Design of a One‐Tube Nested PCR ‐Lateral Flow Immunochromatography Assay for Highly Sensitive Diagnosis of Southern Corn Rust

ABSTRACT Puccinia polysora exhibits a rapid incubation period in maize plant tissues and can lead to extreme outbreaks of southern corn rust under suitable conditions, making the disease difficult to control. We developed a highly sensitive one‐tube nested PCR‐lateral flow immunochromatography assay (OTNPCR‐LFIA) to detect P. polysora DNA using a closed‐tube approach with two primers and two probes that sequentially react with the rDNA internal transcribed spacer (ITS) region. The specificity of the OTNPCR‐LFIA was consistent with the one‐tube nested PCR‐agarose gel electrophoresis (OTNPCR‐AGE). The analytical sensitivity of the OTNPCR‐LFIA approach was 0.1 fg μL −1 for plasmid DNA and 0.46 urediniospores μL −1 , approximately 10‐fold and 25‐fold higher than that of OTNPCR‐AGE, respectively. In field validation tests, OTNPCR‐LFIA achieved a detection rate of 80%, outperforming the OTNPCR‐AGE rate of 70%. The established OTNPCR‐LFIA is simple, with high specificity, sensitivity and visual interpretation. Our novel technology enables rapid detection of P. polysora during the incubation period, supporting early prediction and effective disease management decision‐making.