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

Xuan Lv; Jiarui Jiang; Ziqian Yang; Sishu Lan; Yue Ma; Jie Deng; Congying Zhou; Zhifang Wang; Yuchen Li; Zhanhong Ma

doi:10.1016/j.ijbiomac.2024.137634

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

Int J Biol Macromol. 2024 Nov 21:137634. doi: 10.1016/j.ijbiomac.2024.137634. Online ahead of print.

Authors

Xuan Lv¹, Jiarui Jiang¹, Ziqian Yang¹, Sishu Lan¹, Yue Ma¹, Jie Deng¹, Congying Zhou¹, Zhifang Wang¹, Yuchen Li¹, Zhanhong Ma²

Affiliations

¹ Department of Plant Pathology, Ministry of Agriculture and Rural Affairs (MOA) Key Lab of Pest Monitoring and Green Management, College of Plant Protection, China Agricultural University, Beijing 100193, China.
² Department of Plant Pathology, Ministry of Agriculture and Rural Affairs (MOA) Key Lab of Pest Monitoring and Green Management, College of Plant Protection, China Agricultural University, Beijing 100193, China. Electronic address: mazh@cau.edu.cn.

PMID: 39579823
DOI: 10.1016/j.ijbiomac.2024.137634

Abstract

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 °C within 30 min. The real-time RPA assay required only a 20-min reaction at 39 °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/μL, and that of RPA-LFD was 10⁴ fg/μL. 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.

Keywords: Fast molecular detected methods; Lateral flow dipstick; Puccinia striiformis f. sp. tritici; Real-time RPA; Recombinase polymerase amplification.