Optimization and Validation of Reverse Transcription Recombinase-Aided Amplification (RT-RAA) for Sorghum Mosaic Virus Detection in Sugarcane
Fenglin Wang,
Qinmin Liang,
Rongman Lv,
Shakeel Ahmad,
Mishal Bano,
Guangzhen Weng,
Ronghui Wen
Affiliations
Fenglin Wang
State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Life Science and Technology, Ministry and Province Co-Sponsored Collaborative Innovation Center for Sugarcane and Sugar Industry, Guangxi Key Laboratory of Sugarcane Biology, Guangxi University, Nanning 530004, China
Qinmin Liang
State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Life Science and Technology, Ministry and Province Co-Sponsored Collaborative Innovation Center for Sugarcane and Sugar Industry, Guangxi Key Laboratory of Sugarcane Biology, Guangxi University, Nanning 530004, China
Rongman Lv
State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Life Science and Technology, Ministry and Province Co-Sponsored Collaborative Innovation Center for Sugarcane and Sugar Industry, Guangxi Key Laboratory of Sugarcane Biology, Guangxi University, Nanning 530004, China
Shakeel Ahmad
State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Life Science and Technology, Ministry and Province Co-Sponsored Collaborative Innovation Center for Sugarcane and Sugar Industry, Guangxi Key Laboratory of Sugarcane Biology, Guangxi University, Nanning 530004, China
Mishal Bano
State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Life Science and Technology, Ministry and Province Co-Sponsored Collaborative Innovation Center for Sugarcane and Sugar Industry, Guangxi Key Laboratory of Sugarcane Biology, Guangxi University, Nanning 530004, China
Guangzhen Weng
State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Life Science and Technology, Ministry and Province Co-Sponsored Collaborative Innovation Center for Sugarcane and Sugar Industry, Guangxi Key Laboratory of Sugarcane Biology, Guangxi University, Nanning 530004, China
Ronghui Wen
State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Life Science and Technology, Ministry and Province Co-Sponsored Collaborative Innovation Center for Sugarcane and Sugar Industry, Guangxi Key Laboratory of Sugarcane Biology, Guangxi University, Nanning 530004, China
Sorghum mosaic virus (SrMV) causes sugarcane mosaic disease and has significant adverse economic impacts on the cultivation of sugarcane. This study aimed to develop a rapid isotherm nucleic acid amplification method for detecting SrMV. Specific primers were designed to target the conserved region of the P3 gene of SrMV. The reverse transcription recombinase-aided amplification (RT-RAA) method was developed by screening primers and optimizing reaction conditions. Comparative analyses with RT-PCR demonstrated that the RT-RAA method exhibited superior specificity, sensitivity, and reliability for SrMV detection. Notably, using a standard plasmid diluted 10-fold continuously as a template, the sensitivity of RT-RAA was 100-fold higher than that of RT-PCR. Moreover, the RT-RAA reaction displayed flexibility in a temperature range of 24–49 °C, eliminating the need for expensive and complex temperature control equipment. Thus, this method could be utilized at ambient or even human body temperature. Within a short duration of 10 min at 39 °C, the target sequence of SrMV could be effectively amplified. Specificity analysis revealed no cross-reactivity between SrMV and other common sugarcane viruses detected via the RT-RAA. With its high sensitivity, rapid reaction time, and minimal equipment requirements, this method presents a promising diagnostic tool for the reliable and expedited detection of SrMV. Furthermore, it indicates broad applicability for successfully detecting other sugarcane viruses.
