Optimization and Validation of Reverse Transcription Recombinase-Aided Amplification (RT-RAA) for Sorghum Mosaic Virus Detection in Sugarcane

Abstract

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.

Pyrococcus furiosus Argonaute-mediated porcine epidemic diarrhea virus detection

“…RAA, similar to RPA, has the advantages of rapid nucleic acid amplification at room temperature and simple operation (Wu et al 2022 ). However, due to the need for purification, nucleic acid electrophoresis, and the tendency for false positives to occur when using RAA alone (Chen et al 2022b ; Wang et al 2023 ), we combine PfAgo with RAA to increase the convenience and specificity of detection based on the high sensitivity and specificity of PfAgo cleavage activity. In using RAA and PfAgo, a visual detection approach was developed for PEDV in this article.…”

“…Recombinase-aided amplification (Wang et al 2023 ) is an isothermal amplification technique that combines recombinant enzymes with other enzymes to achieve DNA or RNA amplification in vitro. In this study, the authors employed a combination of recombinase-aided amplification (RAA) and PfAgo to develop a novel detection technique for PEDV.…”

Recombinase polymerase amplification: method’s characteristics and applications in diagnostics of infectious diseases

“…Установлено, что для успешной амплификации минимальная температура реакции должна быть выше 30ºC. В некоторых источниках сообщается о положительном результате РПА при 25ºC [34,35]. Такая относительно низкая температура позволяет отказаться даже от термостата и использовать для поддержания температуры другие источники, например грелку для рук.…”