Development of a reverse transcription loop-mediated isothermal amplification assay for rapid and visual detection of Sugarcane streak mosaic virus in sugarcane

Abstract

Sugarcane mosaic disease (SMD) is one of the most destructive sugarcane diseases that occur around the world. Sugarcane streak mosaic virus (SCSMV) belongs to the genus Poacevirus of the family Potyviridae, and is a novel pathogen that causes SMD. The pathogen spreads primarily through infected sugarcane setts, and hence the use of disease-free planting materials is essential to prevent disease development in the field. In the present study, a species-specific reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the rapid and accurate detection of SCSMV. Based on the conserved polyprotein gene nucleotide sequences of SCSMV isolates that differ from Sugarcane mosaic virus (SCMV) and Sorghum mosaic virus (SrMV), four SCSMV specific primers, namely, P2-F3, P2-B3, P2-FIP, and P2-BIP, were designed and screened, using a panel of sugarcane viruses as controls. After optimization of the reaction conditions, the detection sensitivity of the RT-LAMP assay was 10-fold more sensitive than the conventional RT-PCR. The assay showed a high specificity in discriminating SCSMV from common viral pathogens of sugarcane such as SCMV, SrMV, Sugarcane yellow leaf virus (SCYLV), and sugarcane bacilliform virus (SCBV). The RT-LAMP assay also detected SCSMV in young sugarcane leaves with no visible sugarcane mosaic disease symptoms. The findings of this study provide a visual, simple, highly sensitive, rapid, and reliable technique for the detection of SCSMV, a tool that is useful for sugarcane quarantine and production of SCSMV-free seedcane. This is the first report of a RT-LAMP-based assay for the detection of SCSMV in sugarcane.