SARS-CoV-2 RNA was searched in all the cases on sections from olfactory bulb and tract, postrolandic cortex and rostral medulla as previously described (10). FFPE 10 micron sections were pre-treated using 160 µL Deparaffinization Solution (Qiagen, Hilden, Germany) with 180 μL of tissue lysis buffer (ATL buffer, Qiagen) and 20 μL of protease (Proteinase K solution, Qiagen, Germany). Tissues were incubated overnight at 56 °C and one-hour at 90 °C. Then extraction and amplification of nucleic acids were performed using ELITeInGenius® instrument with ELITeInGenius SP 200 and SARS-CoV-2 ELITe MGB Kit (ELITechGroup, Milano, Italy). The tissue viral load was reported as number of copies/microgram RNA. Quantification of RNA was performed using Qubit Assay (Invitrogen by ThermoFisher). Positive results below the lower limit of quantification (2 copies/reaction) were reported as <15 copies/microgram RNA. The identification of key mutations in SARS-CoV-2 Spike Protein gene (E484K, N501Y, HV69-70 del, L452R, K417T, K417N, W152C) was performed with Allplex SARS-CoV-2 Variants I and II Assay, Seegene.
Sars cov 2 elite mgb kit
Manufactured by ELITechGroup
Sourced in Italy
The SARS-CoV-2 ELITe MGB Kit is a molecular diagnostic kit designed for the detection of the SARS-CoV-2 virus. The kit utilizes real-time PCR technology and a proprietary MGB (Minor Groove Binder) probe chemistry to identify the presence of the virus in clinical samples.
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Lab products found in correlation
Elite ingenius, by ELITechGroup (2 mentions)
Deparaffinization solution, by Qiagen (1 mentions)
Atl buffer, by Qiagen (1 mentions)
Eliteingenius sp 200, by ELITechGroup (1 mentions)
Qubit assay, by Thermo Fisher Scientific (1 mentions)
Proteinase k solution, by Qiagen (1 mentions)
Genexpert dx system, by Cepheid (1 mentions)
Cobas e411, by Roche (1 mentions)
Xpert xpress cov 2 plus, by Cepheid (1 mentions)
Genefinder covid 19 plus realamp kit assay, by ELITechGroup (1 mentions)
Vacutainer k2edta tube, by BD (1 mentions)
Neumodx, by Qiagen (1 mentions)
Cobas 6000, by Roche (1 mentions)
Genexpert, by Cepheid (1 mentions)
9 protocols using sars cov 2 elite mgb kit
1
SARS-CoV-2 Detection and Variant Identification
2
SARS-CoV-2 Real-Time PCR Detection
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We performed SARS-CoV-2 real-time PCR on these samples using the TaqPath COVID-19 CE-IVD RT-PCR kit (Thermo Fisher Applied Biosystems, cat.no. A48067) or the SARS-CoV-2 ELITe MGB kit (ELITechGroup, cat. no. RTS170ING) following the manufacturer’s instructions.
3
Evaluating SARS-CoV-2 Antigen and Molecular Tests
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Data of multiplex respiratory panel tests were integrated with SARS-CoV-2 test results assessed by antigenic or molecular tests. SARS-CoV-2 antigen testing was performed using the electrochemiluminescence immunoassay (ECLIA) Roche Elecsys SARS-CoV-2 Antigen on a Roche Cobas e411 (Roche Diagnostics GmbH, Mannheim, Germany) or the fluorescent immunoassay RADT STANDARD F COVID-19 Ag FIA on an F2400 analyzer (SD BIOSENSOR, Korea). According to the manufacturers’ instructions, a result of cut-off index (COI) ≥ 1.0 was interpreted as reactive for SARS-CoV-2 antigen. Molecular tests were performed for the detection of the SARS-CoV-2 virus through reverse transcription (RT), followed by real-time PCR from RNA extracted from respiratory samples by different assays according to the manufacturers’ instructions. The Cepheid Xpert Xpress CoV-2 plus performed on Cepheid’s GeneXpert®DX system (Cepheid, Sunnyvale, CA, USA) is an assay that amplifies and detects unique sequences in the nucleocapsid (N), envelope (E), and RNA-dependent RNA polymerase (RdRP) genes of the SARS-CoV-2 genome. The SARS-CoV-2 ELITe MGB Kit® (Elitechgroup, Turin, Italy), performed using the ELITe InGenius instrument (Elitechgroup, Turin, Italy), is an assay that amplifies and detects unique sequences in the RdRp and ORF8 genes of the SARS-CoV-2 genome.
4
SARS-CoV-2 Detection in Plasma
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In order to detect SARS-CoV-2 in the bloodstream, blood specimens were collected in BD Vacutainer® K2EDTA tube to obtain plasma. Samples collected from March 2020 to February 2021 were tested for SARS-CoV-2 RNA by means of GeneFinderTM COVID-19 Plus RealAmp Kit assay (targets: E, N and RdRP genes) (ELITechGroup Italy). Starting from March 2021, samples were tested using SARS-CoV-2 ELITe MGB® Kit (targets: RdRP and ORF8 genes) (ELITechGroup Italy). The change of SARS-CoV-2 assay by the manufacturer represents an optimization of SARS-CoV-2 detection through a more appropriate target genes choice. The detection of one target is the minimum condition to define a positive result and the cut-off of the assay is <45 cycle threshold (Ct).
5
SARS-CoV-2 Surveillance in Healthcare Workers
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Naso-pharyngeal swabs were collected and tested for SARS-CoV-2 RNA positivity in subjects with symptoms suggestive of SARS-CoV-2 infection or in case of contact with infected subjects. SARS-CoV-2 RNA real time PCR was performed weekly or every two weeks, using SARS-CoV-2 ELITe MGB® Kit (ELITechGroup, Puteaux, France). Moreover, in compliance with the local healthcare workers’ surveillance protocol, personnel working in clinical wards dedicated to fragile patients underwent a routine screening for SARS-CoV-2 infection every 14 days, while monitoring was scheduled every 30 days for healthcare workers in the other wards. The health condition of all workers was regularly monitored and data on symptoms were collected during an interview by a physician and inserted into a specific database.
6
SARS-CoV-2 Screening and Antibody Detection
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Nasopharyngeal swabs were routinely used to screen the workforce and test symptomatic workers and close contacts of a confirmed COVID case. The SARS-CoV-2 ELITe MGB Kit (Elitechgroup, Italy) was used to detect SARS-CoV-2 RNA extracted from nasopharyngeal swabs. Extraction, detection, and quantification by RT-PCR were performed using a commercial automatized platform (Elite InGenius®, Elitechgroup, Italy).
The electrochemiluminescence immunoassay (ECLIA) Elecsys® anti-SARS-CoV-2 was used to detect immunoglobulins (IgG/A/M) for anti-SARS-CoV-2-S (Roche Diagnostics International Ltd, Rotkreuz, Switzerland). The assay uses a recombinant protein representing the RBD of the S antigen in a double-antigen sandwich assay format from SARS-CoV-2 wild-type (WT) (Wuhan-Hu-1) strain. Serum samples of 20 μL are collected using EDTA tubes. The results are expressed as U/mL, the cut-off was 0.8 U/mL, and the upper limit of detection was 250 U/mL. Since the antibody titers elicited in immunized individuals were very high, we tested all serum samples at a dilution of 1:20, in accordance with Roche, so the upper limit of detection was raised to 5000 U/mL and the dynamic range could be extended.
The electrochemiluminescence immunoassay (ECLIA) Elecsys® anti-SARS-CoV-2 was used to detect immunoglobulins (IgG/A/M) for anti-SARS-CoV-2-S (Roche Diagnostics International Ltd, Rotkreuz, Switzerland). The assay uses a recombinant protein representing the RBD of the S antigen in a double-antigen sandwich assay format from SARS-CoV-2 wild-type (WT) (Wuhan-Hu-1) strain. Serum samples of 20 μL are collected using EDTA tubes. The results are expressed as U/mL, the cut-off was 0.8 U/mL, and the upper limit of detection was 250 U/mL. Since the antibody titers elicited in immunized individuals were very high, we tested all serum samples at a dilution of 1:20, in accordance with Roche, so the upper limit of detection was raised to 5000 U/mL and the dynamic range could be extended.
7
COVID-19 Diagnostic Lab Protocols
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The following measurements were collected and included in statistical analysis: C-reactive protein; white cell; neutrophil; hemoglobin and platelets counts; D-dimer; and IL-6 (electrochemiluminescence immunoassay, Cobas® 6000 Roche, pg/mL). RT-PCR assays were either performed in the multipurpose laboratory of Cannes General Hospital or in the Bioesterel Biogroup laboratory. According to the study period and to the laboratory guidelines, four RT-PCR assays were used: the NeuMoDX® (Qiagen) instrument (targeting N and NSP2 genes), the GeneXpert® (Cepheid, E and N2 genes), the Gene Finder COVID-19 Plus RealAmp kit (GeneFinder, N, E and RdRP genes) or the SARS-Cov-2 ELITe MGB kit (Elitech, ORF8 and RdRP genes). Screening of virus isolate was realized by targeting N501Y, E484K and K417N mutations using the GSD NovaType II SARS CoV-2 and NovaTec Immundiagnostica GmbH assays.
8
SARS-CoV-2 Detection by One-Step RT-qPCR
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Real-time PCR was performed using the SARS-CoV-2 ELITe MGB Kit (ELITechGroup S.p.A.). In this assay, we used a standard reaction mix volume of 30 µL (20 µL of CoV-2 PCR Mix, 0.3 µL of RT Enzyme Mix, and 10 μL of extracted RNA). The real-time PCR protocol included 20 min at 45 °C (RETROTRASCRIPTION), 2 min at 95 °C (DENATURATION), 10 s at 95 °C, 30 s at 60 °C, 10 s at 60 °C, and 20 s at 72 °C (AMPLIFICATION AND DETECTION), repeated for 44 cycles. The one-step fluorescence RT-PCR reaction system allowed us to perform retrotranscription of viral RNA directly during qRT-PCR amplification. Two detection channels were used to amplify ORF8 and RdRp, namely, FAM and ROX, while the VIC channel was used for human RNase P as an internal control (IC). The CFX96™ Real-Time System C1000™ Thermal Cycle (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used to perform this analysis.
9
Rapid SARS-CoV-2 Screening and Confirmation
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Rhino-pharyngeal swabs (RPS) were routinely used to perform fortnightly screening on our personnel as well as to test symptomatic workers and close contacts of a confirmed COVID-19 case. In the case of positive antigenic swabs (cassette), a molecular test was performed to confirm the case. The SARS-CoV-2 ELITe MGB Kit® (Elitechgroup, Turin, Italy) was used for the detection of SARS-CoV-2 virus through reverse transcription (RT) followed by real-time PCR from RNA extracted from RPSs. One-step RT-real-time polymerase chain reaction was used to confirm the presence of COVID-19 by amplification of two regions: the RdRp and ORF8 genes. Extraction, detection, and quantification were performed using a commercial automatized platform (Elite InGenius®, Elitechgroup, Turin, Italy). Briefly, primary RPS samples were loaded directly and processed on the Elite InGenius® system according to the manufacturer’s instructions, and the results were available after a 2.5 h process of 200 μL for each sample. Result interpretation and analysis were automatically performed by the Elite InGenius® system.
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