Quantitative Fluorescent

Quantitative fluorescent PCR (QF-PCR) is based on the principle that the amount of amplicon produced by a PCR in its exponential phase (before the yield has reached its plateau) is proportional to the starting amount of target (53). Thus, such PCRs are carried out with generally no more than 24 cycles. Fluorescent detection of such amplicons, via labeling of one of the primers, facilitates detection and quantitation of the PCR product using a fluorescent DNA analyzer, either gel or capillary based. No processing of the PCRs is necessary, other than the usual preparation of samples for running on such analyzers. Validation of such assays using samples tested by an independent method is necessary.

4.1. USES OF QF-PCR Quantitative fluorescent PCR is well suited to the rapid detection of aneuploidies in antenatal amniocentesis or chorion villus samples and is in wide use (54-57). Fluorescence in situ hybridization (FISH) is perhaps slightly less reliable than QF-PCR for the detection of aneu-ploidy in such situations, and QF-PCR also has the benefit of easy automation. However, although FISH reagents are considerably more expensive than those needed for QF-PCR, the capital cost of a fluorescent microscope is less than a fluorescent DNA analyzer, although neither can be described as inexpensive (58). QF-PCR has been applied to RhD testing, Duchenne muscular dystrophy testing, and analysis of TP53 deletions in Li-Fraumeni syndrome (59-61).

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