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

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.

Fig 1. The qualification of multiplex RT-qPCR for SARS-CoV-2 compared with single RT-qPCR.

(A) Multiplex RT-qPCR detection of SARS-CoV-2 N1 and N2 genes was validated using 10-fold dilutions of viral RNA into pooled negative NP samples. We measured sensitivity and efficiency for 20 replicates. Data are mean ± SD. Individual values are indicated in Table 1. (B) The Ct values for 4 independent COVID-19 inpatients’ NP (n = 2) or saliva (n = 2) samples, 1 negative control, and 1 positive control (P) (10³ virus copies/μl) were compared between single RT-qPCR (FAM only), multicolor single RT-qPCR (Singleplex), and multiplex RT-qPCR (Multiplex). The dotted line indicates the cutoff Ct value of 38. Negative control was undetectable. Individual values are indicated in Table 2. (C) Forty-two RNA templates from NP swabs and saliva samples obtained from COVID-19 inpatients or healthcare workers and positive control (P) (10³ virus copies/μl) were investigated via single and multiplex RT-qPCR. The dotted line indicates the cutoff Ct value of 38. Individual values are indicated in Table 3. COVID-19, coronavirus disease 2019; Ct, cycle threshold; E, amplification efficiency; NP, nasopharyngeal; P, positive control; R², regression coefficient value; RT-qPCR, quantitative reverse transcription PCR; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.