Quantitative Pcr

In the past  two decades, pressure to provide more rapid and  selective virus identification (diagnosis) and quantification has led to the development of molecular assays that do not rely on biological or replicative properties of viruses but  rather on the  simple detection of their genomic sequences. The polymerase chain reaction (PCR) is a method that uses a thermostable DNA polymerase enzyme to copy a defined nucleic acid target sequence (selected by the user) in a step-wise fashion. PCR is a cyclical procedure, and for each cycle the number of copies of the target sequence theoretically doubles. Quantitative PCR (qPCR, sometimes called  real-time PCR) has been used to quantify virus by deter- mining the number of copies of a given virus sequence (the target) that are present after a known number of cycles, and  subsequently estimating the number of target sequences (i.e. virus genomes) that were present in the original test sample. The assay requires accurate  estimation of the  number of copies of the  target sequence and  this  is usually done by comparison with  a series  of diluted control sample targets where the  actual numbers  of molecules have  been calculated. Detection of target sequences requires either a fluorescent intercalating (dsDNA-binding) dye or probe. Both systems require expensive reagents, detection equipment, and interpretation software and provide a particle (strictly speaking, genome) count, not an infectivity measurement. However with newly prepared virus the technique has been shown to be reliable and accurate (compared with infectivity assays), and is much more rapid than the plaque or TCID50 assays. The development, methodology, and applications of PCR are thoroughly reviewed in the Instant Notes Molecular Biology volume.