Real-Time PCR and Ct Values

I am frequently asked what a Ct values means in a PCR (polyermase chain reaction) test result. I will try to explain what the Ct value means.

Real-time PCR is a highly sensitive way to amplify and quantify a specific nucleic acid sequence in real time. In veterinary disease testing, the specific nucleic acid sequence is often from a pathogenic bacteria, virus, fungus, yeast, or parasite.

For years culture techniques were considered the gold standard for detecting a range of pathogens involved in infectious disease in animals. The advent of real-time PCR has made us re-think the gold standard for diagnosis of a range of animal diseases.

Diagnostic samples from livestock are often contaminated from field collection, have undergone autolysis or bacterial overgrowth during transport to the laboratory, or are from animals that have chronic disease. This presents challenges that PCR can help overcome.

In addition, some pathogens are highly fastidious having evolved to grow within the host animal, not a laboratory. This is another challenge that PCR can overcome.

During real-time PCR, the sample to be tested is mixed with a reaction mix. The reaction mix contains a fluorescent dye that binds to DNA either free in the reaction mixture or attached to a sequence specific probe.

The PCR reaction consists of repeated cycles of heating and cooling. During these cycles of heating and cooling pathogen DNA, if present in the sample, is amplified exponentially.

After each cycle of heating and cooling, the amount of pathogen DNA is measured in a real-time PCR machine that detects the fluorescent signal being emitted by the sample reaction mixture. The software running the real-time PCR machine then plots (graphs) the amount of fluorescence against the cycle number.

The Ct value or cycle threshold is the point at which the amplification plot crosses a threshold (i.e. there is a significant detectable increase in fluorescence).

Once the fluorescence from a sample crosses the threshold, the sample is reported as positive.

Real-time PCR reactions are typically run for 35-45 heating and cooling cycles, depending on the assay. If a sample hasn’t crossed the threshold for an assay within this time, it is reported as negative.

Real-Time PCR result showing the threshold in red. The Ct value is where the thick black plot of normalized fluorescence versus cycle crosses the threshold read on the x-axis i.e. a Ct value of approximately 12.5.