Into the Genome: COVID-19 Diagnostics
Controlling the spread of a disease outbreak requires an accurate understanding of the causative agent, transmission mechanism, patient profiles, and available interventions. Unexplained respiratory outbreaks including the recent COVID-19 disease outbreak, previously referred to as 2019 novel coronavirus (2019nCoV), started from initial investigations of a cluster of pneumonia cases in Wuhan. A new type of coronavirus, now called SARS-CoV-2, is then isolated from infected patients early January 2020.
Novelty of the pathogen
Five days after the virus has been isolated, Wuhan scientists published the full genome sequence of SARSCoV-2. This shared information helps in the international community to join in the development of tools to stop the advance of the disease outbreak – from diagnostic tools to preliminary vaccine development. Due to this effort, what took almost six months for the SARS outbreak for assay establishment is completed in just a few weeks for COVID-19.
Through genome analysis, SARS-CoV-2 is recognized as a sister to severe acute respiratory syndrome coronaviruses (SARS-CoVs) and belongs to the genus Betacoronavirus, subgenus Sarbecovirus, species Severe acute respiratory syndrome-related coronavirus. The relatedness of SARS-CoV-2 to MERS-CoV (2012, Betacoronavirus, subgenus Merbecovirus) and SARS-CoV (2002, Betacoronavirus, subgenus Sarbecovirus) greatly helps in the development of diagnostics and therapeutics.
Figure 1. Genome (29.7 kb organization of SARS-CoV-2), Image source: GISAID Genomic epidemiology of SARS-CoV-2
Like other coronaviruses, the positive-strand RNA genome of SARS-COV-2 consists of open reading frames (ORFs) corresponding to predicted proteins required for the pathogenicity of the viruses including polymerase 1a,1b (RNA-dependent RNA polymerase, RdRP), surface proteins - spike protein (S), small membrane protein (E), membrane protein (M), nucleocapsid (N), and other accessory proteins needed by the virus to infect cells.
With the continuous addition of full-length genome sequences being published online, samples show differences and few mutations from a common ancestor, which may have originated between November-December 2019. The sustained human-to-human transmission allows for continued mutation of the virus; as what is also observed for RNA viruses. Despite this, diversity is only observed at select regions in the genome, including ORF1a, ORF3a, and ORF8. This allows the development of PCR-based primers and probes against conserved regions of the genome.
Development of diagnostic kits
Due to the swift action to publish a full genome sequence, research and development labs, as well as the diagnostic industry, quickly jumped into the fast development of diagnostic kits for COVID-19 confirmation.
Real-time PCR kits have been designed based on differentiating regions (to related coronaviruses) in the genome of SARS-CoV-2. The first paper (Zhu et al.) describing the virus used the S gene for analysis. Agency for Science, Technology and Research (A*Star) scientists also developed pre-packed diagnostic kit (approved by Health Sciences Authority) for the coronavirus, distributed to local hospitals and laboratories and later, 10,000 units sent to China.
WHO distributed kits developed by Drosten and team, which based the real-time PCR kit primers and probes on 3 genes, with the RdRP gene used to differentiate SARS-CoV to SARS-CoV-2.
Pan-CoV identification is also included in some developed kits to facilitate better classification of coronaviruses, as well as capturing possible mutations of the virus. In this respect, several variations are being developed including capability for multiplexing to identify detect presence of Betacoronavirus and differentiate different strains of coronavirus.
Solutions for diagnosis and research
PCR workflow for SARS-CoV-2 diagnosis would typically involve RNA extraction, mastermix preparation, and amplification. Diagnostics for SARS-CoV-2 should be conducted in a Biosafety Level 2 (BSL-2) laboratory, according to guidelines set by the Centers for Disease Control and Prevention (CDC). Esco offers range of quality products for laboratories handling SARS-CoV-2 diagnostics.
Figure 2. Esco products for PCR workflow
Esco equipment for PCR workflow assures the health professional and researcher accurate PCR results, less cross-contamination, and protection from harm.
Global efforts to stop the spread of the outbreak include development of rapid diagnostic kits and enhancing diagnostic capacity (providing test kits and training technicians to perform PCR). Faster turnaround times are needed as screening methods in some airports around the world include testing those that are high at risk – with recent travel history to China.
We contribute to the international community efforts by providing equipment enabling technologies to prevent further spread of COVID-19 outbreak. We ensure availability of stocks – as outbreaks have no breaks, and so do we!
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World Health Organization (2020). Novel Coronavirus - China. Last accessed 20 February 2020. Retrieved from https://www.who.int/csr/don/12-january-2020-novel-coronavirus-china/en/
Zhou, P. et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature https:// doi.org/10.1038/s41586-020-2012-7 (2020).