Real-time polymerase chain reaction (PCR) is the ability to monitor the progress of the PCR as it occurs (i.e. in real time). Data are therefore collected throughout the PCR process, rather than at the end of the PCR. This completely revolutionises the way one approaches PCR-based quantitation of DNA and RNA. In real-time PCR (qPCR), reactions are characterised by the point in time during cycling when amplification of a target is first detected rather than the amount of target accumulated after a fixed number of cycles. The higher the starting copy number of the nucleic acid target, the sooner a significant increase in fluorescence is observed.

qPCR includes heating and cooling cycles to make copies of the target DNA. The copies made are detected using fluorescent probes, making it possible to estimate the quantity of nucleic acid in the sample.

Primers designed specifically for the region of interest, a fluorescent probe, a polymerase enzyme and nucleotides to build new DNA chains are required. If amplifying RNA, it is necessary to use the reverse transcriptase enzyme, which converts the RNA into DNA.

Primers are small DNA sequences designed to hybridise to specific regions of the DNA for amplification. Probes are fluorophore-labelled molecules that are used to detect amplification in real time.

The primers (forward and reverse) and probes are designed to complement specific DNA sequences for amplification. Both primers should have a similar melting temperature (Tm) and not generate secondary structures.

VIASURE PCR kits use TaqMan probes. Taqman probes are hydrolysis probes that consist of a fluorophore covalently attached to the 5’-end and a fluorescence quencher at the 3’-end. They are based on the 5’-3’ exonuclease activity of Taq polymerase. As Taq synthesises the new DNA chain in a 5’-3’ direction, it degrades the now hybridised probe, causing the release of fluorophore from the probe, its physical separation from the quencher and resulting in the emission of fluorescence.

Tm is crucial because it affects specificity of the bond between primers and DNA. An adequate Tm ensures specific hybridisation during amplification.

The qPCR assay includes negative controls (no DNA), positive controls and internal controls to verify the quality of the reaction. An extraction control may be used instead of the internal control.

Negative controls guarantee there is no contamination and positive controls verifies that the reaction is working.

It is fundamental to use controls as they make it possible to ensure that the results obtained are valid. VIASURE PCR kits include negative and positive controls that are added as an additional sample to guarantee there is no contamination and to verify that all the reagents of the reaction are working. Each test also includes an internal control (IC). This is a parallel reaction performed in the same reaction well. It is used to verify the assay in each of the samples tested, such as to verify that there are no PCR inhibitors in the eluate. This IC may be replaced by an extraction control (EC).

Real-time PCR results are represented in amplification curves, showing the exponential growth of the level of fluorescence, which is proportional to the number of gene copies obtained in an amplification, plotted on the vertical axis, and the number of reaction cycles required (Ct), plotted on the horizontal axis.

The Ct, or cycle threshold, value is the cycle in which the curve, in its exponential phase, crosses the threshold perpendicularly for each reaction, and amplification is therefore considered to be present. Ct is a semi-quantitative value inversely related to the quantity of nucleic acid in the sample. Therefore, a low Ct value indicates a high pathogen load and vice versa.

Ct (cycle threshold): the fractional cycle number at which the fluorescence passes the fixed threshold NTC (no-template control): a sample that does not contain template. It is used to verify amplification quality.

The threshold is the imaginary line from which a qPCR plot begins to reflect an exponential curve. This exponential shape reflects that the efficiency of the reaction is constant cycle to cycle.

It therefore helps us to distinguish a negative result from a positive result. All curves above this line will be positive, while those below this line will be negative.

The threshold will always depend on our thermal cycler but it is recommendable to observe the value determined automatically by the software.

Answer: Baseline fluorescence is the fluorescence that is present in the initial cycles of the reaction. This fluorescence is not caused by an amplification reaction but by the chemistry of the reagents.

It must be eliminated so that the fluorescence obtained by all the samples in early cycles is zero and the curves can be analysed correctly. It is therefore determined from which value the fluorescence obtained for each channel is high enough to be considered a “real” amplification.

Normally, all software used to analyse results automatically sets the baseline for each curve individually. Nevertheless, this parameter can also be set manually for those curves where automatic analysis has not been satisfactory. It is recommended that it be set during early cycles of the curve, between cycle 3 and cycle 15, to eliminate baseline fluorescence present in initial cycles of the reaction.

Background is non-specific fluorescence in the reaction produced by the chemistry of the reagents. PCR equipment tends to eliminate this signal automatically to show clear results.

Passive reference is a dye that provides an internal reference to which the kit’s fluorophore signal can be normalised during data analysis. Normalisation is necessary to correct for fluorescence fluctuations caused by changes in concentration or volume. Viasure does not use a passive reference.

qPCR results can be quantified by calculating a standard curve showing the quantity of DNA and the Ct value by using samples of known concentration.

Efficiency is calculated from the slope of the amplification curves and is used to determine how many DNA copies are duplicated in each cycle.

DNA/RNA quality and quantity affect the results. Adequate extraction protocols should be followed to ensure high-quality starting material.

qPCR is highly sensitive, quick and quantitative. Its ability to detect subtle changes in gene expression also make it essential for biological research.

Multiplex qPCR is an assay in which several DNA sequences are amplified simultaneously, allowing the detection and differentiation of various targets at the same time. Multiple primers and probes labelled with different fluorophores are used so that their fluorescence can be emitted in the different reading channels. In the case of VIASURE kits, the channels used are FAM, HEX, ROX and Cy5.