Resource Summary Report  New Search Previous Search Results

URL: https://dx.doi.org/DOI:10.17504/protocols.io.bfc8jizw

Authors: Wei-Ting Lu, Ming Jiang, Robert Goldstone, Karen Ambrose, Chris Ekin, Amy Strange, Nnenna Kanu, Paul Grant, Efthymios Fidanis, Jerome Nicod, David Moore, Michael Howell

Group: BGI, Coronavirus Method Development Community, Crick COVID-19 Consortium

Summary: Purpose of examination / Clinical relevanceAt the end of 2019, several pneumonia cases were reported in Wuhan, China and the pathogen was confirmed as a new viral strain. World Health organization has named the newly identified coronavirus as 2019-nCoV, also known as COVID19. The disease developed into a dangerous pandemic, posing major challenges to the NHS. Although more research is necessary to better understand the virus, in response to the emergency, simple and rapid testing is essential to identify the virus in infected individuals. This will aid the implementation of efficient interventions to contain the spread, and distinguish healthcare workers who have been infected, and are required to self-isolate, from those showing similar symptoms but which are not 2019-nCoV associated. The latter category may continue to work, alleviating stress on hard-pressed healthcare resources. 2019-nCoV is an RNA virus, and the diagnostic tests detect viral RNA in swabs from patient airways using a reverse transcriptase PCR assay. Samples are submitted to HSL, an accredited reporting laboratory, and transferred to the Crick for testing. The first step of the process is sample receipt at the Crick.This protocol describes the preparation of the RT-PCR master mix plate, automated addition of RNA template and the reverse-transcriptase quantitative PCR of viral RNA to detect the Orf1ab region of the viral genome, which has high specificity to the 2019-nCoV virus. Furthermore, the protocol describes the analysis of the COVID-19 test results.Principles of ExaminationThe kit is a qualitative in vitro nucleic acid amplification assay to detect the new coronavirus 2019-nCoV identified in China in 2019 using Reverse transcription PCR of RNA derived from specimen of throat swabs and Bronchoalveolar Lavage Fluid (BALF) from individuals suspected to have been exposed to the virus. The kit is based on in vitro RT-PCR with fluorescent probes. Primers and sequence-specific fluorescence probes were designed tailored to the high conservative ORF1ab region of the 2019-nCoV genome. The probes are oligonucleotides with attached fluorophores at the 5' end (FAM as reporter) and at the 3’ end (quencher). In addition, a specific primer and probe set designed to human b-actin is included as an internal reference with fluorophores VIC/HEX attached at 5’ end as reporter. During the PCR procedure, the DNA polymerase cleaves the probe at the 5’ end and separates the reporter dye from the quencher dye when the probes hybridize to the target DNA. This cleavage results in a fluorescent signal generated by the cleaved reporter dye, which is monitored in real-time by the PCR detection system. Monitoring the fluorescence intensities during Real Time allows the qualitative detection of 2019-nCoV in specimens.Analysis of the COVID19 test results will allow the qualitative detection of the 2019-nCoV nucleic acid in specimens from patients with suspected 2019n-nCoV. This process will enable the assessment of the infection in clinical and public health practice. The workflow for reporting results from this test is illustrated below:

Associated Publications: Aitken, J., Ambrose, K., Barrell, S. et al. Scalable and robust SARS-CoV-2 testing in an academic center. Nat Biotechnol (2020). https://doi.org/10.1038/s41587-020-0588-y

Affiliations: The Francis Crick Institute, The Francis Crick Institute, The Francis Crick Institute, The Francis Crick Institute, Health Services Laboratories, The Francis Crick Institute, University College London, Health Services Laboratories, The Francis Crick Institute, The Francis Crick Institute, University College London, The Francis Crick Institute

External URL: https://doi.org/10.1038/s41587-020-0588-y

Version: 1

Publication Date: 2020

Expand All Collapse