A rapid visual detection method for Sugarcane streak mosaic virus based on one-tube RPA-CRISPR/Cas12a

Sugarcane is the most important crop for sugar production. Sugarcane streak mosaic virus (SCSMV) triggered sugarcane mosaic disease can lead to substantial reductions in both yield and sucrose content. In the process of disease prevention and control, target pathogen detection technology is indispensable. However, traditional detection methods are time-consuming and require expensive equipment, making them less efficient for timely disease control and unfavorable to disease resistance breeding. Here, we introduce a novel detection technology that combines recombinase polymerase amplification (RPA) with CRISPR-Cas12a. The method utilizes crude extracts from sugarcane leaves as the reaction template, significantly simplifying and expediting the preparation process. By combining RPA and CRISPR-Cas12a in a single reaction tube, the risk of aerosol contamination has decreased markedly. The entire process, from sample preparation to result interpretation, only takes 50 min, and the reaction equipment only a water bath pot, and results can be blue light spectrometer or UV flashlight assessed visually. Importantly, the method demonstrates high sensitivity, detecting a minimum of 50 copies of the plasmid, which surpasses the sensitivity of reverse transcription polymerase chain reaction (RT-PCR) and is comparable to quantitative RT-PCR (RT-qPCR). The method exhibits excellent specificity, showing no cross-reactivity with other common sugarcane viruses, including Sugarcane mosaic virus, Sugarcane yellow leaf virus, and Sorghum mosaic virus. The practicality of this technique was validated through the detection of leaf crude extracts from 40 field samples. The detection results were consistent with those obtained from RT-PCR and RT-qPCR using leaf RNA as the template, indicating its suitability for laboratory detection and field applications.