Search

To ensure the safety of foodstuffs, widespread non-laboratory monitoring for pathogenic contaminants is in demand. A suitable technique for this purpose is lateral flow immunoassay (LFIA) which combines simplicity, rapidity, and productivity with specific immune detection. This study considered three developed formats of LFIA for Salmonella Typhimurium, a priority pathogenic contaminant of milk. Common sandwich LFIA with all immunoreagents pre-applied to the test strip (format A) was compared with incubation of the sample and (gold nanoparticle—antibody) conjugate, preceding the lateral flow processes (format B), and sequential passages of the sample and the conjugate along the test strip (format C). Under the chosen conditions, the detection limits and the assay times were 3 × 104, 1 × 105, and 3 × 105 cells/mL, 10, 15, and 20 min for formats A, B, and C, respectively. The selected format A of LFIA was successfully applied to test milk samples. The sample's dilution to a fat content of 1.0% causes pathogen detection, with 70–110% revealing and 1.5–8.5% accuracy. The obtained results demonstrate that the developed LFIA allows the detection of lower concentrations of Salmonella cells and, in this way, accelerates decision-making in food safety control. [ABSTRACT FROM AUTHOR]

Pectobacterium brasiliense is a bacterial phytopathogen that causes soft and black rot and actively spreads worldwide. Our study is the first development of immunoassays for detecting P. brasiliense. We immunized rabbits and obtained serum with an extremely high titer (1:108). Isolated polyclonal antibodies were tested by enzyme-linked immunosorbent assay (ELISA) using 18 closely related strains and 5 non-related bacterial pathogens. No cross-reactivity was found concerning the tested pathogens. The ELISA of P. brasiliense was developed in a double-antibody sandwich format with a detection limit of 1.5 × 104 cells/mL. A lateral flow immunoassay (LFIA) for P. brasiliense was also developed in a double-antibody sandwich format with a detection limit of 1 × 105 cells/mL. The results of P. brasiliense cells testing with LFIA in plant matrix showed a high correlation (R2 = 0.932) between concentrations of added and revealed cells. When testing potato seed material, ELISA and LFIA confirmed 75 and 66% of positive samples according to real-time PCR, respectively. For negative samples, ELISA showed 84% coincidence, and LFIA coincided with PCR for 89% of samples. Thus, the developed immunoassays can be used to evaluate plant material in poorly equipped conditions or under field testing. [ABSTRACT FROM AUTHOR]

A promising and sought-after class of nanozymes for various applications is Pt-containing nanozymes, primarily Au@Pt, due to their easy preparation and remarkable catalytic properties. This study aimed to explore the freeze–thaw method for functionalizing Pt-containing nanozymes with oligonucleotides featuring a polyadenine anchor. Spherical gold nanoparticles ([Au]NPs) were synthesized and subsequently used as seeds to produce urchin-like Au@Pt nanoparticles ([Au@Pt]NPs) with an average diameter of 29.8 nm. The nanoparticles were conjugated with a series of non-thiolated DNA oligonucleotides, each consisting of three parts: a 5′-polyadenine anchor (An, with n = 3, 5, 7, 10; triple-branched A3, or triple-branched A5), a random sequence of 23 nucleotides, and a linear polyT block consisting of seven deoxythymine residues. The resulting conjugates were characterized using transmission electron microscopy, spectroscopy, dynamic light scattering, and emission detection of the fluorescent label at the 3′-end of each oligonucleotide. The stability of the conjugates was found to depend on the type of oligonucleotide, with decreased stability in the row of [Au@Pt]NP conjugates with A7 > A5 > 3A3 > 3A5 > A10 > A3 anchors. These [Au@Pt]NP–oligonucleotide conjugates were further evaluated using lateral flow test strips to assess fluorescein-specific binding and peroxidase-like catalytic activity. Conjugates with A3, A5, A7, and 3A3 anchors showed the highest levels of signals of bound labels on test strips, exceeding conjugates in sensitivity by up to nine times. These findings hold significant potential for broad application in bioanalytical systems. [ABSTRACT FROM AUTHOR]

In this study, we developed a sensitive immunochromatographic analysis (ICA) of the Salmonella typhimurium bacterial pathogen contaminating food products and causing foodborne illness. The ICA of S. typhimurium was performed using Au@Pt nanozyme as a label ensuring both colorimetric detection and catalytic amplification of the analytical signal due to nanozyme peroxidase-mimic properties. The enhanced ICA enabled the detection of S. typhimurium cells with the visual limit of detection (LOD) of 2 × 102 CFU/mL, which outperformed the LOD in the ICA with traditional gold nanoparticles by two orders of magnitude. The assay duration was 15 min. The specificity of the developed assay was tested using cells from various Salmonella species as well as other foodborne pathogens; it was shown that the test system detected only S. typhimurium. The applicability of ICA for the determination of Salmonella in food was confirmed in several samples of milk with different fat content, as well as chicken meat. For these real samples, simple pretreatment procedures were proposed. Recoveries of Salmonella in foodstuffs were from 74.8 to 94.5%. Due to rapidity and sensitivity, the proposed test system is a promising tool for the point-of-care control of the Salmonella contamination of different food products on the whole farm-to-table chain. [ABSTRACT FROM AUTHOR]

CRISPR/Cas12a is a potent biosensing tool known for its high specificity in DNA analysis. Cas12a recognizes the target DNA and acquires nuclease activity toward single-stranded DNA (ssDNA) probes. We present a straightforward and versatile approach to transforming common Cas12a-cleavable DNA probes into enhancing tools for fluorescence anisotropy (FA) measurements. Our study involved investigating 13 ssDNA probes with linear and hairpin structures, each featuring fluorescein at one end and a rotation-slowing tool (anchor) at the other. All anchors induced FA changes compared to fluorescein, ranging from 24 to 110 mr. Significant FA increases (up to 180 mr) were obtained by adding divalent metal salts (Mg2+, Ca2+, Ba2+), which influenced the rigidity and compactness of the DNA probes. The specific Cas12a-based recognition of double-stranded DNA (dsDNA) fragments of the bacterial phytopathogen Erwinia amylovora allowed us to determine the optimal set (probe structure, anchor, concentration of divalent ion) for FA-based detection. The best sensitivity was obtained using a hairpin structure with dC10 in the loop and streptavidin located near the fluorescein at the stem in the presence of 100 mM Mg2+. The detection limit of the dsDNA target was equal to 0.8 pM, which was eight times more sensitive compared to the common fluorescence-based method. The enhancing set ensured detection of single cells of E. amylovora per reaction in an analysis based on CRISPR/Cas12a with recombinase polymerase amplification. Our approach is universal and easy to implement. Combining FA with Cas12a offers enhanced sensitivity and signal reliability and could be applied to different DNA and RNA analytes. [ABSTRACT FROM AUTHOR]