VALIDATION OF LAMP ASSAY FOR KLEBSIELLA AEROGENES DETECTION IN THREE VEGETABLE SPECIES

VALIDATION OF LAMP ASSAY FOR KLEBSIELLA AEROGENES DETECTION IN THREE VEGETABLE SPECIES

Marija Pavlović2, Mila Djisalov1, Ljiljana Šašić Zorić1, Ljiljana Janjušević1, Željko D. Popović2, Ivana Gadjanski1

1 BioSense Institute, University of Novi Sad, Dr Zorana Đinđića 1, 21000 Novi Sad, Serbia; 2 Department of Biology and Ecology, Faculty of Sciences, University of Novi Sad, Trg Dositeja Obradovića 3, 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.

Methods: We selected three distinct vegetable types (carrot, cucumber, and lettuce) that were artificially contaminated (spiked) with a bacterial suspension containing 1.5 x 109 CFU/mL of K. aerogenes. DNA extraction from the samples was performed using two approaches: Chelex approach for fast extraction, and Plant/Fungi DNA Isolation Kit (Norgen Biotek Corp., Canada) for spin-column extraction. The total DNA extracted from both methods was subsequently diluted to a concentration of 1 ng/μL and used for the LAMP assay. 

Results: The results indicated successful detection of K. aerogenes by LAMP using total DNA from both extraction approaches. DNA isolated with a commercial kit reaches the exponential phase of the reaction approximately five minutes earlier compared to DNA obtained by the Chelex method. This can be related to the fact that fast DNA extracts often contain nucleic acid amplification inhibitors.

Conclusion: 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.

Key words: LAMP, Klebsiella aerogenes, foodborne pathogens, molecular diagnostics

Acknowledgements: This research was funded by MSCA-RISE H2020 project IPANEMA (grant agreement number 872662) and by the Ministry of Science, Technological Development, and Innovations of the Republic of Serbia (grant agreement number 451-03-47/2023-01/200358).