[PROTOCOL] 2019nCoV: Detection of 2019 novel coronavirus (2019-nCoV) in suspected human cases

by RT-PCR


This protocol is designed to detect 2019-nCoV in human clinical specimens. It was developed by Daniel Chu and Leo Poon, Division of Public Health Laboratory Sciences (HKU), and Malik Peiris, Co-Director at HKU-Pasteur.

The two monoplex assays described here are reactive with coronaviruses under the subgenus Sarbecovirus that includes 2019-nCoV, SARS-CoV and bat SARS-like coronaviruses. The rationales for using this detection approach are: 1) the genetic diversity of 2019-nCoV in humans and animals is yet to be fully determined and 2) many laboratories lack positive controls for 2019-nCoV. Viral RNA extracted from SARS-CoV can be used a positive control in the assays below. As SARS was eliminated in humans, suspected cases that are positive in these RT-PCR assays should be considered to be infected by the 2019-nCoV. The N gene RT-PCR is recommended as a screening assay and the Orf1b assay as a confirmatory one. In the event of a positive PCR result, sequence analyses of the amplicons will further help to confirm the result and to distinguish between SARS-CoV and 2019-nCoV. An N gene positive/Orf1b negative result should be regarded as indeterminate and the case is recommended to be referred to a WHO reference lab for further testing.

These assays have been evaluated using a panel of controls and only the positive control (SARS- CoV RNA) is tested positive in these assays. NB. Synthetic oligonucleotide positive controls or equivalents for 2019-nCoV is not available at present but will be available shortly.

Suitable biosafety precautions should be taken for handling human clinical specimens suspected to be 2019-nCoV infections.

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