2.6 Analytical sensitivity: singleplex assays
The LOD of the singleplex assays was determined using serial dilutions of the PACs (plasmid DNA or in-vitro transcribed RNA) or the viral DNA/RNA in nuclease-free water as template. The viral DNA/RNA extracted from CC grown viruses were used as template except for PaPV which was extracted from CS of an infected cattle (FADDL/DSS accession ID 15-020595). The LOD of the PACs were expressed as copy number per assay where each copy represents one genome copy of the virus particle. The copy number was calculated using the Avogadro’s number (6.022x1023 molecules/mole) using the following formula:
Copy number = [mass (g) × 6.022×1023]/[length (bp) × 109 x 650 Da], or, using the web link http://cels.uri.edu/gsc/cndna.html.When using the viral DNA/RNA as template, the LODs were expressed either as the highest detectable dilution or the lowest detectable virus titer (TCID50/ml) per assay.
The amplification efficiency (AE) and correlation coefficient (R2) of the multiplex assays were determined from the standard curves prepared by plotting the fluorescence threshold cycle (Ct) values against log10 serial dilution of the PACs or the viral DNA/RNA used as template.