S-49. Rapid and Real-time Detection of Chikungunya virus by Reverse Transcription Loop mediated isothermal amplification Assay

A Chikungunya virus (CHIKV) outbreak of unprecedented magnitude was recorded in India during 2006–2007 after a period of 32 years causing 1.3 million cases. In spite of its huge economical and public health importance, molecular assays for rapid and real time detection are not available. In the present study, we report a novel isothermal gene amplification method known as RT-LAMP for early, rapid and real-time detection as well as quantification of CHIKV genomic RNA in acute phase patient serum samples through by targeting the E1 gene. Optimal assay conditions with zero background were established for detection of low levels of CHIKV in acute phase patient serum samples. The feasibility of CHIKV RT-LAMP for clinical diagnosis was validated with patient sera samples of ongoing epidemic in Southern India. The comparative evaluation of RT-LAMP assay with acute phase patient serum samples demonstrated exceptionally higher sensitivity by correctly picking up 21 additional positive borderline cases that were missed by conventional RT-PCR (P< 0.0001) with a detection limit of 20 copies. The quantification of virus load in patient sera samples was also determined from the standard curve based on their time of positivity and was found to be in the range of 2x 10 8 – 20 copy numbers. In addition, field applicability of RT- LAMP assay was also demonstrated by standardizing SYBR Green I based RT-LAMP wherein the amplification was carried out in a water bath at 63ºC for 60 min followed by monitoring of gene amplification with naked eye through colour change. These findings demonstrated that RT-LAMP assay is a valuable tool for rapid, real-time detection as well as quantification of CHIKV in acute phase serum samples without requiring any sophisticated equipments and has potential usefulness for clinical diagnosis and surveillance of CHIKV in developing countries.