One-step colorimetric LAMP (cLAMP) assay for visual detection of Xanthomonas oryzae pv. oryzae in rice

Abstract

Xanthomonas oryzae pv. oryzae (Xoo) is a major pathogen of rice, leading to tremendous rice yield reduction about 20–50% both temperate and tropical rice-growing regions. Here, to develop a rapid and reliable method for Xoo detection, a one-step colorimetric loop-mediated isothermal amplification (cLAMP) assay was established. The conditions for cLAMP using both LpXoo80 and LpXoo4009 primer sets were optimized. The specificity and sensitivity of cLAMP using both primers were determined under the optimal conditions of 60 °C for 40 min that the results can be visualized by naked eye. As a result, cLAMP using LpXoo4009 primer amplifying in only Xoo exhibited higher specificity than LpXoo80 primer, generating a cross-reaction of LAMP amplification in both Xoo and other Xanthomonas species (Xag and Xac). cLAMP using LpXoo80 primer showed higher sensitivity than the LpXoo4009 primer, with consistently amplified positive cLAMP products under detected thresholds of at least 2 × 103 and 2 × 105 cfu/mL, respectively. This developed cLAMP assay was applied to detect Xoo artificially inoculated rice leaf (2.5 × 108 cfu/mL). Positive LAMP product was clearly detected over the first 2 days post-inoculation, while external BLB leaf symptoms disappeared. This cLAMP assay also showed high performance of Xoo detection (>80%) in Xoo naturally infected rice leaf samples, harvested from suspected BLB symptomatic leaf in cultivated rice fields. The work reported a high specificity, sensitivity, and simplicity of the cLAMP assay to detect various Xoo isolates identified from the lower north region of Thailand. This method provided feasible point-of-care testing for Xoo surveillance in fields and long-term BLB disease management.