QPCR fact sheet
What is QPCR
Quantitative real-time PCR (QPCR) is a method of quantifying the amount of a specific DNA present in a sample. Samples can be blood, tissue or cells collected on a swab. The target DNA can be of viral, bacterial or host origin and present at high or low levels.
What are the advantages of QPCR over conventional PCR?
Diagnostic QPCR offers major advantages over older conventional PCR techniques and, as such, we no longer offer conventional PCR tests for diagnostic work. The advantages of QPCR are:
Agarose gel image showing that it is not possible to quantify the amount of pathogen using conventional PCR - all the bands look identical despite the samples having very different numbers of pathogens
- its ability to quantify the amount of pathogen present in the submitted sample, rather than just reporting its presence or absence
- the dramatic reduction in the potential for false positive results due to the "closed tube" system used in QPCR
- its far quicker turnaround time with higher sample throughput, reducing the time between sample receipt and result reporting
What is a threshold cycle value?
The threshold cycle (Ct) value denotes how many cycles of PCR are required for the amount of PCR product (measured by fluorescence) to reach a defined threshold value. The more target DNA present in a sample, the lower the Ct value will be, as the threshold is reached sooner. For more information see here.
Ct values aid the veterinary surgeon in interpreting results by providing information on the level of target DNA present. For example, sequential samples can be used to monitor response to antibiotic treatment and retrovirus loads can be measured. Where relevant, QPCR results will be reported by stating the Ct value for the sample.
What is an internal amplification control?
An internal amplification control (IAC) is a target DNA that is amplified simultaneously with the pathogen DNA. The IAC can be a host gene or an artificial DNA molecule added to each sample at the DNA extraction stage. The use of an internal amplification control ensures that any problems with sample collection, submission or laboratory processing are quickly identified and false negative results avoided.