Highly sensitive detection of SARS-CoV-2 RNA by multiplex rRT-PCR for molecular diagnosis of COVID-19 by clinical laboratories

Abstract

Background: The detection of SARS-CoV-2 RNA by real-time reverse transcription-polymerase chain reaction (rRT-PCR) is used to confirm the clinical diagnosis of COVID-19 by molecular diagnostic laboratories. We developed a multiplex rRT-PCR methodology for the detection of SARS-CoV-2 RNA.

Methods: Three genes were used for multiplex rRT-PCR: the Sarbecovirus specific E gene, the SARS-CoV-2 specific N gene, and the human ABL1 gene as an internal control.

Results: Good correlation of C_q values was observed between the simplex and multiplex rRT-PCR methodologies. Low copies (<25 copies/reaction) of SARS-CoV-2 RNA were detected by the novel multiplex rRT-PCR method.

Conclusion: The proposed multiplex rRT-PCR methodology will enable highly sensitive detection of SARS-CoV-2 RNA, reducing reagent use and cost, and time required by clinical laboratory technicians.

Keywords: COVID-19; High-sensitive; Molecular diagnosis; Multiplex; SARS-CoV-2; rRT-PCR.