Lack of canine COVID-19 data fuels persisting concerns over dog-humaninteractions LifeAtPurdue
Bst 2.0 DNA polymerase, 0.6 U/µL WarmstartU/µL Antarctic Thermolabile UDG, 200 mM Phenol red, 0.2% Tween 20, pH 8.8@25 °C) , 2.5 µL of a 10 × LAMP primer mixture , 5 µL of DNA-free water, and 5 µL of DNA or mucus containing solution. Reactions were pipetted into wells of clear 96-well FrameStarskirted flat optical bottom PCR plates .
$${\text{Colorimetric}}\,{\text{absorbance}}\,{\text{ratio}}=\frac{{{\text{Absorbance at }}\,430{\text{ nm}} - {\text{Absorbance at }}\,620{\text{ nm}}}}{{{\text{Absorbance at }}\,520{\text{ nm}} - {\text{Absorbance at }}\,620{\text{ nm}}}}$$ An image of the plate was taken using an Epson Perfection V800 Photo scanner with settings set to professional mode, 48-bit color image type, and 720 dpi resolution. The image of the sample closest to the threshold value was processed using ImageJ to find the RGB values. Those RGB values were in turn used to calculate Hue Saturation Values values. The Hue threshold was used to determine positive versus negative results in other assays.
Primer sets were scored by first calculating the maximum colorimetric absorbance ratio and reaction time for each replicate at each template concentration for a given primer set. The average and standard deviation of these values were then calculated for each template concentration for a given primer set. Reaction time was defined as the first time point at which the absorbance ratio was greater than 3.0.
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