A stem–loop-mediated reverse transcription real-time PCR for the selective detection and quantification of the replicative strand of an RNA virus

A stem–loop-based method to quantify the replicative strand of a model system, dengue virus, with high specificity and sensitivity is described. The high specificity of this approach is achieved at two levels: the use of a reverse transcription primer folded into a stem–loop structure with optimal energetics and the use of specific PCR primers to the loop structure. This approach has exceptional specificity to the replicative RNA as compared with the genomic sequence (>105-fold difference), with a detection sensitivity of 10 copies. The high correlation to the biological “gold standard” plaque assay, used to quantify infectious virus, renders this method a useful quantitative tool that can replace the time-consuming, labor-intensive, and low-throughput plaque-based assays. The method has been extended to the detection of replicative strands of other RNA viruses (West Nile virus and human respiratory syncytial virus) with similar results. This real-time PCR method is reliable, simple to perform, and easily adaptable to different targets. The ability to detect and rapidly quantify replicating viruses is an important step in the elucidation of pathogenesis and is also useful for the evaluation of drugs designed to inhibit viral replication.