Journal of the science of food and agriculture · 2022
Development of an isothermal recombinase-aided amplification assay for the rapid and visualized detection of Klebsiella pneumoniae.
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Hou Laiwang, Li Darong, Zhang Ni, Zhao Jiayi, Zhao Yong, Sun Xiaohong
Аннотация
BACKGROUND: Klebsiella 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.
RESULTS: The 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%).
CONCLUSIONS: The 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.
RESULTS: The 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%).
CONCLUSIONS: The 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.