Establish a visual loop-mediated isothermal amplification (LAMP) method for rapid detection avian colibacillosis

Abstract

Objective To develop a rapid visual loop-mediated isothermal amplification (LAMP) assay for the detection of pathogenic Escherichia coli in poultry-associated samples. Methods A total of 650 samples were collected from 11 large-scale poultry farms in Jilin Province, China, from which 632 bacterial isolates were recovered. Four sets of LAMP primers were designed based on conserved genomic regions identified by sequence alignment. Reaction temperature, incubation time, and Bst DNA polymerase dosage were optimized. The limit of detection, visual colorimetric performance, and cross-reactivity were evaluated, and the clinical diagnostic performance was compared with conventional PCR. Results The optimized LAMP assay produced detectable amplification within 45 min at 63 °C, with a limit of detection of 3.6 CFU/mL. Positive reactions turned yellow-green, whereas negative reactions remained orange, enabling naked-eye discrimination. No cross-reactivity with non-target bacterial species was observed. The LAMP assay exhibited a 1,000-fold lower limit of detection than conventional PCR, required neither thermal cycling nor gel electrophoresis, and showed complete agreement with PCR results for clinical samples. Conclusion This colorimetric LAMP assay is reliable and holds considerable promise as a rapid on-site screening tool for pathogenic E. coli in poultry-associated samples.