Real-time Thermal Cycler (qPCR)

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Thermal cycler for real-time PCR / qPCR

Real-time PCR or quantitative real-time PCR (qPCR) is a further development of classical PCR. Thanks to this method the amplification cycles of the target DNA can be monitored and analyzed in real time. Thermal cyclers for real-time PCR / qPCR are now standard instruments in almost all molecular biology laboratories around the world. The qPCR thermal cyclers of the qTOWER³ series from Analytik Jena are among the most powerful and reliable instruments on the market. 

Special features of real-time PCR / qPCR

The method is used to qualitatively detect specific nucleic acids in a sample. The basic methodology of PCR is also used in real-time PCR / qPCR. If a nucleic acid is also detected quantitatively, i.e. its concentration is determined, this is also referred to as quantitative real-time PCR or qPCR. Nucleic acids such as DNA or RNA transcripts are amplified in three essential steps – denaturation, primer annealing and elongation – which are repeated several times. However, compared to standard PCR, real-time PCR / qPCR makes it possible to monitor and quantify the amplification process in real time. 

For this purpose, special dsDNA fluorescent markers that bind to the nucleic acids – for example SYBR Green I – are used. Fluorescence can be utilized to quantify the amplified nucleic acids after each cycle. The greater the amount of DNA replicated, the higher the fluorescence. The fluorescence signal increases in proportion to the amount of DNA. 

Another method for quantification and real-time analysis is real-time PCR / qPCR with fluorescence probes. Here, a distinction can be made between hydrolysis and hybridization probes. In hydrolysis probes, also called TaqMan probes, a reporter dye is excited with a light source. The resulting light is first quenched by another dye and only when the DNA is successfully amplified (when the probe is hydrolyzed) is it emitted and can be measured. Hybridization probes, known as FRET probes. These use the physical principle of Förster resonance energy transfer: here, the energy of a dye (donor) excited by a light source is transferred to a second dye (acceptor). If the distance between donor and acceptor increases, the fluorescence signal of the acceptor decreases. The corresponding signal of the donor increases. Different types of FRET probes exist, including TaqMan probes, LoopTaq probes, or hybridization probes. Both TaqMan and FRET probes are preferred in diagnostics due to their high specificity.  

Finally, to be able to make statements on quantification, the Ct value (cycle threshold) is used. This indicates how many cycles of real-time PCR / qPCR are necessary to exceed a predefined limit value of the measurement signal. If the sample already contains a larger amount of target DNA before amplification, fewer cycles are necessary than for smaller amounts. From the Ct value, conclusions can be drawn about the original amount of DNA to be amplified in the sample and thus, for example, statements can be made about a specific viral load. 

Design of thermal cyclers for real-time PCR / qPCR

The main component of qPCR thermal cyclers is also the heating block. The uniform heating and cooling of the samples is an essential step in the process of amplification. The basic principle is the Peltier effect, which causes the rapid change of heating and cooling by reversing the direction of current between two semiconductors. Especially in terms of material, the heating blocks of thermal cyclers for real-time PCR / qPCR sometimes differ significantly. Most devices use an aluminum block with a special alloy. Modern qPCR thermal cyclers, such as Analytik Jena's qTOWER³ series, use improved heating block technologies. The qTOWER³, for example, relies on silver blocks with a gold coating. Thanks to this innovation, much faster heating and cooling rates are possible. Gradient functions for particularly exact scaling of different temperatures inside the sample block are also found in many recent qPCR thermal cyclers.  

For real-time monitoring and quantification, real-time PCR or qPCR thermal cyclers also have optical components for fluorescence excitation and measurement. Usually, a light source excites the fluorescent dyes in the sample. Multiplex applications with multiple fluorescent dyes are also possible with newer devices. 

In recent years, two aspects of thermal cycler design have become increasingly important: Automation and software. Laboratories are faced with an ever-increasing number of samples, but rarely have more human or time resources available. The key lies in automating analytical processes in the laboratory. Robotized systems for pipetting, labeling and transport – to name just a few – can now be found in even the smallest laboratories. Thermal cycler designs must also take this development into account, i.e., be able to integrate easily into these automated setups and provide interfaces to other equipment necessary for the analysis process. Directly related to this is the second aspect. Due to the automation trend, the software of a qPCR thermal cycler today must have an open and flexible design in order to communicate with different laboratory applications and devices. In addition, the demands of laboratory personnel using the software have changed significantly. Today, graphical user interfaces, simple modeling of processes, and an intuitive user experience are required to make work more efficient and to quickly familiarize untrained or non-specialist personnel with the equipment.

Target industries and applications of qPCR thermal cyclers

Thermal cyclers for real-time PCR / qPCR have a wide range of applications. PCR or real-time PCR / qPCR is known to the general public primarily for diagnostic applications. However, the detection of pathogens in a wide variety of sample materials is only one of many areas in which PCR and qPCR are used. Target industries range from pharma and chemicals to the food industry and environmental analysis. Real-time PCR / qPCR thermal cyclers are also widely used in university and institutional research. 

Thermocyclers for real-time PCR / qPCR are also used, for example, to produce proteins or for research into the properties of certain proteins in the pharmaceutical industry. Numerous applications also exist in food and agriculture analysis. For example, real-time PCR / qPCR is used for species and origin analysis of certain foods or for the detection of genetically modified organisms (GMOs). Another field that has recently relied more and more on real-time PCR / qPCR is environmental analysis. In addition to the long-established detection of chemical elements or sum parameters, analyses of pathogens in water or soil are playing an increasingly important role. Wastewater-based epidemiology is particularly noteworthy in this context. Due to the Corona pandemic, this discipline has received a lot of attention in recent years. Targeted testing of wastewater for bacterial or viral pathogens makes it possible to anticipate widespread disease outbreaks, providing authorities and other public health institutions with much earlier indications of potentially critical situations. During the Corona pandemic, the efficiency of this early warning system was already demonstrated in test trials. Real-time PCR / qPCR plays an essential role in this. Wastewater samples from treatment plants or sewerage can be analyzed with it in a very short time. This is not only interesting for the early identification of coronavirus hotspots, but also for the large-scale monitoring of other biological parameters, E.coli bacteria for example. Analytik Jena is a pioneer in this field and supports numerous customers worldwide in establishing these early warning systems with know-how and technological solutions.