QPCR theshold cycle (Ct) values explained
The state-of-the-art PCR facilities in the Molecular Diagnostic Unit use a new advanced form of PCR technology called quantitative real-time PCR (QPCR), to detect target DNA. QPCR differs from conventional PCR in that it is quantitative; enabling the veterinary surgeon to know how much target DNA is present in a sample.
During QPCR, the amount of PCR product formed is measured each cycle and reported in fluorescence units. The more target DNA present in a sample, the more quickly the PCR product (and therefore fluorescence) is generated. A sample is positive if the amount of fluorescence produced rises above the threshold level. The threshold cycle (Ct) value denotes how many PCR cycles are required for the sample fluorescence to reach the threshold level. Thus, the more target DNA present in a sample, the lower the Ct value will be, as the threshold is reached sooner.
QPCR results are reported by stating the Ct value for the sample. This helps the veterinary surgeon interpret results by providing information on the amount of target DNA present. For example, retrovirus proviral loads can be measured and repeat samples used to monitor a cat’s progress; or response to haemoplasma treatment can be assessed by measuring haemoplasma loads after treatment.
An example
The figure below illustrates how Ct values are generated. The QPCR traces for two samples are shown. Sample A contains 250 times more target DNA than Sample B, so it reaches the threshold level at a lower cycle number (20) than Sample B (28). Samples containing no target DNA never reach the threshold level.
