Screening for novel coronavirus with one-step RT-qPCR: getting ahead of the outbreak

Multiple deaths, disrupted air travel to and from China, and the quarantine of potentially exposed individuals has prompted the World Health Organization (WHO) to declare the novel coronavirus (2019-nCoV) outbreak that originated in Wuhan, China, a global health emergency. The New York Times has reported that, as of January 31, 2020, the respiratory illness caused by 2019-nCoV has resulted in a least 132 deaths worldwide and caused about 6,000 people to become ill.

Coronaviruses are a group of viruses that can cause upper- and lower-respiratory tract illnesses and have a characteristic spiky appearance that resembles the sun's corona. Although many common coronaviruses usually cause mild to moderate illnesses, some can cause severe illnesses that can progress to an often-fatal pneumonia, which occurred during the MERS-CoV and SARS-CoV outbreaks of 2015 and 2003, respectively. Since there is a 2% death rate from 2019-nCoV and it may be transmitted by exposed individuals who show no symptoms, effective screening tests for the virus are vitally important to controlling this outbreak.

The authors of the first publication that identified 2019-nCoV (Zhu et al. 2020) developed a screening test for the virus that uses Takara Bio's One Step PrimeScript RT-PCR Kit (Perfect Real Time) to detect specific viral sequences they obtained using RNA-seq technology to sequence and classify 2019-nCoV. This group also visualized the viral particles and observed cytopathic effects in 2019-nCoV-infected human airway epithelial cell cultures using light microscopy and transmission electron microscopy. Another publication (Zhou et al. 2020) has identified the virus as 96% identical at the whole-genome level to a bat coronavirus. The pairwise protein sequence analysis of seven conserved nonstructural proteins showed that this virus belongs to the species of SARSr-CoV. Importantly, they confirmed that 2019-nCoV uses the same cell entry receptor, ACE2, as SARS-CoV. Takara Bio's SMARTer RACE 5'/3' Kit was used to determine the 5' ends of the genomes. Another research group (Letko and Munster 2020) studied the cell-entry mechanism of 2019-nCoV by using Takara Bio's In-Fusion Cloning technology to clone the spike coding sequences of the virus.

Similar approaches have since been used by many groups to identify other parts of this specific virus. A 2019-nCoV detection method using the one-step PrimeScript RT-PCR kit has been recommended by the Chinese Center for Disease Control and Prevention and is being used throughout China. The World Health Organization (WHO) and Centers for Disease Control and Prevention (CDC) have developed similar methods, and the WHO will be making recommendations for viral detection methods as more cases are reported. We are moving quickly to meet the demand for this kit in China as well as in other countries where demand for 2019-nCoV screening is increasing.

References

Letko, M. & Munster, V. Functional assessment of cell entry and receptor usage for lineage B β-coronaviruses, including 2019-nCoV. bioRxiv 2020.01.22.915660 (2020).

What Is the Coronavirus? Symptoms, Treatment and Risks - The New York Times. at <https://www.nytimes.com/article/what-is-coronavirus.html>

Zhou, P. et al. Discovery of a novel coronavirus associated with the recent pneumonia outbreak in humans and its potential bat origin. bioRxiv 2020.01.22.914952 (2020).

Zhu, N. et al. A novel coronavirus from patients with pneumonia in China, 2019. N. Engl. J. Med. NEJMoa2001017 (2020).