Rapid detection of Puccinia striiformis f. sp. tritici from wheat stripe rust samples using recombinase polymerase amplification combined with multiple visualization methods

Puccinia striiformis f. sp. tritici (Pst), the airborne fungal pathogen of wheat stripe rust, causes a decline in wheat quality and severe yield loss. Effective field management of wheat stripe rust heavily relies on timely and accurate monitoring of incoming fungal sources. Therefore, rapid and accurate detection of Pst is invaluable in disease control programs. In this study, three assays using recombinase polymerase amplification (RPA) combined with different visualization methods, including RPA followed by agarose gel electrophoresis, RPA lateral- flow dipstick (RPA-LFD) technology, and real-time RPA, were developed for rapidly and sensitively detecting Pst. The RPA and RPA-LFD assays were conducted at an isothermal temperature of 39 degrees C within 30 min. The realtime RPA assay required only a 20-min reaction at 39 degrees C. The specificities of the three assays were determined based on the lack of cross-reactivity with the other seven control wheat pathogens. The detection limits of RPA and real-time RPA were 105 fg/mu L, and that of RPA-LFD was 104 fg/mu L. Additionally, the three RPA assays detected Pst in field leaf samples. Among the 73 samples examined, 44 (60.3 %), 45 (61.6 %), and 60 (82.2 %) tested positive in the RPA, real-time RPA, and RPA-LFD assays, respectively. The result suggested that RPA-LFD was the most sensitive in detecting Pst from field samples. Giving the simple, rapid, and practical nature of RPA assays, this method can serve as a promising molecular diagnostic tools for accurate and rapid detection of Pst.