Division of Virology, Defence R & D Establishment (DRDE), Jhansi Road, Gwalior-474002.
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.
Dengue virus is the most widely prevalent a mosquito-borne flavivirus in tropical and sub tropical regions of Asia, Africa and Central and South America. The virus isolation and antibody detection have less impact on patient management and control measures exercised by medical and public health personnel. Therefore, there is a great demand for the rapid detection of dengue virus infection in the acute phase of illness in order to provide timely clinical treatment and etiologic investigation and disease control. In the present study, we report the development and validation of a one-step, real-time and quantitative dengue virus serotype specific RT-LAMP assay targeting the 3’- non-coding region for rapid detection and differentiation of dengue virus serotypes. The RT-LAMP assay is very simple and rapid wherein the amplification can be obtained in 30 min under isothermal condition at 63°C by employing a set of four serotype specific primer mix through real-time monitoring in an inexpensive turbidimeter. The evaluation of RT-LAMP assay for clinical diagnosis with limited number of confirmed patient serum samples of each serotype revealed 100% concordance with RT-PCR. Moreover, the RT-LAMP assay revealed higher sensitivity by picking up 37% samples as positive as compared to 33% and 30% by RT-PCR and virus isolation respectively. The sensitivity and specificity of RT-LAMP assay for detecting viral RNA in patient serum samples with reference to virus isolation was 100% and 91% respectively. Optimal assay conditions with zero background having no cross-reaction with other closely related members of Flavivirus group as well as within the four serotypes of dengue virus was established. None of the healthy serum samples screened in this study showed any cross-reaction with any of the four dengue virus serotype specific RT-LAMP primers. These findings demonstrated that RT-LAMP assay has the potential clinical application for detection and differentiation of dengue virus serotypes especially in developing countries.
