The advantages of real-time TaqMan PCR over conventional quantitative PCR
The titration assay based on competitive PCR was first described in 1990 [25]. Though this method is very accurate for DNA measurements, there are several pitfalls that should be considered when using this technique for the quantification of low abundance mRNA [14]. Differences in reverse transcriptase (RT) efficiency can vary up to 50% and are overcome by the addition of an homologous internal control RNA to the RT reaction. Any modification of this technique involves two rounds of PCR, which include a titration and a quantification assay [45,68]. By using the results obtained from the initial titration assay, a known quantity of RNA sample is mixed with the internal control so that the quantities of both molecules are equal. This mixture is then reverse transcribed and a PCR is performed on samples that have been diluted serially. The PCR products are separated by gel-electrophoresis and the band intensities are quantified by video imaging and densitometry. This assay is accurate and sensitive, but involves the definition of very stringent limits [61].

In contrast, the kinetic ELISA-PCR is based on the measurement of the amount of amplicons produced by PCR during each successive cycle. It is a technique that has been applied in many fields [3,63,65] and even analysis at the single cell level has been carried out [31]. It is based on a liquid-phase hybridization step after PCR amplification for the detection and quantification of the PCR products, and therefore internal standards can be used. Biotinylated primers, incorporated in the PCR products, are caught by avidin bound to the ELISA-plate. After binding of amplification products, quantification is achieved by using digoxigenin-labelled internal probes. A colour reaction is induced by adding an anti-digoxigenin alkaline phosphatase-coupled antibody (anti-DIG-AP) and paranitrophenyl (PNP) as the substrate. The optical density (OD) can be measured in any ELISA reader, at a wavelength of 405 nm. This method allows direct quantification of PCR products and provides the results in a digital format.

Kinetic real-time TaqMan PCR, implemented in the ABI Prism 7700, is the method of choice for quantitative PCR because it uses internal probes for the quantification of PCR products; the hybridisation step is carried out during amplification and does not require post-amplification handling and thus reduces the overall manual handling and the risk of carry-over. Real-time TaqMan PCR, on the other hand, has the wide dynamic range and robustness of kinetic PCR and the advantages of the liquid hybridization assay, but lacks the time consuming post-amplification steps involved in kinetic ELISA PCR. The TaqMan system was thought do be less sensitive [15], but in our hands, and in many others, it will repeatedly achieve an absolute sensitivity of 5-10 molecules [26,28,40,67].

The greatest problem facing the diagnostic application of PCR is the production of false-positive results. They are attributable to contamination by nucleic acids, particularly from previously amplified material (carry-over). Any contaminant, even the smallest airborne remnant carried over from the previous PCR procedure or from a strongly positive sample (contamination), may be multiplied and produce a false-positive result. In the TaqMan system, the problem of carry-over is significantly reduced because of the real-time measuring principle, which is based on a closed-tube detection system. The probability of carry-over can be decreased further, or even eliminated, by inclusion of the AmpErase UNG system [47].

Taken together, the quantitative real-time TaqMan PCR technique has several advantages over the classical quantitative PCR system. The use of fluorescent dye-labelled probes increases the sensitivity of the system by at least 7 orders of magnitude and gives rise to a linear relationship between copy number and CT values. In addition, the liquid hybridization assay adds further specificity to the system, comparable to hybridization techniques using blotted PCR products. The elimination of post-amplification steps increases reliability and reproducibility of the assay [26,32]. A major factor responsible for the accuracy of the kinetic PCR method is the determination of the CT value within the logarithmic phase of the amplification reaction, instead of the endpoint determination used by conventional systems. The Sequence Detection Software (SDS) calculates the CT value when amplification of PCR products is first detected, in other words at the beginning of the exponential phase of amplification, when accumulation of inhibitory PCR products is unlikely to occur. This system offers great potential for automation. Standardisation is achieved by using specific software for primer-probe design. The default settings of the Primer Express Software (Applied Biosystems, Foster City, CA), designs oligonucleotide triplets (two primers and a matching TaqMan probe) that can all be amplified with the same protocol and universal mastermix.

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