Table 4.
Analytical specificity of the 5-plex PCR examined by mixtures of different viruses.
| Sample No. | Sample Detail (Volume Mixing Ratio) | PEDV/PDCoV/TGEV/SADS-CoV/XIPC 5-Plex PCR Ct Value | ||||
|---|---|---|---|---|---|---|
| PEDV (JUN) | TGEV (VIC) | PDCoV (ABY) | SADS-CoV (FAM) | XIPC (Cy5) | ||
| Sample #1 | PEDV USA/IN19338/2013 | 15.208 | ≥40 | ≥40 | ≥40 | 31.359 |
| Sample #2 | TGEV Purdue (VR-763) | ≥40 | 13.874 | ≥40 | ≥40 | 31.658 |
| Sample #3 | PDCoV USA/IL/2014 | ≥40 | ≥40 | 16.55 | ≥40 | 31.891 |
| Sample #4 | SADS-CoV N-gene IVT RNA | ≥40 | ≥40 | ≥40 | 26.057 | 25.991 |
| Sample #5 | PEDV & TGEV (1:1) | 17.245 | 16.413 | ≥40 | ≥40 | 31.079 |
| Sample #6 | PEDV & PDCoV (1:1) | 17.375 | ≥40 | 17.332 | ≥40 | 32.060 |
| Sample #7 | TGEV & PDCoV (1:1) | ≥40 | 15.993 | 17.834 | ≥40 | 32.358 |
| Sample #8 | PEDV & SADS-CoV (1:1) | 15.646 | ≥40 | ≥40 | 26.771 | 32.073 |
| Sample #9 | TGEV & SADS-CoV (1:1) | ≥40 | 15.076 | ≥40 | 26.870 | 32.533 |
| Sample #10 | PDCoV & SADS-CoV (1:1) | ≥40 | ≥40 | 16.936 | 27.144 | 32.941 |
| Sample #11 | PEDV & TGEV & PDCoV (1:1:1) | 18.434 | 15.574 | 17.216 | ≥40 | 33.345 |
| Sample #12 | PEDV & TGEV & SADS-CoV (1:1:1) | 17.539 | 16.991 | ≥40 | 26.980 | 32.119 |
| Sample #13 | PEDV & PDCoV & SADS-CoV (1:1:1) | 17.215 | ≥40 | 17.22 | 27.434 | 32.812 |
| Sample #14 | TGEV & PDCoV & SADS-CoV (1:1:1) | ≥40 | 16.454 | 18.261 | 27.469 | 32.630 |
| Sample #15 | PEDV & TGEV & PDCoV & SADS-CoV (1:1:1:1) | 19.124 | 16.782 | 17.869 | 28.350 | 35.304 |
| Sample #16 | Nuclease-free water without XIPC | ≥40 | ≥40 | ≥40 | ≥40 | ≥40 |
Notes: For samples #5–#15, the respective viruses were manually mixed at the equal volume ratio; the PEDV probe was labeled with fluorescence dye JUN; the TGEV probe was labeled with fluorescence dye VIC; the PDCoV probe was labeled with fluorescence dye ABY; the SADS-CoV probe was labeled with fluorescence dye FAM; XIPC is an internal positive control and its probe was labeled with fluorescence dye CY5.