Upon receipt of Thermo Scientific AerosolSense 2900 air sampler cartridge at City of Milwaukee Health Department Laboratory (MHDL), collection substrates were aseptically removed and transferred to a 5 mL sterile screw-cap tube filled with 1 mL of Remel viral transport medium. Samples were kept frozen at −70°C until total nucleic extraction was performed using 200 μL elute and Applied Biosystems™ MagMAX™ Viral/Pathogen Nucleic Acid Isolation Kit using ThermoFisher Scientific KingFisher Flex instrument. Real-time RT-PCR setup was performed using 10 μL of extract and approved Applied Biosystems TaqPath™ COVID-19 Combo Kit containing three primer/probe sets specific to different SARS-CoV-2 genomic regions (open reading frame 1ab (ORF1ab), spike (S) protein, and nucleocapsid (N) protein-encoding genes) and primers/probes for bacteriophage MS2 which served as internal process control for nucleic acid extraction. RT-PCR assay was performed on Applied Biosystems 7500 Fast Dx Real-Time PCR System according to the TaqPath™ COVID-19 Combo Kit protocol. PCR results were interpreted using the Applied Biosystems COVID-19 Interpretive Software, and results were reported as positive, negative, or inconclusive for the detection of SARS-CoV-2 RNA following successful quality control checks.
Taqpath covid 19 combo kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, Spain, Canada
The TaqPath COVID-19 Combo Kit is a real-time RT-PCR test for the detection of SARS-CoV-2, the virus that causes COVID-19. The kit is designed to detect three different genetic targets within the SARS-CoV-2 virus, providing reliable and accurate results.
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Lab products found in correlation
Magmax viral pathogen nucleic acid isolation kit, by Thermo Fisher Scientific (11 mentions)
Cobas sars cov 2 test, by Roche (11 mentions)
Kingfisher flex, by Thermo Fisher Scientific (8 mentions)
Abi 7500 fast, by Thermo Fisher Scientific (6 mentions)
Magmax viral pathogen 2 nucleic acid isolation kit, by Thermo Fisher Scientific (5 mentions)
Cobas 6800 system, by Roche (5 mentions)
Magmax viral rna isolation kit, by Thermo Fisher Scientific (4 mentions)
Novaseq, by Illumina (4 mentions)
Nextseq, by Illumina (4 mentions)
Covidseq test kit, by Illumina (4 mentions)
Xpert xpress sars cov 2, by Cepheid (4 mentions)
Kingfisher flex purification system, by Thermo Fisher Scientific (3 mentions)
Taqpath covid 19 ce ivd rt pcr kit, by Thermo Fisher Scientific (3 mentions)
Design and analysis software, by Thermo Fisher Scientific (3 mentions)
Quantstudio 7 flex real time pcr system, by Thermo Fisher Scientific (3 mentions)
Cobas sars cov 2 6800 8800, by Roche (3 mentions)
7500 fast dx real time pcr instrument, by Thermo Fisher Scientific (3 mentions)
Applied biosystems 7500 fast dx real time pcr system, by Thermo Fisher Scientific (3 mentions)
Exiprep 96 lite, by Bioneer (3 mentions)
Applied biosystems 7500 fast dx real time pcr instrument, by Thermo Fisher Scientific (2 mentions)
88 protocols using taqpath covid 19 combo kit
1
SARS-CoV-2 Detection Protocol in Air Samples
2
SARS-CoV-2 Detection Using TaqPath Combo Kit
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The TaqPath COVID-19 combo kit (Thermo Fisher Scientific, Waltham, MA) was used for the detection of SARS-CoV-2 RNA in clinical samples according to the manufacturer’s instructions outlined in the EUA. Sample and assay preparation was done using the Hamilton Star, an automated liquid handler (Hamilton Company, Reno, NV). Nucleic acid (NA) extraction was performed using the MagMax viral/pathogen nucleic acid isolation kit and the KingFisher Flex purification system (Thermo Fisher Scientific). RNA extraction from specimens was then used as a template for the real-time RT-PCR, which targets the SARS-CoV-2 S (spike), N (nucleocapsid), and ORF1ab (polyprotein) genes, performed on the Applied Biosystems 7500 Fast DX real-time PCR instrument (Thermo Fisher Scientific). RT-PCR data were analyzed using the COVID-19 Interpretive Software v2.5, and qualitative results were provided by the software. Positive results were based on detection of at least 2 of the 3 SARS-CoV-2 targets. Cycle threshold (CT) values for each gene were also obtained using the interpretive software for each sample identified as positive.
3
Longitudinal Study of SARS-CoV-2 in Marine Recruits
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The COVID-19 Health Action Response for Marines (CHARM) study has been previously described [18 (link),19 (link)], but in brief, U.S. marine recruits were quarantined for two weeks prior to basic training, and within 48 h of arrival, offered the opportunity to volunteer in this longitudinal, prospective study. The study protocol was approved by the Naval Medical Research Center Institutional Review Board in compliance with all applicable federal regulations governing protection of human subjects. All participants provided written informed consent for participation. On day 0, mid-turbinate nares swabs collected in virus transport media (VTM) were assessed for SARS-CoV-2 by real-time reverse transcriptase polymerase chain reaction (RT-RT-PCR), with additional swabs assessed at study days 7, 14, 28, 42, and 56 for all participants using the FDA Emergency Use Authorization (EUA) TaqPath COVID-19 Combo Kit (Thermo Fisher Scientific; Waltham, MA, USA). If positive, more intensive sampling was performed twice per week for the first two weeks and then biweekly thereafter for the subsequent six weeks. Serum samples were also collected at the same time points, and presence of SARS-CoV-2-specific IgG and IgM antibodies was determined by ELISA.
4
SARS-CoV-2 Lineage Surveillance in Nursing Homes
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Clinical samples were received in The Jackson Laboratory Clinical Genomics Laboratory (CGL) as part of a statewide (Connecticut) COVID-19 surveillance program, with the majority of samples representing asymptomatic screening of nursing home and assisted living facility residents and staff. Total nucleic acids were extracted from anterior nares swabs in viral transport media or saline (200 μL) using the MagMAX Viral RNA Isolation kit (ThermoFisher) on a KingFisher Flex purification system. Samples were tested for the presence of SARS-CoV-2 RNA using the TaqPath COVID-19 Combo Kit (ThermoFisher). For this analysis, only the CT values from the N gene primer/probe set were used (Figure 4 ).
Samples with CT values ≤ 30 for the N gene target were prepared for sequencing using the Illumina COVIDSeq Test kit. Sequencing was performed on an Illumina NovaSeq or NextSeq in the CGL. Data analysis was performed using the DRAGEN COVID Lineage App in BaseSpace Sequence Hub. Sequences with >80% of bases with non-N basecalls and ≥1500-fold median coverage were considered successful and were submitted to GISAID. Lineages were assigned using pangolin v.2.4.235 (link) and the most current version of the pangoLEARN assignment algorithm.
Samples with CT values ≤ 30 for the N gene target were prepared for sequencing using the Illumina COVIDSeq Test kit. Sequencing was performed on an Illumina NovaSeq or NextSeq in the CGL. Data analysis was performed using the DRAGEN COVID Lineage App in BaseSpace Sequence Hub. Sequences with >80% of bases with non-N basecalls and ≥1500-fold median coverage were considered successful and were submitted to GISAID. Lineages were assigned using pangolin v.2.4.235 (link) and the most current version of the pangoLEARN assignment algorithm.
5
Comparative Evaluation of Rapid Antigen and RT-PCR Tests for COVID-19 Diagnosis
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Two nasopharyngeal samples were taken per patient, one in each choana. One of them was taken with the swab provided by ClinitestRT, and the other one with a suitable swab for taking a virus sample including a universal transport medium for RT-PCR (Copan flocked swabs with UTM™, Universal Transport Medium). ClinitestRT was performed immediately, under point-of-care conditions (regulations of quality systems ISO 15,189) [3 ,4 (link)], and according to the manufacturer's instructions (lot number 2011072) by physicians and nurses from emergency and primary care services trained by microbiology specialists. The second swab was used for a molecular diagnostic (RT-PCR) by each hospital according to its standard procedures for COVID-19 diagnosis. The commercial RT-PCR methods used for the participants hospitals in this study were Allplex™ 2019-nCoV Assay (Seegene, Seoul, South Korea), GENOMICA S.A.U. (Madrid, Spain), TaqPath COVID-19 Combo Kit (Thermo Fisher, Waltham, MA, USA), GeneXpert (Cepheid, Sunnyvale, CA, USA), and Cobas 6800 (Roche, Indianapolis, USA) Due to the high diagnostic demand, in some hospitals different PCR techniques were used.
6
SARS-CoV-2 Detection via RT-qPCR
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Nucleic acid extraction was performed from 400 μL PBS eluate using the MagMAX™ Viral/Pathogen Nucleic Acid Isolation Kit (Thermo Fisher). RT-qPCR was performed on the QuantStudio™ 7 Flex 96-well Fast (Thermo Fisher) using 5 μL RNA and the TaqPath COVID-19 Combo Kit according to the EUA Instructions For Use, 14March2020; Pub. No. MAN0019181 Rev. A.0 (https://www.thermofisher.com/content/dam/LifeTech/Documents/PDFs/clinical/taqpath-COVID-19-combo-kit-full-instructions-for-use.pdf ). Raw cycle thresholds were obtained using the QuantStudio™ Real-Time PCR Software v1.3 and the following thresholds: 40,000 for ORF1ab, 25,000 for N, and 38,000 for S. Samples with indeterminate or spurious amplification signals were designated a cycle threshold (Ct) value of 40 cycles. For the experiments using human matrix, samples were also tested using the TaqMan® RNase P assay (Thermo Fisher) on the QuantStudio™ Dx Real-Time Instrument.
7
COVID-19 diagnosis using RT-PCR on saliva
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For HCWs tested at MSKCC, the COVID-19 diagnosis was made through detection of viral RNA in nasopharyngeal swabs or saliva samples using two real-time reverse transcriptase PCR tests: the Taq Path COVID-19 Combo Kit (Thermo Fisher Scientific, Waltham, MA) or the Cobas SARS-CoV-2 test (Roche Molecular Diagnostics, Indianapolis, IN). The performance of these real-time reverse transcriptase PCR on saliva samples was previously described [8 (link),9 (link)]. The samples were reported as positive per manufacturers' instructions.
8
COVID-19 Diagnostic Test Protocol
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Samples were extracted using the MagMAX™ Viral/Pathogen Nucleic Acid Isolation Kit (Thermofisher, Warrington, UK) and Kingfisher Flex extraction platform (ThermoFisher). PCR amplification was carried using the TaqPath™ COVID-19 Combo Kit (Thermofisher) with 384 well format on a Quantstudio Flex 7 System (Thermofisher) and analysed by FastFinder® PCR analysis software (UgenTec, Hasselt, Belgium). FastFinder® PCR analysis software (UgenTec, Hasselt, Belgium) used an artificial intelligence algorithm (AI) and semi-automated analysis to assign Cq value to the amplification curves of the assay targets. In concert with human review of results, FastFinder® assigned positive, negative, inconclusive, or invalid test results based on a clinical decision tree, which considered Cq values of the three assay targets, the internal MS2 control and the positive and negative controls on the PCR plate.
9
SARS-CoV-2 Detection in Saliva Samples
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Paired nasopharyngeal swabs and saliva samples were sent to the Clinical Virology Laboratory at Children’s Hospital Los Angeles. Total nucleic acid was extracted from 250 μl of undiluted saliva samples using the Thermo Fisher KingFisher Flex specimen processing system with the Applied Biosystems MagMAX viral/pathogen nucleic acid isolation kit (Thermo Fisher, Waltham, MA) and eluted to 50 μl of total nucleic acid. Real-time quantitative reverse transcriptase PCR (qRT-PCR) was performed using the TaqPath COVID-19 Combo kit (Thermo Fisher). A positive result for SARS-CoV-2 detection was determined by amplification of at least one of the three genes targeted (N gene, S gene, or ORF1ab gene) using a cutoff threshold cycle (CT) value of <40. When multiple targets were detected in a sample, the CT values for those targets were averaged (18 (link)). When a single target was positive, the exact CT value was used. A valid negative result for SARS-CoV-2 detection was determined by amplification of MS2 internal control using a cutoff CT value of <32.
10
SARS-CoV-2 Variant Detection Protocol
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Samples were received in partnership with various clinical laboratories as either purified RNA or original nasal swab in viral transport media. Samples were screened for S-gene target failure (SGFT) using the Thermo Fisher TaqPath COVID-19 Combo Kit diagnostic assay prior to receipt at Yale. Nucleic acid was extracted from original samples (300 μL) using the MagMAX viral/pathogen nucleic acid isolation kit (Thermo Fisher) and eluted into 75 μL. All RNA was then screened again using an assay developed by our laboratory that is specific for variants of concern (Vogels et al., 2021 (link)). Samples identified by the screen as potential variants were then prioritized for sequencing. Multiple extraction controls were included for each RNA extraction batch and tested negative for SARS-CoV-2 RNA by the same assay.
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