Wang Fenglin, Liang Qinmin, Lv Rongman, Ahmad Shakeel, Bano Mishal, Weng Guangzhen, Wen Ronghui
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.
Pathogens. 2023 Aug 18;12(8):1055. doi: 10.3390/pathogens12081055.
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.
高粱花叶病毒（SrMV）引发甘蔗花叶病，对甘蔗种植造成重大经济负面影响。本研究旨在开发一种用于检测SrMV的快速等温核酸扩增方法。设计了针对SrMV P3基因保守区域的特异性引物。通过筛选引物和优化反应条件，建立了逆转录重组酶介导的扩增（RT-RAA）方法。与RT-PCR的对比分析表明，RT-RAA方法在检测SrMV时具有更高的特异性、灵敏度和可靠性。值得注意的是，以连续10倍稀释的标准质粒为模板时，RT-RAA的灵敏度比RT-PCR高100倍。此外，RT-RAA反应在24-49°C的温度范围内表现出灵活性，无需昂贵且复杂的温度控制设备。因此，该方法可在环境温度甚至人体温度下使用。在39°C下短短10分钟内，就能有效扩增SrMV的靶序列。特异性分析表明，通过RT-RAA检测，SrMV与其他常见甘蔗病毒之间无交叉反应。该方法灵敏度高、反应时间短且设备要求低，是一种用于可靠、快速检测SrMV的有前景的诊断工具。此外，这表明它在成功检测其他甘蔗病毒方面具有广泛的适用性。
