Reverse transcriptase loop-mediated isothermal amplification and reverse transcriptase recombinase amplification assays for rapid and sensitive detection of cardamom vein clearing virus

Abstract

In the present study two isothermal molecular assays viz. reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) and reverse transcriptase recombinase amplification (RT-RPA) were developed to detect the cardamom vein clearing virus (CdVCV) infecting cardamom. Assays were optimized for parameters like duration, temperature and concentration of magnesium sulfate, and betaine in the case of RT-LAMP and magnesium acetate in the case of RT-RPA. Detection limits of both assays were determined and compared with conventional RT-PCR and SYBR Green-based real-time RT-PCR. RT-LAMP was found 10000 times additional sensitive than RT-PCR and one-tenth that of real-time RT-PCR. RT-RPA was found 1000 times additional sensitive than RT-PCR and one-hundredth that of real-time RT-PCR. Both assays were specific, rapid, and sensitive for detecting CdVCV. Compared to real-time RT-PCR, these assays are economical and can be employed in large scale screening of cardamom plants against CdVCV for the selection of virus-free plants.