A Comprehensive Visual Detection Strategy: Versatile LAMP Assay with Phenol Red and Lateral Flow Dipstick for On-Site...

Riemerella anatipestifer (RA) is the primary causative agent of infectious serositis in ducks, causing significant economic losses. In this study, a rapid and visual loop-mediated isothermal amplification (LAMP) assay targeting the conserved region of the ompA gene was developed. Specific primers and a FAM-labeled probe were designed, and amplification products were visualized using phenol red-based colorimetric detection and a lateral flow dipstick (LFD) system. Among the five candidate primer sets, primer set 2 was selected because it showed the highest amplification efficiency and specificity, with no cross-reactivity detected against 12 common waterfowl pathogens. Under optimal conditions, the phenol red-based LAMP assay yielded visible results after incubation at 65 °C for 30 min, while the LAMP-LFD assay required an additional 3~5 min probe hybridization step, with detection limits of 7.76 × 10² copies/μL for the phenol red-based method and 7.76 × 10⁰ copies/μL for the LAMP-LFD method. Thirty clinical samples suspected of RA infection were analyzed using conventional PCR and the developed visual LAMP assays. The positive detection rates obtained with the LAMP-LFD and phenol red-based LAMP methods were 63.3% and 60%, respectively, showing high concordance with conventional PCR (56.7%). In conclusion, the LAMP assay integrating phenol red visualization and lateral flow dipstick detection is rapid, sensitive, and easy to perform, and both detection formats show potential for point-of-care or on-site applications, and can be used for the early diagnosis and detection of RA.