Application of Molecular Diagnostic Techniques for Viral Testing

Cobo, Fernando

In this method, the target amplification and detection steps occur simultaneously. These methods require special thermal cyclers that can monitor the fluorescence emission from the sample. The computer software supporting the thermal cycler monitors the data at every cycle and generates an amplification plot for each reaction [19] . The PCR product is detected by using fluorescent dyes that preferentially bind to double-stranded DNA. The specificity of real-time PCR can also be increased by using fluorescent resonance energy transfer (FRET) probes in the reaction mixture. Another approach to real-time PCR is the use of dual hybridization probes that uses two specially designed sequence-specific oligonucleotide probes. Finally, detection and quantitation of amplification products can be carried out with molecular beacons. Real-time PCR decrease the time required to perform nucleic acid assays because there are no post-PCR processing steps. The main advantages of these methods are also the decrease of contamination and the possibility for quantitative applications.