Quantitative PCR and RT-PCR in virology.

I n the last few years, molecular hybridization methods have been used extensively in basic and applied virology because of their technical flexibility and high specificity. Using these techniques, the detection of DNA and RNA viruses directly from clinical specimens, the analysis of the specific transcriptional activity of viral genes in vitro and in vivo, and the study of virus-host relationships have all been carried out at the molecular level. However, although these methods are efficient for many purposes, only development (~) and optimization (z) of PCR amplification have dramatically improved sensitivity. Currently, PCR is the method of choice for the detection of viral nucleic acids present at very low amounts in biological samples, and it allows the molecular study of most acute and persistent viral infections. Impressive achievements that are rapidly emerging from the application of PCR to virology have also indicated that quantitative evaluation of the target sequence is necessary in several cases. In particular, methods for viral nucleic acid quantitation seem to be important, both in measuring the level of viral activity in most persistent human infections and in assessing the efficacy of specific compounds active at different steps of the viral replication cycle in vivo and in vitro. Accordingly, in the last few years, several strategies and protocols based on different methodological and technical approaches have been adopted for the semiquantitative or quantitative analysis of viral nucleic acids. This review will focus on the strategies proposed for viral nucleic acid quantitation using PCR and reverse transcription (RT)-PCR, and the applications of these methods in basic and diagnostic virology. THE IMPACT OF PCR AMPLIFICATION IN VIROLOGY