Figure 2个  Table 4个
    • Virus (target) Target gene Primer Sequence ConcentrationA(nmol/L) Reference
      Multiplex PCR #1
      HCoV-NL63 Nucleoprotein Forward AGGACCTTAAATTCAGACAACGTTCT 100 Theamboon-Alers et al., 2007
      Reverse GATTACGTTTGCGATTACCAAGACT 50
      Probe FAM-TAACAGTTTTAGCACCTTCCTTAAGCAACCCAAACA-TAMRA 25
      HCoV-229E Nucleoprotein Forward CGCAAGAATTCAGAACCAGAG 50 Adapted from Theamboon-Alers et al., 2007
      Reverse GGCAGTCAGGTTCTTCAACAA 75
      Probe HEX-CCACACTTCAATCAAAAGCTCCCAAAATG-TAMRA 25
      SARS-CoV Nucleoprotein Forward TGGACCCACAGATTCAACTGA 50 Adapted from Theamboon-Alers et al., 2007
      Reverse GCTGTGAACCAAGACGCAGTAT 50
      Probe CY5-TAACCAGAATGGAGGACGCAATGAG-BHQ2 25
      Multiplex PCR #2
      HCoV-OC43 Nucleoprotein Forward GCTCAGGAAGGTCTGCTCC 50 Theamboon-Alers et al., 2007
      Reverse TCCTGCACTAGAGGCTCTGC 25
      Probe FAM-TTCCAGATCTACTTCGCGCACATACC-TAMRA 25
      MERS-CoV Nucleoprotein Forward GGCACTGAGGACCCACGTT 50 Adapted from Lu et al., 2014
      Reverse TTGCGACATACCCATAAAAGCA 50
      Probe CY5-CCCCAAATTGCTGAGCTTGCTCCATACA-BHQ2 25
      HCoV-HKU1 Replicase 1b Forward CCTTGCGAATGAATGTGCT 50 Adapted from Dare et al., 2007
      Reverse TTGCATCACCACTGCTAGTACCAC 375
      Probe HEX-TGTGTGGCGGTTGCTATTATGTTAAAGCCTG-TAMRA 25

      Table 1.  Primers for two-tube multiplex real-time RT-PCR.

    • Target Two-tube multiplex real-time RT-PCR One-tube multiplex real-time RT-PCR Awith inline electrophoresis
      Range of Ct values Positive samples (%) Positive samples (%)
      HCoV-NL63 25-33.5 5 (1.46) 5 (1.46)
      HCoV-229E 26-34 10 (2.89) 10 (2.89)
      SARS-CoV 0 0
      HCoV-OC43 26-35 6 (1.73) 5 (1.46)
      MERS-CoV 0 0
      HCoV-HKU1 28-34.5 3 (0.87) 1 (0.29)
      Co-infectiona 3 (0.87) 3 (0.87)
      Total 25-35 24 (6.94) 21 (6.1)
      Note: a HCoV-NL63 and HCoV-229E (n = 1), HCoV-OC43 and HCoV-229E (n = 2).

      Table 2.  Detection of coronavirus in 346 clinical samples.

    • Samples Ct values
      Monoplex real-time RT-PCRa Two-tube multiplex real-time RT-PCR
      Rhesus monkeys challenged with MERS-CoV
      Nasal swab 1 day post infection 33.86 34.00
      Nasal swab 2 days post infection 32.46 32.50
      Nasal swab 1 day pre infection
      Throat swab 2 days post infection 30.13 30.00
      Throat swab 1 day pre infection
      Anal swab 1 day post infection 34.05 34.20
      Anal swab 2 days pre infection
      Human patient with suspected MERS
      Pharyngeal swab 1 31.21
      Pharyngeal swab 2 31.27
      Pharyngeal swab 3 30.76
      Whole blood 36.50
      Note: a In-house monoplex real-time PCR for MERS-CoV N genes. The linear relationship can be described by y = –2.78x + 34.2.

      Table S1.  Two-tube multiplex real-time RT-PCR of clinical specimens.

    • Sample(Culture hours) Ct values
      Monoplex real-time RT-PCRa Two-tube multiplex real-time RT-PCR
      0 31.74 30.91
      2 27.22 27.14
      24 25.59 25.78
      48 25.03 25.32
      96 23.86 24.34
      120 25 25.29
      144 25.02 25.31
      168 25.56 25.76
      Uninfected cells
      2 × TCID50 of HCoV-HKU1 25 25.29
      Note: a In-house monoplex real-time PCR for HCoV-HKU1 replicase 1b. The linear relationship can be described by y = –3.2x + 33.4.

      Table S2.  Detection of HCoV-HKU1 in the supernatant of infected cultures of human airway epithelial cells.