Reading PIKA Test Kit Amplification Curves

The Open qPCR software provides more detail for your kit runs by generating an Amplification Curve, which shows how many fluorescence units there are in the two fluorescence channels. Fluorescence indicates the presence of targeted DNA for each channel. To view the amplification curves for your experiment, select “View Full Results” at the bottom of your results screen. Make sure the “Baseline Subtraction” option at the top left of your curve is toggled on. You can toggle between the channels next to the “Choose Channel” option at the top right of your curve. Highlight wells below the curve to view individual well results. 

The dual channel Open qPCR allows for multiple target detection. The target detected by Channel 1 is the beer spoiler DNA, which will be present in the Positive Control and any contaminated samples. Channel 2 detects the Internal Amplification Control in your negative control and clean beer samples. This channel functions as a quality control to detect potential PCR inhibition.

You can also analyze your results by looking at the Cq values in Channel 1 and Channel 2 in the table to the right of the Amplification Curve. The Cq value is inverse to the amount of target present in your reaction. A low Cq value indicates a high quantity of your target, while a high Cq value indicates a low target amount.

Channel 1: The positive control well should always amplify and generate a Cq Ch 1 value. The negative control should not amplify and should never generate a Cq Ch 1 value. Samples with trace amounts of beer spoiler will amplify and generate Cq Ch1 values; the more spoiler presence there is, the earlier the curve will amplify and the lower the Cq Ch1 value will be. Clean samples will have flat amplification lines and will not generate Cq Ch1 values. The curve below is from a successful run in which a customer ran a positive control, negative control, and two clean beer samples. The single amplified blue curve is the positive control well in Channel 1. 

Channel 2: Wells that do not amplify in Channel 1 should amplify in Channel 2 and generate Cq Ch2 values. This includes the negative control and wells containing clean samples. If these wells do not generate Cq Ch2 values, your experiment experienced inhibition. Your positive control well may also sometimes generate a Cq Ch2 value. For cases when there is Channel 1 amplification, Channel 2 sometimes may not amplify; this is acceptable and does not indicate inhibition. In the curve below, the Channel 2 amplified curves are from the positive control, negative control, and beer spoiler wells, while the flat lines are wells that contain no tubes.