FluoroType®-Technology - Innovative Technology with highest Sensitivity and maximum Specificity

Our fluorescence-based PCR test systems guarantee rapid and valid results for efficient diagnostics in your laboratory. Nucleic acids are isolated from sample material in a first step, followed by amplification and detection using fluorescence-labeled probes. Amplification and detection are carried out with the FluoroCycler®The FluoroType® technology is based on different methods:

PCR with hybridization probes

Detection via fluorescence-labeled probes takes place directly after amplification of the target sequences. 
The probes are marked with fluorophores and are complementary to the amplified nucleic acids.

The fluorophore cannot be excited to emit fluorescence if the probes are unbound. 

 
After hybridization with the amplicon’s target sequence, excitation of the fluorescence dye is possible and therefore an increase in fluorescence can be detected. 

During the subsequent melting curve analysis, the probes dissociate from the amplicon at their specific temperature, leading to an immediate decrease in fluorescence.  

PCR with hydrolysis probes

In this variant of real-time PCR, the detection of the amplicon is performed using hydrolysis probes.        The probes are labeled with possess a fluorophore and a quencher.  In unbound state, no fluorescence emission occurs, since both molecules - fluorophore and quencher - are in close proximity. 

During amplification, the probes hybridize with the target sequences and are hydrolytically degraded by the DNA polymerase during the elongation phase of the PCR.

Fluorophore and quencher of the degraded probes are released into the medium and therefore are no longer in close proximity – the fluorescence signal can now be detected.

The increase in fluorescence intensity can thereby be tracked in real-time in parallel to the increase of free fluorophore. Moreover, this innovative technology offers the possibility for quantification of the target sequences. 

 Asymmetric excess PCR with Lights-On/-Off-Probes

Asymmetric excess PCR is an optimized asymmetrical PCR, where one of the primers is present in excess.   The second primer is only present in a limited concentration. Because of this limitation, a linear increase of single-stranded nucleic acid occurs after a short exponential phase (with both primers in action) – thus creating excess single-stranded amplicons. Afterwards the detection is carried out using Lights-On/-Off probes. 

These probes contain either only a quencher (Lights-Off probe) or both fluorophore and quencher (Lights-On probe). The quencher of the Lights-Off probe eliminates the fluorescence emitted by the Lights-On probe, if both probes are hybridized in close proximity on the amplicon. 


During a melting curve analysis, the Lights-On/-Off probes detach from the amplicon at a temperature specific for the respective probe. In the unbound state, the fluorophore of the Lights-On probes cannot be excited to emit fluorescence. In the unbound state, the fluorophore of the Lights-On probes cannot be excited to emit fluorescence.
Therefore, fluorescence is either emitted or suppressed, which is reflected through a characteristic fluorescence pattern in the melting curve analysis. 

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