Colorimetric aptasensor for detecting Salmonella spp., Listeria monocytogenes, and Escherichia coli in meat samples

Abstract

In this study, we successfully developed a simple and rapid method for simultaneous detection of Salmonella spp., Listeria monocytogenes, and Escherichia coli using gold nanoparticles and the aptamer aptasensor. We screened 25 specific DNA aptamer candidates against these pathogens using whole-cell Systematic Evolution of Ligands by EXponential enrichment. Among them, Ap6 was selected due to its low energy minimization values of −12.25 and −27.67 kcal/mol derived from MFold and RNAFold analysis, respectively. The assay presented in this study allowed the visual colorimetric detection of labeled colloidal gold nanoparticles as well as determination of UV absorbance at 625 and 525 nm under optimized conditions. The detection limit of this aptasensor was as less as 105 CFU/ml. A random investigation of 50 meat samples, including ham and chicken sausages, collected from the local market revealed 96% accuracy, 96% specificity, and 100% sensitivity of the assay. The colorimetric aptasensor can accomplish one-step detection without pre-culture, DNA extraction, and amplification. Hence, it is an easy, rapid, specific, and qualitative assay that can be used as a point-of-care testing to directly detect multiplex foodborne pathogens.

Rapid point‐of‐need detection of bacteria and their toxins in food using gold nanoparticles

“…Water soluble salts, such as MgSO 4 , NaCl, or MgCl 2 , are usually used to adjust the ionic strength of the buffer and optimize the sensors' analytical parameters (H.-S. Kim et al, 2017;Y.-J. Kim et al, 2018;Ledlod et al, 2020). Salts neutralize the repulsive forces between AuNPs, which perturbs the equilibrium between van Der Waals attractive and electrostatic repulsive forces, and favors particle aggregation (Gao et al, 2012;Ledlod et al, 2020).…”

“…Kim et al, 2018;Ledlod et al, 2020). Salts neutralize the repulsive forces between AuNPs, which perturbs the equilibrium between van Der Waals attractive and electrostatic repulsive forces, and favors particle aggregation (Gao et al, 2012;Ledlod et al, 2020). A higher ionic straight results in faster aggregation and lower sensitivity (Hianik et al, 2007).…”

“…However, pH 3 was not found to be an optimal pH in the majority of publications. For instance, Kim et al determined pH 5.5 as the most suitable pH since it promoted DNA adsorption and improved stability of conjugates, while other authors asserted a pH range from neutral to slightly basic (pH 7.3-8.2) as the most suitable due to the stabile equilibrium between attractive and repulsive forces at the surface of AuNPs (Hianik et al, 2007;Ledlod et al, 2020). It appears, thus, that the most suitable pH of the solution cannot be standardized but should be optimized for each test and each food matrix.…”

“…AuNPs for detection of bacteria and toxins in meat were either purchased choosing an average size of 15-20 nm (Y.-J. Kim et al, 2018;Wachiralurpan et al, 2018), or synthesized using the citrate reduction method with an average size of 15-30 nm (Cho & Irudayaraj, 2013;Ledlod et al, 2020;H. b. Liu et al, 2019).…”

Annotation of miRNAs in COVID-19 coronavirus

“…The extended sequences were folded with RNAfold [ [26] , [27] , [28] ] to form secondary structures and to identify a hairpin structure sequence containing a mature miRNA sequence on one arm for each different secondary structure. Finally, 4 sequences were identified.…”