Sensitivity of Loop-Mediated Isothermal Amplification in Comparison to Digital Droplet PCR for Identification of...

According to the EFSA Report on Zoonoses (2024), yersiniosis was classified as the fourth most commonly reported zoonosis in humans in 2023, with a 13.5% increase in yersiniosis infections compared to 2022. In 2024, the findings were consistent with the 2020-2023 trend. Isolation and identification of enteropathogenic Yersinia is difficult and time consuming, especially when examining food and environmental samples. Among them, Y. pseudoturbeculosis poses a challenge due to the lack of a single selective medium for all bioserotypes. Therefore, faster methods for the detection of Yersinia spp. need to be implemented into the praxis. Rapid identification of pathogens in food or at the time and location of the epidemiological outbreak (point-of-care testing) enables either prevention of the outbreak or early stage diagnosis and prompt decisions. The loop-mediated isothermal amplification (LAMP) is increasingly coming to scientists' attention as a robust and rapid methodology for pathogen detection in laboratories with limited resources and equipment. The aim of current study is to evaluate, for the first time, the sensitivity of the LAMP protocol based on colorimetric detection in the visible spectrum in comparison with that of the digital droplet PCR (ddPCR). For this aim, a series of decimal logarithmic dilutions of the pathogen Y. pseudotuberculosis in artificially contaminated raw goat milk was used. One commercial LAMP kit with two different dyes (one dsDNA-binding and one Mg²⁺-sensitive) was compared to the sensitivity of the detection to ddPCR. The results obtained revealed a high sensitivity of the kit for detection of DNA isolated from artificially contaminated milk samples in the following range: visible detection based on visible color change-3.1 × 10⁴ mL (violet dye) and 3.4 × 10³/mL (blue dye); detection with gel electrophoresis-2.0 × 10¹/mL (violet dye) and 3.4 × 10²/mL (blue dye). The enumeration of the DNA copies in the same samples was performed with ddPCR, with a detection limit of 2.0 × 10¹/mL. Our results indicate the potential and the possible applicability of the LAMP method for rapid and sensitive visual detection of Y. pseudotuberculosis in raw goat milk. The presented ddPCR protocol can be used for highly sensitive identification and enumeration of Y. pseudtuberculosis in raw goat milk. In conclusion, the conducted comparison is of importance for future implementation of LAMP protocols for on-field analysis near the sampling site and point-of-care or laboratory diagnostics of Y. pseudtuberculosis after the successful validation procedure of an appropriate LAMP protocol.