Quantification of Coronavirus Infection Using a Dual-Reporter Assay

Take a culture of genetically modified epithelial cells expressing two reporter proteins: a constitutively active Renilla luciferase or Rluc, which acts as a control, and a firefly luciferase or FFluc, rendered inactive by insertion of a protease-cleavable oligopeptide.

Infect the cells with a coronavirus.

The virus releases its RNA and expresses the viral protease, which cleaves the oligopeptide, activating FFluc.

Replace the media with a buffer containing a detergent and the FFluc substrate.

The detergent lyses the cells, releasing intracellular contents.

The FFluc interacts with its substrate and emits luminescence.

Transfer the lysate to an opaque plate and record the luminescence.

Add a buffer containing an inhibitor and an Rluc substrate.

The inhibitor binds to FFluc, quenching the luminescence.

The Rluc substrate reacts with Rluc, emitting luminescence.

Record the luminescence and calculate the FFluc-to-Rluc activity ratio. A higher ratio indicates a high level of coronavirus infection.