The application of real-time PCR to food and agricultural systems. A review

Real-time PCR (RT-PCR) allows each cycle of DNA amplification to be observed on a computer screen throughout the sequence of thermal cycling, hence the designation "real-time." RT-PCR assays have been developed for a variety of target sequences of food borne microbial pathogens, plant pathogens, and genetically modified foods. A major and highly sensitive aspect of RT-PCR is the use of fluorescent reporter dyes that undergo enhanced fluorescence when bound to DNA. Among the fluorescent reporter systems employed are SYBR green, and the molecular probes designated TaqMan, FRET, molecular beacons, and variations of these systems. A major advantage of RT-PCR involves the amplification of short DNA target sequences of 60-70-bp, which allows greatly reduced extension times, which when coupled with the advanced technology of RT thermal cyclers with reduced ramp times, greatly reduces cycle times so that amplified targets can be recognized within 30 min of amplification in some cases. Another major advantage is that agarose-gel electrophoresis is not required to visualize amplified target DNA which greatly reduces assay time.