Comparison of reverse-transcription qPCR and droplet digital PCR for the detection of SARS-CoV-2 in clinical specimens of hospitalized patients

Abstract

Accurate detection of severe acute respiratory syndrome coronavirus 2 is not only necessary for viral load monitoring to optimize treatment in hospitalized coronavirus disease 2019 patients, but also critical for deciding whether the patient could be discharged without any risk of viral shedding. Digital droplet PCR (ddPCR) is more sensitive than reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and is usually considered the superior choice. In the current study, we compared the clinical performance of RT-qPCR and ddPCR using oropharyngeal swab samples from patients hospitalized in the temporary Huoshenshan Hospital, Wuhan, Hubei, China. Results demonstrated that ddPCR was indeed more sensitive than RT-qPCR. Negative results might be caused by poor sampling technique or recovered patients, as the range of viral load in these patients varied significantly. In addition, both methods were highly correlated in terms of their ability to detect all three target genes as well as the ratio of copies of viral genes to that of the IC gene. Furthermore, our results evidenced that both methods detected the N gene more easily than the ORF gene. Taken together, these findings imply that the use of ddPCR, as an alternative to RT-qPCR, is necessary for the accurate diagnosis of hospitalized coronavirus disease 2019 patients.

Keywords: COVID-19; Clinical performance; Quantitative detection; RT-qPCR; SARS-CoV-2; ddPCR.

Fig. 1

Comparison of the distribution of results obtained with RT-qPCR and ddPCR. (A) CT values of the IC gene in negative, suspected, and positive samples determined using RT-qPCR. (B) -log2 values of the IC gene in negative, suspected, and positive samples determined using ddPCR. (C) CT values of the ORF and N genes determined using RT-qPCR. (D) CT values of the ORF and N genes determined using ddPCR. P values are included in the box. CT = cycle threshold; ddPCR = digital droplet PCR; IC gene = internal control gene; RT-qPCR = real-time polymerase chain reaction.

Fig. 2

Correlation analysis between the CT values and -log2 values determined by RT-qPCR and ddPCR, respectively, for the IC gene (A), ORF gene (B), and N gene (C). Comparison of deviations of values obtained for each sample ran using the same method. (A) CT values of the three target genes determined using RT-qPCR. (B) -log2 values of the three target genes determined using ddPCR. Correlation analysis between the ORF and N genes. (A) CT values determined with RT-qPCR. (B) -log2 values determined by ddPCR. CT = cycle threshold; ddPCR = digital droplet PCR; IC gene = internal control gene; RT-qPCR = real-time polymerase chain reaction.

Fig. 3

Correlation analysis among ratios of the three target genes determined using the same or different method. (A) Correlation between CT_IC/ CT_ORF and CT_IC/ CT_N determined using RT-qPCR. (B) Correlation between −log2(value_IC/ value_ORF) and -log2(value_IC/ value_N) determined using ddPCR. (C) Correlation between CT_IC/ CT_ORF and −log2(value_IC/ value_ORF) determined by RT-qPCR and ddPCR, respectively. (D) Correlation between CT_IC/ CT_N and −log2(value_IC/value_N) determined by RT-qPCR and ddPCR, respectively. (E) Correlation between CT_N/ CT_ORF and −log2(value_N/value_ORF) determined by RT-qPCR and ddPCR, respectively. CT = cycle threshold; ddPCR = digital droplet PCR; IC gene = internal control gene; RT-qPCR = real-time polymerase chain reaction.