Understanding Cq values in PCR
The Takeaway: A positive reaction in a polymerase chain reaction (PCR) assay is detected by an accumulation of fluorescent signals. The cycle threshold, which goes by a number of names and symbols, is the number of cycles required for that fluorescent signal to exceed background levels and “cross the threshold.” These levels are inversely proportional to the amount of target nucleic acid in a sample.
What is a Cq value?
Cq stands for cycle threshold and refers to the number of times a machine must copy a piece of genetic material before it can be detected in a PCR test. The value represents the amount of viral genetic material in a particular sample at a specific point in time. In general, the lower the Cq value, the higher the viral load that is found in the sample, while the higher the Cq value, the lower the viral load is.
Cycle threshold can be called by several different names that all mean basically the same thing. They include:
- Ct: threshold cycle
- Cp: Crossing point
- Cq: Quantification cycle
- TOP: Take-off point
While “Ct” is used widely in research settings, standards outlined more than a decade ago suggest the use of Cq.
What factors impact Cq values?
Although the Cq value is an indication of how much genetic material is present in a particular specimen, many factors can influence the overall result. Cq values played an important role in SARS-CoV-2 testing in recent years, although the value itself is not an indication of how sick a person is. Factors that can impact a Cq value include:
- What type of test was used
- Other substances present in the sample
- How long sample was stored prior to PCR testing and what the storage conditions were
- What type of sample was collected
- How much virus was present when the sample was collected
Common problems in determining Cq values
While the factors listed above can impact Cq values, how the sample is prepared, how the PCR experiment is run, and what PCR components are used all play roles as well. Common problems include:
- Using a poor-quality master mix with incorrect pH and salt concentrations, which can change fluorescence emission
- Using a poor-quality master mix with poor PCR reaction efficiency
- Issues with reporter dye ratios
- PCR inhibition
- Insufficient amounts of template
- Degradation of RNA or cDNA due to workplace contamination, handling behavior, or storage issues
Can Cq values be used to determine contagiousness?
Cq values play important roles in PCR tests, and were cited commonly during the height of SARS-CoV-2 testing during the pandemic. But can the values be used to tell health experts how contagious a person is? The answer is not clear.
While there is an indirect correlation between Cq values and viral presence, and viral presence is an indicator of contagiousness, there are many caveats. Poor sampling may impact Cq values, and as noted above, there can be problems with both pre-analytic and analytic variables. A Cq value by itself does not have great significance, but it can be used in conjunction with other factors such as exposure history, vaccination status, and others.
What can Cq values reveal?
Cq values of less than 30 are strong and indicative that there is abundant target nucleic acid in the sample. Values of up to 37 indicate moderate amounts of target nucleic acid. Anything higher is classified as weak and shows minimal amounts of target nucleic acid.
Do you have the right master mix?
Getting a high-quality master mix is key to obtaining accurate Cq values. IDT has a collection of premium master mixes for qPCR that offer advanced capabilities including the ability to amplify from crude or purified samples.
