Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Assay for Rapid Detection of Cotton Leafroll Dwarf Virus (CLRDV) in Cotton

Cotton leafroll dwarf disease caused by Cotton leafroll dwarf virus (CLRDV) is an emerging viral disease in cotton in the U.S. Infection with the virus shows varying degrees of symptoms causing it difficulty in proper early diagnosis. Conventional reverse transcription-polymerase chain reaction (RT-PCR) is the most common diagnostic method for CLRDV from symptomatic tissues, which is relatively complex and time consuming. To overcome these shortcomings, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of CLRDV and its specificity and sensitivity were compared with conventional RT-PCR assay. For this assay, four sets of six primers to amplify the partial P1 (ORF1) and P0 (ORF0) region of the viral genome were designed. The primers were used for amplification at isothermal temperature with total RNA extracted from CLRDV infected cotton leaf tissues, and the optimum primer concentrations, reaction temperature, and assay time were determined. The amplified products were visualized by gel electrophoresis. Two primers sets designed for detecting CLRDV were successful in RT-LAMP assay at the concentrations of 0.4 μM internal primers, 0.1 μM external primers, and 0.2 μM loop primers at 65⁰C for 45 minutes. The RT-LAMP specificity was similar to RT-PCR; however, RT-LAMP was able to detect viral dilutions up to 1 x10^-6 which was 10³ times more sensitive than the RT-PCR assay. These results suggest that CLRDV RT-LAMP is a simple, rapid, and sensitive diagnostic tool suitable for easy detection of CLRDV.