Development of Crude Leaf Extract-Based Duplex RT-RPA Assay for Detection of Sugarcane Streak Mosaic Virus and Sugarcane Yellow Leaf Virus and its Application in Planting Material Certification
摘要
Sugarcane streak mosaic virus (SCSMV) and sugarcane yellow leaf virus (ScYLV) are the two major viruses associated with sugarcane causing severe yield loss. Limiting the spread of these viral pathogens is necessary to improve the quality of planting material. In this study, duplex reverse transcription-recombinase polymerase amplification (RT-RPA) assay was developed, which could detect SCSMV and ScYLV simultaneously in 30 min at 40 ºC with limited laboratory resources. The technique employed a simple crude leaf extract-based template preparation in 50 mM NaOH and 2.5 mM EDTA (pH 8.0) in (1:1) ratio that eliminated the need of complex nucleic acid extraction procedures. No cross reactivity was observed in specificity test with another virus infecting sugarcane, sugarcane mosaic virus (SCMV), demonstrating that the assay is highly specific for the simultaneous detection of SCSMV and ScYLV. Moreover, the assay could detect both the viruses simultaneously up to the dilution level of 10–5 in sensitivity evaluation test. All symptomatic samples were detected positive for SCSMV and ScYLV in the validation experiments using duplex RT-RPA and conventional duplex RT-PCR assays. The developed duplex RT-RPA assay would be highly valuable for mass indexing of viruses associated with sugarcane in certification programs and effective disease management to prevent the further spread of these viruses across the country.