AbstractBACKGROUNDKlebsiella pneumoniae is a zoonotic opportunistic pathogen, leading to severe infections in dairy cows and humans. Efficient, on‐site and accurate detection of K. pneumoniae is necessary to reduce the harm of cow mastitis and human infections. The objective of this study was to establish a recombinase‐aided amplification (RAA) method combined with lateral flow dipstick (LFD) for rapid detection of K. pneumoniae.RESULTSThe primer concentration, incubation temperature and incubation time of the RAA reaction were optimized. When the primer concentration was 100 nmol L–1, the strongest band could be obtained by incubation at 37 °C for 20 min. The RAA‐LFD method had high specificity to K. pneumoniae and showed no cross‐reaction with other pathogens. In addition, the detection limit of RAA‐LFD for K. pneumoniae was 20 fg genomic DNA and 2.5 × 102 CFU mL−1 of bacteria in pure culture, which is 100 times higher than that of polymerase chain reaction (PCR) detection. Moreover, the RAA‐LFD method can detect K. pneumoniae at initial concentrations as low as 2.5 CFU per 25 mL in artificially spiked milk samples after at least incubation for 6 h. Importantly, RAA‐LFD had a high agreement with a test accuracy of 96.9%, compared with the biochemical identification method. Also, the detection accuracy of RAA‐LFD was higher than that of the PCR assay (95.3%).CONCLUSIONSThe results demonstrated that the RAA‐LFD assay is an accurate, sensitive, simple and point‐of‐use detection method for K. pneumoniae, which could be used as a potential application in the research laboratory and for disease diagnosis. © 2021 Society of Chemical Industry