Division of Virology, Defence R & D Establishment (DRDE), Jhansi Road, Gwalior-474002, Madhya Pradesh.
Abstracts of the papers presented at the 16th Annual Convention and International Symposium of Indian Virological Society on “Management of Vector-Borne Viruses” at International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru-502324, Hyderabad, India, February 7–10, 2006.
RT-LAMP is a novel method of gene amplification that amplifies the nucleic acid with high specificity, efficiency and rapidity under isothermal condition employing a set of six specially designed primers spanning eight distinct sequences of target. The whole procedure is very simple and rapid wherein the amplification can be obtained in less than one hour (30 min) by incubating all the reagents in a single tube with reverse transcriptase and Bst DNA polymerase at 63°C.The detection of gene amplification could be accomplished by agarose gel electrophoresis as well as by real-time monitoring in an inexpensive turbidimeter. In addition, the gene amplification can also be visualized either as turbidity in the form of white precipitate or by employing a fluorescent intercalating dye (SYBR Green I) through UV lamp. Being an isothermal amplification, RT-LAMP does not require any thermal cycler and thus can be performed even with heating block and/or water bath. A one-step single tube real–time accelerated reverse transcription loop mediated isothermal amplification (RT-LAMP) assays for rapid detection of some of the recently emerged human viral pathogens viz; West Nile, SARS and Dengue viruses have been developed and evaluated. On comparison to conventional RT-PCR, RT-LAMP assay demonstrated 10 to 100 fold more sensitivity with a detection limit of 0.01 to 10 PFU of virus in all these cases. Thus the RT-LAMP assay reported here has the advantages of rapid amplification, simple operation and easy detection with potential usefulness for clinical diagnosis and surveillance of viral diseases in developing countries, as it does not require sophisticated equipment or skilled personnel.
