche, Basel, Switzerland).20 Selective amplification of the target nucleic acid from the sample was achieved with the use of target-specific forward and reverse primers for ORF1ab non-structural region, which is unique to SARS-CoV-2. Another conserved region in the structural protein envelope E-gene was selected for pan-sarbecovirus detection.
We reviewed the cycle threshold (Ct) values for both gene targets for all RT-PCR tests that were performed on NP swabs or endotracheal aspirates when the patients were mechanically ventilated, as described in a previous study.21 (link) The RT-PCR results were expressed in terms of the Ct value, and the samples were considered positive when the Ct value was ≤40. Viral dynamics were calculated on the basis of the Ct value of the SARS-CoV-2-specific target (ORF1ab). The infectious viral shedding time was defined as the time from symptom onset to the first day of obtainment of a Ct value beyond 30.22 (link)
,23 (link) The patients without data on Ct values beyond 30 during their hospital stay were right-censored at the time of their last Ct value assessment.
We reviewed the cycle threshold (Ct) values for both gene targets for all RT-PCR tests that were performed on NP swabs or endotracheal aspirates when the patients were mechanically ventilated, as described in a previous study.21 (link) The RT-PCR results were expressed in terms of the Ct value, and the samples were considered positive when the Ct value was ≤40. Viral dynamics were calculated on the basis of the Ct value of the SARS-CoV-2-specific target (ORF1ab). The infectious viral shedding time was defined as the time from symptom onset to the first day of obtainment of a Ct value beyond 30.22 (link)
,23 (link) The patients without data on Ct values beyond 30 during their hospital stay were right-censored at the time of their last Ct value assessment.
8
SARS-CoV-2 Antibody and PCR Testing
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An electrochemiluminescence immunoassay, Roche Elecsys® Anti-SARS-CoV-2 (99.5% sensitivity,7 (link) 99.8% specificity;7 (link),8 Roche, Switzerland, was used to identify antibodies in the serological samples. Reactive for optical density (a proxy for antibody titer) cutoff index 1.0 and non-reactive for cutoff index 1.0, according to manufacturer instructions.8 PCR testing of aliquots of Universal Transport Medium UTM) used for nasopharyngeal and oropharyngeal swab collection was utilized to assess current infection (Huachenyang Technology, China).
Aliquots were extracted using the QIAsymphony platform (QIAGEN, USA) and evaluated using the TaqPathTM COVID-19 Combo Kit (100% sensitivity and specificity;9 Thermo Fisher Scientific, USA) on an ABI 7500 FAST (Thermo Fisher, USA). On a Hamilton Microlab STAR (Hamilton, USA), samples were extracted using a custom protocol10 (link) and tested using the AccuPower SARS-CoV-2 Real-Time RT-PCR Kit (100% sensitivity and specificity)11 (link) on an ABI 7500 FAST, or directly loaded into a Roche cobas® 6800 system to be assayed. All laboratory testing was done at the HMC Central Laboratory according to defined procedures.12–15 (link) The laboratory is accredited by the College of American Pathologists.
Aliquots were extracted using the QIAsymphony platform (QIAGEN, USA) and evaluated using the TaqPathTM COVID-19 Combo Kit (100% sensitivity and specificity;9 Thermo Fisher Scientific, USA) on an ABI 7500 FAST (Thermo Fisher, USA). On a Hamilton Microlab STAR (Hamilton, USA), samples were extracted using a custom protocol10 (link) and tested using the AccuPower SARS-CoV-2 Real-Time RT-PCR Kit (100% sensitivity and specificity)11 (link) on an ABI 7500 FAST, or directly loaded into a Roche cobas® 6800 system to be assayed. All laboratory testing was done at the HMC Central Laboratory according to defined procedures.12–15 (link) The laboratory is accredited by the College of American Pathologists.
9
SARS-CoV-2 Detection in Cell Culture
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Viral RNA was detected in nasopharyngeal swabs and serum on an automated Roche Cobas 6800 system (Roche, Mannheim, Germany) with the Roche cobas® SARS-CoV-2 Test (Roche, Mannheim, Germany).
To confirm the presence of SARS-CoV-2 in the cell culture supernatant, RNA was isolated from 100 μL of the supernatant which was added to 300 μL of a TriPure Isolation Reagent (Roche, Mannheim, Germany). After the addition of 95–100% ethanol (100 μL), the samples were incubated at room temperature for 5 min and then centrifuged through the QIAamp Mini Spin Columns (Qiagen, Hilden, Germany) for 1 min at 6000 ×
g. Further isolation was performed following a Qiagen QIAamp® Viral RNA Mini Kit (Qiagen, Hilden, Germany) protocol. Viral RNA was detected by the real-time RT-PCR [21] from 5 μL of the eluate using a TaqMan Fast Virus 1-Step Master Mix (Thermo Fisher Scientific, Waltham, MA USA) in a 20 μL reaction mixture on an Applied Biosystems StepOnePlus thermocycler (Thermo Fisher Scientific, Waltham, MA USA).
To confirm the presence of SARS-CoV-2 in the cell culture supernatant, RNA was isolated from 100 μL of the supernatant which was added to 300 μL of a TriPure Isolation Reagent (Roche, Mannheim, Germany). After the addition of 95–100% ethanol (100 μL), the samples were incubated at room temperature for 5 min and then centrifuged through the QIAamp Mini Spin Columns (Qiagen, Hilden, Germany) for 1 min at 6000 ×
g. Further isolation was performed following a Qiagen QIAamp® Viral RNA Mini Kit (Qiagen, Hilden, Germany) protocol. Viral RNA was detected by the real-time RT-PCR [21] from 5 μL of the eluate using a TaqMan Fast Virus 1-Step Master Mix (Thermo Fisher Scientific, Waltham, MA USA) in a 20 μL reaction mixture on an Applied Biosystems StepOnePlus thermocycler (Thermo Fisher Scientific, Waltham, MA USA).
10
SARS-CoV-2 Detection Protocols and Platforms
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Nasopharyngeal and/or oropharyngeal swabs (Huachenyang Technology, China) were collected for PCR testing and placed in Universal Transport Medium (UTM). Aliquots of UTM were: extracted on a QIAsymphony platform (QIAGEN, USA) and tested with RT-qPCR using TaqPath™ COVID-19 Combo Kits (100% sensitivity and specificity [25 ]; Thermo Fisher Scientific, USA) on an ABI 7500 FAST (ThermoFisher, USA); extracted using a custom protocol [26 (link)] on a Hamilton Microlab STAR (Hamilton, USA) and tested using AccuPower SARS-CoV-2 Real-Time RT-PCR Kits (100% sensitivity and specificity [27 (link)]; Bioneer, Korea) on an ABI 7500 FAST; or loaded directly into a Roche cobas® 6800 system and assayed with a cobas® SARS-CoV-2 Test (95% sensitivity, 100% specificity [28 ]; Roche, Switzerland). The first assay targets the viral S, N, and ORF1ab regions. The second targets the viral RdRp and E-gene regions, and the third targets the ORF1ab and E-gene regions.
All tests were conducted at the HMC Central Laboratory or Sidra Medicine Laboratory, following standardized protocols.
All tests were conducted at the HMC Central Laboratory or Sidra Medicine Laboratory, following standardized protocols.
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