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

Analytical Biochemistry
Azlinda AnwarHeng Phon Too

Abstract

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 (>10(5)-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 ...Continue Reading

References

May 26, 1999·Proceedings of the National Academy of Sciences of the United States of America·G BonnetF R Kramer
Jun 6, 2000·Trends in Microbiology·H Bielefeldt-Ohmann
Jan 5, 2002·Clinical and Diagnostic Laboratory Immunology·Gilberto VaughanAlejandro Escobar-Gutiérrez
May 15, 2002·Trends in Biotechnology·Natalia E Broude
Jul 27, 2002·Annual Review of Microbiology·Margo A Brinton
Jun 26, 2003·Nucleic Acids Research·Michael Zuker
Jan 13, 2004·Journal of Virological Methods·Florence Komurian-PradelPatrice André
Apr 2, 2004·The Journal of Clinical Investigation·Alan L Rothman
Apr 16, 2004·Clinical Microbiology Reviews·Andrew J EastonHelene F Rosenberg
Jun 3, 2004·Human Immunology·Giuseppe GernaM Grazia Revello
Aug 28, 2004·Journal of Virology·Fabienne BrilotCheryl A Stoddart
Oct 21, 2004·Journal of Clinical Virology : the Official Publication of the Pan American Society for Clinical Virology·Guy Vernet
May 5, 2005·Applied and Environmental Microbiology·Jian-Wen He, Sunny Jiang
Jun 17, 2005·The Journal of General Virology·Heli HarvalaTimo Hyypiä
Oct 6, 2005·Nature Reviews. Microbiology·J Lindsay WhittonRalph Feuer
Oct 15, 2005·Development·Tingting Du, Phillip D Zamore

❮ Previous
Next ❯

Citations

Oct 23, 2009·PloS One·Nicole E PlaskonKevin M Myles
Mar 24, 2009·Transfusion Medicine Reviews·Wandong ZhangHoward Njoo
Oct 13, 2006·Biomaterials·David Tai LeongDietmar Werner Hutmacher
Oct 10, 2006·Pediatric Clinics of North America·Jiang FanKelly J Henrickson

❮ Previous
Next ❯

Related Concepts

Related Feeds

Antivirals (ASM)

Antivirals are medications that are used specifically for treating viral infections. Discover the latest research on antivirals here.

Antivirals

Antivirals are medications that are used specifically for treating viral infections. Discover the latest research on antivirals here.