Feasibility of aptamer-based biosensor design by fluorescence method for rapid identification and counting of Yersinia enterocolitica in red meat

Abstract

Abstract Introduction: Yersinia enterocolitica is known as one of the most important food pathogens that cause yersiniosis transmitted from meat products, which can cause pseudo-appendicitis, septicemia, acute gastroenteritis, and lymphadenitis in humans. For this purpose, rapid and sensitive identification of this bacterium in food products is necessary. Objective: An innovative 5-FAM-labeled aptamer with high affinity was developed for the detection of Yersinia enterocolitica bacteria using graphene oxide (GO) as a quenching platform. Method: First, the optimal concentrations of different parameters (aptamer, GO, time and pH) were calculated in the study. The selectivity of the prepared system was evaluated in the presence of common symbiotic bacteria such as Yersinia pseudotuberculosis and other bacteria, Staphylococcus aureus, Listeria monocytogenes (as gram-positive bacteria), Escherichia coli, and Salmonella typhimurium (as gram-negative bacteria). The calibration graph was calculated at different concentrations of the target bacteria. The quenching effect of GO was investigated in the aptasensory system. Results: The results showed that in the absence of Yersinia enterocolitica, FAM-labeled aptamer was linked with GO and caused the fluorescence to be relatively weak. After the addition of Yersinia enterocolitica, the aptamer is released from the GO surface and binds to the target bacteria, significantly increasing the fluorescence intensity at an excitation wavelength of 410 nm and an emission wavelength of 530 nm. Due to the wide range of detection of Y. enterocolitica, the fluorescence aptasensor can be useful in the evaluation of food contamination by pathogenic bacteria. Based on comparison with other available methods, detection range and LOD for Y. enterocolitica were very favorable compared to published methods. In this study, the fluorescence intensity value of the recovery was linearly proportional to the logarithm of the bacterial concentration, which resulted in a strong correlation (R2=0.99) between the fluorescence intensity value and Y. enterocolitica measurement. Finally, LOD and LOQ were found to be 3 CFU•mL-1 and 9.7 CFU•mL-1, respectively. Conclusion: This fluorescence-based biosensor system showed that the aptamers designed with GO for Y. enterocolitica bacteria can be successful in whole-cell forms for rapid and sensitive detection of the target bacteria. Also, this designed system showed that it has the ability to identify Y. enterocolitica bacteria in red meat food samples, which can be used on a commercial scale by examining the available characteristics. Key words: Aptamer, biosensor, fluorescence, Yersinia enterocolitica, graphene oxide, red meat