EURORealTime: Direct detection of infectious agents using real-time PCR (IVD)

Amplification and detection of pathogenic DNA: real-time polymerase chain reaction

Real-time PCR amplification curves of quantification standards (10², 10³, 10⁴, 10⁵ copies) and one sample with an unkown number of copies of the pathogen genome (top figure). Standard curve, generated using the standards, for determination of the number of pathogens in the sample (bottom figure).
  • For tests based on the RNA sequence, the RNA is first transcribed into complementary DNA (cDNA) using reverse transcription. For tests based on the DNA sequence, this step is not required.
  • In the next step, the relevant characterised sections of the DNA/cDNA are amplified millionfold using polymerase chain reaction (PCR).
  • Two starter DNA molecules (primers) define the section to be copied. If the patient sample contains the respective DNA section (target sequence), the primers bind to it and a copy of the target sequence is made.
  • The reaction is repeated several times until the DNA region between the primers is amplified exponentially.
  • During each PCR cycle, specific fluorescence-labelled DNA probes bind to the target sequence, which only produce a fluorescence signal if the DNA has been amplified.
  • At the end of a PCR cycle the fluorescence intensity is measured to allow the DNA amplification to be tracked 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 there is no increase in the fluorescence signal (see top left figure).
  • By including the corresponding quantification standard the method allows quantification of the amount of DNA in the original sample (see top right figure).
  • Furthermore, the use of several fluorescence dyes with different excitation and emission wavelengths enables the detection of different DNA sequences in one reaction.

Advantages of the EURORealTime system

  • Specific and highly sensitive direct detection of pathogens even in early infection stages
  • Complete detection in one reaction vessel even of RNA viruses
  • Ready-to-use PCR components
  • Convenient guidance through the entire work process (EURORealTime Analysis software)
  • Simple quantification of the number of pathogens in the sample material possible*
  • Prevention of errors using automatically generated pipetting schemes (EURORealTime Analysis software)
  • Fully automated, standardised evaluation, generation of result reports and documentation (EURORealTime Analysis software)
  • Complete process validated and CE-labelled in accordance with the IVD directive
  • LIMS connection prepared

*Only for quantitative tests.