Magnetic Multiplex Loop Mediated Isothermal Amplification (MM-LAMP) technique for simultaneous detection of dengue and chikungunya virus.

ABSTRACT

Dengue and chikungunya viruses are arthropod borne virus spread through common vector instigating infection in human. There has been an increased recognition that more attention needs to be paid to similar sympotoms caused by both of the virus as they spread in the same region at same time. It warrants need of cost effective, user friendly and rapid multiplex diagnostic technique which could simultaneously diagnose and identify between two virus diseases in resource poor setting. A magnetic multiplex loop mediated isothermal amplification (MM-LAMP) technique was developed by coupling multiplex LAMP with magnetic particle-based naked eye visualization to overcome the shortcoming of simultaneous detection of both diseases. In recent years this technology has emerged as a particularly attractive candidate as amplification reaction process completes within 45 min. The first step involves multiplexing biotin and digoxigenin coated dengue and chikungunya primers respectively in LAMP reaction followed by precipitation of the amplified DNA with polyethylene glycol (PEG) buffer and finally clumping with streptavidin and anti-digoxigenin coated magnetic particle for virus discrimination and naked eye visualization. The DNA detection limit of MM LAMP visualization was 51.65 ng/µl which is comparable to the electrophoresis base UV light visualization. The results showed potential superiority over standard methods polymerase chain reaction (PCR). This current advancement empowers multiplex LAMP utility in resource limited setting without using any of the florescent dyes, turbidimeter, or the sophisticated quantitative PCR machine etc which restrict multiplex LAMP technique to laboratorial use only. We have proposed a novel method without such limitations. This technique has potential as a point of care technique for simultaneous detection of two diseases.