Introduction
The polymerase chain reaction (PCR), first described in 1985, is a highly sensitive and specific technique used for the detection of nucleic acids [55]. The inventor of this technology earned a Nobel prize for his achievement [43,44], which has revolutionised research and diagnostic possibilities. Qualitative PCR is a well established and straightforward technology, but quantification of specific nucleic acids present in a sample is a demanding task. Accurate quantification is hampered by a number of variations that may occur during sample preparation, storage or the course of the reaction. Even minor variations in reaction conditions are greatly magnified by the exponential nature of the PCR amplification. These variations may partly be overcome by normalising the amount of PCR products of the specific template with respect to an internal reference template. Considering the hundreds of papers published on the use of quantitative PCR, it is not surprising that a great variety of protocols exist. These methods are almost exclusively restricted to use in research because of two factors they have in common: they are difficult to perform and are costly to run.

In the need for faster, more accurate and more economic systems with a high throughput capacity, three keywords have become important for the development of the next-generation of PCR systems: automation, standardisation and miniaturisation. The development process was accelerated by combining computer-assisted PCR with laser technology so that now the laser-guided detection of PCR products, with the help of a so-called TaqMan probe, and the real-time accumulation of fluorescent data points for every PCR cycle virtually replace the need for a time-consuming post-amplification step. In addition, using an internationally standardised 96-well microtitre plate format enables large numbers of samples to be screened within a few hours. The TaqMan principle is implemented in an Applied Biosystems (ABI) Prism Sequence Detection device (Applied Biosystems, Foster City, California, USA), which is one of the most sophisticated technologies currently available and offers a unique platform for further development.

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