With RNA-based detection, the RNA is first converted into complementary DNA (cDNA) by reverse transcription. With DNA-based detection, this step is unnecessary. Every EURORealTime test kit contains all PCR reagents ready for use, including reverse transcriptase and/or DNA polymerase and the validated specific primers and probes. In this way, the number of pipetting steps is reduced: the ready-for-use PCR reagents are pipetted together followed by the addition of DNA/RNA.
The polymerase chain reaction (PCR) amplifies the relevant characteristic DNA/cDNA sequences a million-fold. Here, two starter DNA molecules (primers) define the region that requires amplification. If the patient sample contains the corresponding DNA sequence, the primers can bind to it. This helps produce a copy of the target sequence. This reaction undergoes many repetitions producing millions of copies of the DNA region between the primers.
During each PCR cycle, specific fluorescent-dye labeled DNA probes bind to the target sequence. These only cause a fluorescence signal when the DNA undergoes amplification. At the end of a PCR cycle, you can measure the fluorescence intensity to follow the DNA amplification in real-time.
If the target sequences are not present in the patient sample, the primers and probes cannot bind to them. Thus, the DNA is not amplified and the fluorescence signal does not increase. By including the corresponding quantification standards, this method also allows the quantification of the DNA in the original sample. Moreover, it allows you to detect different DNA sequences in one reaction. This involves the use of several fluorescence dyes with different excitation and emission wavelengths.