Reading PIKA Test Kit Amplification Curves

The Open qPCR software provides more detail for your kit runs by generating Amplification Curves, which show the fluorescence measurements for each target fluorescence channel. An increase in fluorescence indicates the presence of your target DNA for each channel. 

To view the amplification curves for your experiment, select “Amplification Curve” from the menu of your experiment results screen. Keep the “Baseline Subtraction” option at the top of your curve toggled on. You can toggle between the channels under the “Targets” window to the right of your curve. Highlight individual or multiple wells at the bottom as desired to view the curves for each well. 

The dual channel Open qPCR allows for multiple target detection. The target detected by Channel 1, the FAM fluorophore detection channel, is your beer spoiler DNA, which will be present in the Positive Control and any contaminated samples. Channel 2, the HEX fluorophore detection channel, detects the Internal Control (IPC) which should be present in all control and sample tubes. This channel functions as a quality control to detect potential PCR inhibition and prevent false negatives.

You can also analyze your results by looking at the Cq values for each channel in the table to the right of the Amplification Curve. The Cq value is the cycle during the 40-cycle PCR run where there is a detectable amount of your specific target (whether your beer spoiler DNA or Internal Control). 

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.

Here is what to expect in each of your control and sample results: 

Positive Control: 

Channel 1 (blue): The Positive Control should always amplify and generate a Cq value for your beer spoiler target. 

Channel 2 (purple): A Cq value may or may not be generated for the Internal Control (IPC) target. Either is acceptable and does not indicate inhibition.

Negative Control: 

Channel 1 (blue): The beer spoilage target should not amplify and should never generate a Cq value for the Negative Control. A Cq value here indicates contamination at some point during reaction setup.

Channel 2 (purple): A Cq value should always be present for the Internal Control (IPC). Lack of a Cq value indicates PCR inhibition or incorrect PCR reaction setup.

Beer samples with positive result: 

Channel 1 (blue): Samples with trace amounts of beer spoiler will amplify and generate Cq values for the beer spoilage target. The more spoiler presence there is, the earlier the curve will amplify and the lower the Cq value will be. 

Channel 2 (purple): Samples with trace amounts of beer spoiler may or may not amplify the Internal Control (IPC). Either way is acceptable and does not indicate inhibition.

Beer samples with negative result:

Channel 1 (blue): Clean samples will have flat amplification lines and will not generate Cq values for the beer spoilage target.

Channel 2 (purple): A clean sample should always have a Cq value present for the Internal Control (IPC). Lack of a Cq value indicates PCR inhibition or incorrect PCR reaction setup.