A Rapid and Simple Loop-Mediated Isothermal Amplification Assay for the Detection of Pseudomonas aeruginosa From Equine Genital Swabs

• We developed a simple assay for rapid detection of P. aeruginosa directly from swabs.
• Pseudomonas aeruginosa can be directly detected from a swab without prior enrichment.
• As few as 11 copies of the target may be detected in less than 15 minutes.

Pseudomonas aeruginosa is a causative agent of infectious reproductive disease in horses. Conventional methods for identification of this organism from equine genital swabs can be time consuming, laborious, and may rely on the presence of live bacteria. However, the use of loop-mediated isothermal amplification (LAMP) provides a simple and rapid detection method. We aimed to develop and evaluate a simple-to-use and interpret LAMP assay, based on the P. aeruginosa ecfX gene, capable of detecting P. aeruginosa from DNA extracted directly from equine genital swabs without culture or prior enrichment. The specificity of the assay was determined using DNA extracted from 29 different bacterial species, as well as in silico. The diagnostic sensitivity of the assay was determined through analysis of DNA extracted directly from 46 equine genital swabs. A P. aeruginosa–specific LAMP assay that could detect as few as 11 copies of its target in less than 15 minutes was developed. The reaction amplicon could be detected by real-time fluorescence or using a simple lateral flow device. The assay was specific to its target, showing no cross-reactivity or interference when tested against DNA extracted from 29 other bacterial species, as well as in silico. The diagnostic sensitivity of the assay was 86.8% (39 of 46) and 88.5% (40 of 46), when the amplicon was detected by real-time fluorescence and lateral flow device technology, respectively. The P. aeruginosa–specific LAMP assay provides a simple and rapid means of detecting the presence of viable and nonviable P. aeruginosa directly from equine genital swabs.