Development of a Reverse Transcription-Recombinase Polymerase Amplification Assay for Detection of Sugarcane Yellow Leaf Virus
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Sugarcane yellow leaf virus (SCYLV, genus Polerovirus) is a harmful agent that causes sugarcane yellow leaf disease. Current methods for SCYLV detection present some limitations. In this study, a novel isothermal amplification assay, namely reverse transcription-recombinase polymerase amplification (RT-RPA), was developed to promote SCYLV detection for disease management. The assay was evaluated in terms of specificity, sensitivity, reliability, temperature limit, and time limit. The developed RT-RPA assay was highly specific and non-cross-reactive with Potato leafroll virus, which is a type of species in Polerovirus. This assay can also detect at least 103-fold diluted cDNA from a SCYLV-infected sugarcane leaf, and its sensitivity is tenfold lower than that of RT-polymerase chain reaction (PCR). The reliability of the proposed assay was examined by detecting field sugarcane samples via RT-RPA and RT-PCR assays. The RT-RPA assay showed the same results as those of RT-PCR assay, indicating that the former was highly reliable for SCYLV detection. Analysis of the temperature and time limits revealed a wide operating temperature range from 27 to 45 °C, which was easily reached, and a rapid assay duration of 20 min. In summary, the developed RT-RPA assay was rapid, specific, and reliable with acceptable sensitivity and easily reachable operating temperature and thus could be a promising tool for SCYLV diagnosis in sugarcane.
KeywordsRecombinase polymerase amplification Sugarcane Sugarcane yellow leaf virus Yellow leaf disease
Loop-mediated isothermal amplification
Nucleic acid sequence-based amplification
Polymerase chain reaction
Potato leafroll virus
Recombinase polymerase amplification
Sugarcane yellow leaf virus
This study was funded by the National Natural Science Foundation of China (31771865) and Sugar Crop Research System (CARS-170301). The authors would like to thank Professor Cheng-Gui Han from China Agricultural University for generously donating PLRV-infected N. benthamiana leaves for the cross-reactivity study.
Shu-Zhen Zhang devised the experiments. Xiao-Yan Feng operated the experiments, analyzed the results, and wrote the manuscript. Lin-Bo Shen collected field sugarcane samples. Wen-Zhi Wang, Jun-Gang Wang, Zheng-Ying Cao, Cui-Lian Feng, and Ting-Ting Zhao revised the manuscript. All authors have read and approved the manuscript.
Compliance with Ethical Standards
Conflict of interest
The authors declare that they have no conflict of interest.
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