This set of reagents for highly sensitive, specific detection of the nucleic acid of Influenza A is based on the Polymerase Chain Reaction in real time.
The reaction set up of our products is very convenient – simply combine the Master Mix (5 μl) with the Oligo Mix (5 μl) and add a clinical sample (10 μl).
Our products require a very short cycling profile, delivering results in less than 1 hour.
All our kits are designed with the same fluorophores – FAM, HEX, ROX and Cy5. They are therefore compatible with all the common PCR devices.
All our kits can be run using the same cycling conditions which allows combining different tests in one run.
To monitor all the steps of the procedure (sample collection, nucleic acid extraction, etc.), an endogenous internal control is detected, therefore no additional pipetting step for adding the internal control is required.
This test detects all the known strains of Influenza A.
PCR kits from Poly Diagnostics – offer the reverse transcription and the PCR reaction in one tube, that offers a greater speed from a sample to results (~1h).
The primers and probes are designed to detect very conservative genome regions of all the reported targets.
All of our kits use some of the most common fluorophores – FAM, HEX, ROX та Cy5 which are compatible with all the real-time PCR devices, for example:
These recommendations are supplementary to the Instruction for Use of the product.
To analyze the obtained PCR data a threshold line has to be set up. In the software of certain PCR devices this procedure is done automatically. Nevertheless, it is advisable to inspect the outcome of this procedure, as inappropriately set threshold may adversely influence interpretations of the data. In case the software set the threshold incorrectly, it has to be done manually. The threshold has to be set for each fluorophore separately. The threshold is set above the baseline fluorescence. The baseline fluorescence (“noise”) is observed in the early PCR cycles (usually between cycles 2 and 12) when low quenched reporter signal is detected. If the amplification target is detected, than enough of cleaved probes will be produced and the amplification signal will cross the baseline fluorescence level.
There are two types of PCR data representation – linear and logarithmic.
Setting up the threshold is not the exact science and especially when set manually different operators might do it differently. However, it is advisable to set the threshold such that it crosses the beginning of the exponential phase of the fluorescence curve when the data is depicted linearly (see Graph 1).
Graph 1. Setting the threshold and the baseline fluorescence.
The threshold line can also be set in the logarithmic data presentation. In this case a correctly set line has to cross the middle of the exponential phase of the fluorescence curve.
In all the kits produced by Poly Diagnostics the internal control is detected on the Cy5 channel. If amplification curves are detected in the negative sample in any other channel than Cy5 this might hint towards a contamination. In this case all the PCR results obtained in the run are considered not valid (Graph.2). The whole run has to be repeated. Please follow PCR laboratory practice guidelines to avoid contamination.
Graph.2 Presence of FAM fluorescent signal in the negative control.
In case there is no amplification curve in the Cy5 channel or the curve is considerably shifted to the right the results are considered equivocal (Graph 3). It is recommended to perform the test again starting from the extraction.
Graph.3 Amplification of the internal control
For a simultaneous detection of multiple pathogens several different fluorophores are used – FAM, HEX, ROX, Cy5. Each fluorophore is detected in a separate channel, therefore the threshold has to be set for each fluorophore separately. It is important to notice that every fluorophore will have its own intensity. When all fluorophores are depicted at the same time the software will try to scale the fluorescence using the channel with the greatest fluorescence. This, in turn, might lead to a false assumption that some curves look “weaker” than the others. However, when the results are interpreted only the Ct values are taken into account and not the relative fluorescence (Graph 4).
Graph 4. Relative intensities of different fluorophores. Note, that a sample might have different fluorophores intensities, but almost the same Ct values.
If no fluorescent signal is observed in all the channels we recommend to check all the hardware parts of your PCR device, as well as the software settings again and make sure that the reading takes place. In case the hardware and the software are in order we recommend to run PCR reactions again with a freshly extracted sample. It is very important to make sure that the sample and the reagents were stored and handled properly.