Development of a loop‐mediated isothermal amplification‐lateral flow dipstick ( <scp>LAMP</scp> ‐ <scp>LFD</scp> ) assay and on‐site rapid detection evaluation for <i>Rodentibacter heylii</i> and <i>Rodentibacter pneumotropicus</i>

Abstract Background Rodentibacter pneumotropicus and Rodentibacter heylii are important exclusion pathogens in specific pathogen‐free (SPF) laboratory animals. However, routine detection methods such as traditional bacterial culture remain labor‐intensive and time‐consuming. The loop‐mediated isothermal amplification‐lateral flow dipstick (LAMP–LFD) assay provides a self‐contained format for nucleic acid testing that combines speed, sensitivity, and specificity while minimizing aerosol contamination, making it well‐suited for point‐of‐need applications. Objectives This study aimed to develop a streamlined, on‐site detection platform capable of rapidly identifying both pathogens with high analytical accuracy, operational simplicity, and suitability for routine laboratory surveillance. Methods A LAMP–LFD system was established for the detection of the two pathogens using primers designed against the conserved 16S–23S rRNA ITS region. The assay's specificity, sensitivity, repeatability, and limit of detection (LoD) were evaluated. Results The assay achieved detection limits of 1 × 10 −4 ng/μL for R. pneumotropicus and 1 × 10 −5 ng/μL for R. heylii , demonstrating significantly higher sensitivity than PCR. No cross‐reactivity was observed with 15 non‐target bacteria. Among 28 infected samples, the method detected 19 positives, compared with 21 by multiplex PCR and 23 by qPCR. In 842 laboratory animal samples, culture‐based, qPCR, and LAMP–LFD detected 2, 41, and 37 positive samples, respectively. The LoDs were 10 2 and 10 1 CFU/mL in fecal samples and 10 1 and 10 0 CFU/mL in throat swab samples for R. pneumotropicus and R. heylii , respectively. Conclusions This method provides an approach for monitoring the health status of laboratory mice by enabling the detection of these two target pathogens.