Development of multiplex RT-ddPCR assays for detection of SARS-CoV-2 and other common respiratory virus infections

Abstract

Background: Measures for mitigation of Coronavirus Disease 2019 (COVID-19) were set to reduce the spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). SARS-CoV-2 and other respiratory viruses share similar transmission routes and some common clinical manifestations. Co-circulation of SARS-CoV-2 and other common respiratory viruses is imminent. Therefore, development of multiplex assays for detecting these respiratory viruses is essential for being prepared for future outbreaks of respiratory viruses.

Methods: A panel of three reverse transcription droplet digital PCR (RT-ddPCR) assays were developed to detect 15 different human respiratory viruses. Evaluations of its performance were demonstrated. A total of 100 local and 98 imported COVID-19 cases in Hong Kong were screened for co-infection with other common respiratory viruses.

Results: All detected viral targets showed distinct signal clusters using the multiplex RT-ddPCR assays. These assays have a broad range of linearity and good intra-/inter-assay reproducibility for each target. The lower limits of quantification for all targets were ≤46 copies per reaction. Six imported cases of COVID-19 were found to be co-infected with other respiratory viruses, whereas no local case of co-infection was observed.

Conclusions: The multiplex RT-ddPCR assays were demonstrated to be useful for screening of respiratory virus co-infections. The strict preventive measures applied in Hong Kong may be effective in limiting the circulation of other human respiratory viruses. The multiplex assays developed in this study can achieve a robust detection method for clinical and research purposes.

Keywords: COVID-19; RT-ddPCR; SARS-CoV-2; co-infection; human respiratory viruses.

FIGURE 1

RT‐PCR droplets found positive for respiratory viruses in Assay 1. The x and y axes are the fluorescence amplitude in the HEX and FAM channels, respectively. Signals of RNA samples (up to 10³ copies per reaction) that are positive for a specific virus are shown as indicated (panels A–F). A, Influenza A virus (M gene); P1, PIV1 (HN gene); S, SARS‐CoV‐2 (RdRp gene); Ad, Adv (Hexon gene); R, RSV (N); N, Negative

FIGURE 2

RT‐PCR droplets found positive for respiratory viruses in Assay 2. The x and y axes are the fluorescence amplitude in the HEX and FAM channels, respectively. Signals of RNA samples (up to 10³ copies per reaction) that are positive for a specific virus are shown as indicated (panels A–H). B, Influenza B virus (M gene); P2, PIV2 (HN gene); P3, PIV3 (HN gene); P4, PIV4 (N gene); ER, EV/RV (5′‐UTR); M, HMPV (N gene); N, Negative

FIGURE 3

RT‐PCR droplets found positive for respiratory viruses in Assay 3. The x and y axes are the fluorescence amplitude in the HEX and FAM channels, respectively. Signals of RNA samples (up to 10³ copies per reaction) that are positive for a specific virus are shown as indicated (panels A–D). E, HCoV‐229E (N gene); L, HCoV‐NL63 (N gene); H, HCoV‐HKU1 (N gene); O, HCoV‐OC43 (N gene); N, Negative

FIGURE 4

Classification of clusters for the assays: (A) Assay 1, (B) Assay 2 and (C) Assay 3 in 2D graphs where the x and y axes are the fluorescence amplitude in the HEX and FAM channels, respectively. Each graph showed the cluster distributions when all targets were added in each assay. Each target was added with not more than 10³ copies per reaction. The table below the 2D graphs demonstrated the location of clusters and corresponding amplitudes for the specific targets of each assay. Abbreviations: A, Influenza A virus (M gene); P1, PIV1 (HN gene); S, SARS‐CoV‐2 (RdRp gene); Ad, Adv (Hexon gene); R, RSV (N); B, Influenza B virus (M gene); P2, PIV2 (HN gene); P3, PIV3 (HN gene); P4, PIV4 (N gene); ER, EV/RV (5′‐UTR); M, HMPV (N gene); E, HCoV‐229E (N gene); L, HCoV‐NL63 (N gene); H, HCoV‐HKU1 (N gene); O, HCoV‐OC43 (N gene); N, Negative

FIGURE 5

Classification of clusters for co‐infection samples: (A) case A; (B) Case B; (C) Case C; (D) Case D; (E) Case E and (F) Case F. The graph titles showed the reactions were done on the designated assay. The results were shown in 2D graphs where the x and y axes are the fluorescence amplitude in the HEX and FAM channels, respectively. Abbreviations: A, Influenza A virus (M); S, SARS‐CoV‐2 (RdRp); ER, EV/RV (5′‐UTR); E, HCoV‐229E (N); O, HCoV‐OC43 (N); N, Negative