Taqpath covid 19 ce ivd rt pcr kit

Manufactured by Thermo Fisher Scientific

Sourced in United States, France, Canada

The TaqPath COVID-19 CE-IVD RT-PCR kit is a real-time reverse transcription-polymerase chain reaction (RT-PCR) assay designed for the qualitative detection of nucleic acid from SARS-CoV-2, the virus that causes COVID-19. The kit uses specific primers and probes to target three different SARS-CoV-2 gene regions.

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RT-PCR kit (189 citations) RT kits (5 citations) TaqPath COVID-19 CE-IVD RT-PCR (3 citations)

68 protocols using taqpath covid 19 ce ivd rt pcr kit

TaqPath COVID-19 RT-PCR Assay Protocol

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Reaction mixtures were prepared using the ThermoFisher TaqPath COVID-19 CE-IVD RT-PCR kit protocol (12 ). RT-PCR of reaction mixtures was performed using the Applied Biosystems QuantStudio 5 real-time PCR instrument. Subsequent EDT files were transferred to a computer with QuantStudio Design and Analysis desktop software v2.5.1 for analysis of exponential curves. The TaqPath COVID-19 assay coamplifies three target genes: ORF1ab, N gene, and S gene. Results were classified as positive with respect to at least 2 single-target genes (ORF1ab, N, and S) provided the raw C_T (cycle threshold) values were below 37 for single gene target signals. Bacteriophage MS2 RNA was added to each sample as an internal positive control for each sample and to monitor potential sample inhibition. A negative control (distilled water [dH₂O]) is included on every plate. The SGTF of the TaqPath COVID-19 CE-IVD RT-PCR kit was considered a proxy for the presence of Δ69/70 in the S gene of SARS-CoV-2.

Ashford F., Best A., Dunn S.J., Ahmed Z., Siddiqui H., Melville J., Wilkinson S., Mirza J., Cumley N., Stockton J., Ferguson J., Wheatley L., Ratcliffe E., Casey A., Plant T., Quick J., Richter A., Loman N, & McNally A. (2022). SARS-CoV-2 Testing in the Community: Testing Positive Samples with the TaqMan SARS-CoV-2 Mutation Panel To Find Variants in Real Time. Journal of Clinical Microbiology, 60(4), e02408-21.

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SARS-CoV-2 Genomic Surveillance in Cyprus

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The Department of Molecular Virology of the Cyprus Institute of Neurology and Genetics was assigned as the reference laboratory for SARS-CoV-2 by the Cyprus Ministry of Health of the Republic of Cyprus. During the study period of November 2021 until April 2022, approximately 110,000 samples from public health services were received and analyzed, from which 14,190 were positive for SARS-CoV-2. Following an agreement of the Government with ECDC, 96 samples were sent on a bi-weekly basis to Eurofins Genomics Europe Sequencing GmbH for full-genome sequencing. For sample selection, a random, unbiased approach was taken without pre-screening for variants of interest to avoid sampling bias. The viral RNA had been previously detected using the Thermo Fisher TaqPath™ COVID-19 CE-IVD RT-PCR kit and all samples selected showed a cycle threshold value (Ct) lower than 30. The study has been approved by the Cyprus National Bioethics Committee (EEBK 21.1.01.03). According to the approval, the Bioethics Committee waived the requirement for informed consent as samples were completely anonymized.

Richter J., Koptides D., Tryfonos C., Alexandrou D, & Christodoulou C. (2022). Introduction, Spread and Impact of the SARS-CoV-2 Omicron Variants BA.1 and BA.2 in Cyprus. Microorganisms, 10(9), 1688.

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SARS-CoV-2 Detection via RT-PCR

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Respecting preventive safety measures, samples were collected by qualified personnel in sample collection tubes with an InActiv Blue^® virus-inactivating and RNA-stabilizing transport medium (Fertipro NV, Beernem, Belgium). The virus was further inactivated by incubation for 30 min at 74 °C. After vortexing the sample, 200 µL of transport medium was transferred into a 96 Deepwell plate (Thermo Fisher Scientific^TM, Waltham, Massachusetts, USA) and the bacteriophage MS2, a single stranded RNA virus, was added as the internal control. RNA was extracted using the MagMAX™ Viral/Pathogen II Nucleic Acid Isolation Kit on the KingFisher^TM Flex Purification System. The TaqPath™ COVID-19 CE-IVD RT-PCR kit (Thermo Fisher Scientific^TM, Waltham, Massachusetts, USA) was used for the PCR in the QuantStudio^TM 5 instrument (ThermoFisher). This assay is designed to detect three SARS-CoV-2 target genes: ORF1ab, S, and N. FastFinder software (v4.6.3, UgenTec, Hasselt, Belgium) was used for the analysis and interpretation of the results.

De Meyer J., Goris H., Mortelé O., Spiessens A., Hans G., Jansens H., Goossens H., Matheeussen V, & Vandamme S. (2022). Evaluation of Saliva as a Matrix for RT-PCR Analysis and Two Rapid Antigen Tests for the Detection of SARS-CoV-2. Viruses, 14(9), 1931.

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Viral Load Assessment by RT-qPCR

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For the assessment of the viral loads, biomolecular examinations were conducted using real-time RT-qPCR. Aliquots of 200 µL were collected from UTM, cell culture and sub-culture cryo-lysates with the addition of 200 µL TRIzol (Ambion, Life Technologies, Carlsbad, CA, US) to perform nucleic acid extraction and purification with KingFisher Flex (Thermo Fisher Scientific, Waltham, MA, USA), using the MVP_2Wash_200_Flex programme, following the manufacturer’s instructions. The elution was carried out in a 50-µL final volume and stored at –80°C until use. RT-qPCR analysis was conducted using TaqPath COVID-19 CE-IVD RT–PCR kit (Thermo Fisher Scientific, Waltham, MA, USA). Amplification settings, thermal profile, and viral loads were performed according to the protocol already described by Cardillo and colleagues [26 (link)].
The association of the development of CPE and the increase in viral loads over time by RT-qPCR allowed us to assess the stability and viability of the virus, which was titrated using the TCID50/mL assay.

Fusco G., Picazio G., Cardillo L., Pucciarelli A., Marati L., Di Maggio F., Nunziato M., Brandi S., De Carlo E., de Martinis C, & Salvatore F. (2023). A comparative study on the persistence and viability of SARS-CoV-2 wild-type and omicron variant on artificially contaminated surfaces: the role of fomites. Emerging Microbes & Infections, 12(2), 2239941.

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SARS-CoV-2 Detection by TaqPath RT-PCR

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Viral RNA was purified from 200 µL of UTM by the MagMax Core Nucleic Acid Purification Kit (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA) in automated sample preparation workstation MagMAX Express 96 (Applied Biosystems), according to the manufacturer’s instructions.
Samples were tested by TaqPath™ COVID-19 CE-IVD RT-PCR Kit (Thermo Fisher) using the 7500HT Fast Real-Time PCR System (Applied Biosystems) according to manufacturer’s instructions. The kit is a multiplex assay that allows amplification of conserved regions in the S, N, and ORF1ab genes of the SARS-CoV-2 genome. Bacteriophage MS2 was used as an internal control to prevent false-negative results due to inhibition factors. Ct values were generated using the 7500 Software SDS 2.3 (Applied Biosystems).
Data analyses were performed by the Applied Biosystem^TM COVID-19 Interpretive Software, v.1.3 (Thermo Fisher Scientific, Waltham, MA, USA).

Rocchigiani A.M., Ferretti L., Ledda A., Di Nardo A., Floris M., Bonelli P., Loi F., Idda M.L., Angioi P.P., Zinellu S., Fiori M.S., Bechere R., Capitta P., Coccollone A., Coradduzza E., Dettori M.A., Fattaccio M.C., Gallisai E., Maestrale C., Manunta D., Pedditzi A., Piredda I., Palmas B., Salza S., Sechi A.M., Tanda B., Madrau M.P., Sanna M.L., Cherchi S., Ponti N., Masala G., Sirica R., Evangelista E., Oggiano A., Puggioni G., Ligios C, & Dei Giudici S. (2023). Origin, Genetic Variation and Molecular Epidemiology of SARS-CoV-2 Strains Circulating in Sardinia (Italy) during the First and Second COVID-19 Epidemic Waves. Viruses, 15(2), 277.

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SARS-CoV-2 Detection and Quantification

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We extracted and purified viral RNA from 300 μL of nasopharyngeal exudates with the KingFisher instrument (ThermoFisher Scientific, https://www.thermofisher.com). This process was followed by RT-PCR using the TaqPath COVID-19 CE-IVD RT-PCR kit (ThermoFisher Scientific), which targets open reading frame 1ab, nucleoprotein, and spike genes. We performed serum antibody determinations by specific quantitative detection of SARS-CoV-2 IgG by using a chemiluminescent microparticle immunoassay on the ARCHITECT system (SARS-CoV-2 IgG II Quant Reagent Kit; Abbott Laboratories, https://www.abbott.com).

Rodríguez-Grande C., Alcalá L., Estévez A., Sola-Campoy P.J., Buenestado-Serrano S., Martínez-Laperche C., Manuel de la Cueva V., Alonso R., Andrés-Zayas C., Adán-Jiménez J., Losada C., Rico-Luna C., Comas I., González-Candelas F., Catalán P., Muñoz P., Pérez-Lago L, & García de Viedma D. (2022). Systematic Genomic and Clinical Analysis of Severe Acute Respiratory Syndrome Coronavirus 2 Reinfections and Recurrences Involving the Same Strain. Emerging Infectious Diseases, 28(1), 85-94.

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RT-PCR and Nanopore Sequencing of COVID-19 Samples

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RT-PCR of nasopharyngeal swabs were carried out on samples using TaqPath COVID-19 CE-IVD RT-PCR kit (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. Nanopore sequencing was carried out as previously described [16 (link)]. Briefly, the extracted RNA of 56/190 samples were subjected to nanopore sequencing according to the manufacture’s instruction using the SQK-RBK110.96 rapid barcoding kit (ONT, Oxford, UK). 1200 bp tiled PCR amplicons were generated with midnight primers as described in Freed et al., 2020 [17 (link)]. All the thermal cycling steps were carried out in a QuantStudio™ 5 Real-Time PCR Instrument (Applied biosystems, Singapore). Barcodes were attached to resulting amplicons and pooled together before the clean-up step. Finally, 800ng of library was loaded into R9.4.1 flow cell and sequenced on the Oxford Nanopore Minion Mk1C. The run was stopped once desired number of reads (~15,000 reads per sample) were achieved.

Jeewandara C., Aberathna I.S., Dayarathna S., Nimasha T., Ranasinghe T., Jayamali J., Kamaladasa A., Karunanada M., Perera L., Ogg G.S, & Malavige G.N. (2022). Comparison of the kinetics and magnitude of antibody responses to different SARS-CoV-2 proteins in Sinopharm/BBIBP-CorV vaccinees following the BNT162b2 booster or natural infection. PLoS ONE, 17(10), e0274845.

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SARS-CoV-2 Detection and Variant Screening

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SARS-CoV-2 was detected using the MGIEasy Nucleic Acid Extraction kit on a MGISP-960 system (Beijing Genome Institute) to extract virus RNA, followed by an RT-PCR with the TaqPath COVID-19 CE-IVD RT-PCR kit (ThermoFisher Scientific) running on the QuantStudio 5 System (Applied Biosystems), or the Aptima SARS-CoV-2 Transcription-Mediated Amplification (TMA) Assay on the Panther System (Hologic). The TaqPath assay targets 3 viral genes including the S gene, whose detection is hampered in the case of del69/70. We initially used this characteristic of SGTF/SGTL detection for variant screening, to discriminate the first Omicron VOC 21K/BA.1 from the Delta VOC (30 (link), 31 (link)). We later screened for variant-specific mutations using the ID SARS-CoV-2/VOC Revolution Pentaplex assay (ID-Solutions) after viral RNA extraction on the MGISP-960 system.

Trémeaux P., Latour J., Ranger N., Ferrer V., Harter A., Carcenac R., Boyer P., Demmou S., Nicot F., Raymond S, & Izopet J. (2023). SARS-CoV-2 Co-Infections and Recombinations Identified by Long-Read Single-Molecule Real-Time Sequencing. Microbiology Spectrum, 11(4), e00493-23.

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SARS-CoV-2 Detection and Variant Screening

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SARS-CoV-2 Variant Tracking in Switzerland

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Throughout the pandemic, Dr. Risch laboratories served all six regions of Switzerland as well as the principality of Liechtenstein with SARS-CoV-2 PCR testing. Referred samples originated from every canton and were mostly nasopharyngeal swabs or saliva samples. Alongside other testing methods, routine PCR testing was performed using the TaqPath COVID-19 CE-IVD RT-PCR Kit by ThermoFisher Scientific, Lucerne, Switzerland (TaqPath). All positive samples, starting from calendar week 37 of 2020 up to calendar week 47 of 2022, tested with the TaqPath Kit were included in the study, spanning a period encompassing the SARS-CoV-2 variant waves of B.1.1.7 (Alpha), B.1.617.2 (Delta), as well as Omicron variants BA.1, BA.2, and BA.4/5. Omicron variants BA.4 and BA.5 were summarized due to their concurrent presence and identical SGTF pattern.

Hilti D., Wehrli F., Berchtold S., Bigler S., Bodmer T., Seth-Smith H.M., Roloff T., Kohler P., Kahlert C.R., Kaiser L., Egli A., Risch L., Risch M, & Wohlwend N. (2024). S-Gene Target Failure as an Effective Tool for Tracking the Emergence of Dominant SARS-CoV-2 Variants in Switzerland and Liechtenstein, Including Alpha, Delta, and Omicron BA.1, BA.2, and BA.4/BA.5. Microorganisms, 12(2), 321.

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