# Detection of SARS-CoV-2 RNA by multiplex RT-qPCR

## TL;DR

This paper introduces a simplified RT-qPCR test for SARS-CoV-2 that reduces reagent use and labor while maintaining accuracy.

## Contribution

The novel multiplex RT-qPCR assay detects SARS-CoV-2 RNA in a single reaction, saving resources compared to traditional methods.

## Key findings

- The multiplex RT-qPCR assay detected SARS-CoV-2 RNA with cycle threshold values comparable to the standard single assay.
- Low copy numbers (≥500 copies/reaction) of SARS-CoV-2 RNA were consistently detected using the new method.

## Abstract

The current quantitative reverse transcription PCR (RT-qPCR) assay recommended for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing in the United States requires analysis of 3 genomic targets per sample: 2 viral and 1 host. To simplify testing and reduce the volume of required reagents, we devised a multiplex RT-qPCR assay to detect SARS-CoV-2 in a single reaction. We used existing N1, N2, and RP primer and probe sets by the Centers for Disease Control and Prevention, but substituted fluorophores to allow multiplexing of the assay. The cycle threshold (Ct) values of our multiplex RT-qPCR were comparable to those obtained by the single assay adapted for research purposes. Low copy numbers (≥500 copies/reaction) of SARS-CoV-2 RNA were consistently detected by the multiplex RT-qPCR. Our novel multiplex RT-qPCR improves upon current single diagnostics by saving reagents, costs, time, and labor.

The current RT-qPCR assay recommended for SARS-CoV-2 testing in the United States requires analysis of three genomic targets per sample: two viral and one host. This study describes a multiplex PCR that saves both reagents and labor while accurately detecting SARS-CoV-2 RNA.