VALIDATION OF LAMP ASSAY FOR Klebsiella aerogenes DETECTION IN THREE VEGETABLE SPECIES Marija Pavlović1, Mila Djisalov2, Ljiljana Šašić Zorić2, Ljiljana Janjušević2, Željko D. Popović1, Ivana Gadjanski2 1Department of Biology and Ecology, Faculty of Sciences, University of Novi Sad, Trg Dositeja Obradovića 3, 21000 Novi Sad, Serbia; 2BioSense Institute, University of Novi Sad, Dr Zorana Đinđića 1, 21000 Novi Sad, Serbia INTRODUCTION Klebsiella aerogenes is a foodborne pathogen that contaminates fruits and vegetables. Detecting it in a timely manner is essential to prevent its transmission through contaminated food sources. In this regard, the LAMP method proved to be a robust, highly sensitive and highly specific method for real-time pathogen detection, and thus method of choice for detection of K. aerogenes. In order to check efficiency of LAMP methodology with fast DNA extraction we conducted LAMP-based K. aerogenes detection in vegetables spiked with bacteria. We selected three distinct vegetable types (carrot, cucumber, and lettuce) that were previosly tested for natural contamination by K. aerogenes. Since the test results came in negative, the tested vegetables were then artificially contaminated (spiked) with a bacterial suspension containing 1.5 x 108 CFU/mL of K. aerogenes in the final sample volume. DNA extraction from the samples was performed using two approaches: Chelex approach for fast extraction to obtain “crude” DNA extract, and Plant/Fungi DNA Isolation Kit (Norgen Biotek Corp., Canada) for spin column extraction to obtain “pure” DNA extract (Fig 1). The total DNA extracted from both methods was subsequently diluted to a final concentration of 1 ng/μL. MATERIALS & METHODS The results indicated successful detection of K. aerogenes by LAMP using total DNA from both extraction approaches. DNA isolated with a commercial kit showed better results compared to the DNA obtained by the Chelex method. For the commercial kit, all three spiked samples were detected: spiked carrot and spiked lettuce around 10 minutes later compared to the positive control, and around 15 minutes later for spiked cucumber (Fig 3a). On the other hand, for the samples obtained with the Chelex method, two of three samples were detected (spiked lettuce and spiked carrot), while the spiked cucumber sample was not detected (Fig 2a). This can be related to the anatomy of the sampled cucumber tissue, which contained the exocarp and mesocarp. K. aerogenes contamination usually occurs on the surface of the cucumber (exocarp) and doesnʹt go further into the tissue (mesocarp). The exocarp contains resin and is therefore harder to homogenize. Regarding the non spiked extracts, the absence of the amplification signal implied that contamination by pathogenic K. aerogenes was not present before the spiking. As for the difference observed in the melting curves between the samples, the low peaks of the curve can be a consequence of the low amount of product obtained in the shorter amplification time for these samples (Fig 2b and 3b). The overall lower efficiency of the Chelex approach can be related to the fact that fast DNA extracts often contain nucleic acid amplification inhibitors. RESULTS ✔The presented research highlights the remarkable potential of the LAMP methodology that can be efficiently combined with fast DNA extraction methods for in-field detection of pathogens. ✔Improvements for the Chelex method could be made in light of sample selection, different sampling approaches, homogenization methods (specifically the intensity and duration of homogenization) and removal of potential reaction inhibitors from the obtained “crude” extracts. ✔The commercial kit showed better compatibility with LAMP for sample preparation and the Chelex method has shown great potential for point-of-need use. CONCLUSION Acknowledgments: This research was funded by MSCA-RISE H2020 project IPANEMA (grant agreement number 872662) and by the Ministry of Science, Technological