Development of Cu-doped CeO2 nanospheres mimic nanozyme-based immunoassay for the specific screening of Bacillus cereus.

Due to the highly similar genetic background, it is difficult to distinguish Bacillus cereus (B. cereus) with other members of B. cereus group. Herein, an antibody-based colorimetric immunoassay using Cu-doped CeO2 nanospheres as peroxidase mimics was developed for the detection of B. cereus in food. First, monoclonal antibodies (mAbs) and polyclonal antibody (pAb) with good specificity to B. cereus were prepared and characterized. Second, the regular-shaped hollow Cu/CeO2 nanospheres with highly catalytic activity and biocompatibility were synthesized as mimic nanozymes to capture secondary antibody. Finally, a sandwich colorimetric immunoassay for the specific and sensitive detection of B. cereus was developed, showing linear detection range from 3.2 × 102 to 1 × 105 CFU/mL and a limit detection of 1.7 × 102 CFU/mL. The developed immunoassay holds great potential as an effective tool for detecting B. cereus in food poisoning.

An RPA-Assisted CRISPR/Cas12a Assay Combining Fluorescence and Lateral Flow Strips for the Rapid Detection of Enterotoxigenic Bacillus cereus.

Bacillus cereus (B. cereus) is a foodborne pathogen that can produce tripartite enterotoxins, which can cause a variety of diseases after infection. It is critical to rapidly and accurately detect strains with enteropathogenic potential to safeguard human health. In this study, a dual-signal visualized detection platform with fluorescence assay and paper-based lateral flow assay (LFA) based on recombinase polymerase amplification (RPA), CRISPR/Cas12a system, and self-developed CRISPR nucleic acid test strips was constructed for enterotoxigenic B. cereus. The genes that encode two tripartite enterotoxins─nheA, nheB, and nheC for nonhemolytic enterotoxin and hblA, hblC, and hblD for hemolysin BL─were utilized as detection targets. The platform was capable of detecting six enterotoxin genes at the same genomic DNA level. The limits of detection for each gene were 10-3 ng/µL in fluorescence assay and 10-4 ng/µL in LFA. Furthermore, 101-102 CFU/mL of B. cereus in pure culture was detected. Additionally, a smartphone miniprogram could assist in evaluating the results in LFA. The platform demonstrated good utility by detecting B. cereus in food samples, including milk and rice. The results indicate that our RPA-CRISPR/Cas12a dual-signal visualized detection platform can quickly and easily detect B. cereus with three-component enterotoxin-producing potentials. The whole analytic process took less than 60 min without complex operation or expensive equipment.

Universal and Naked-Eye Diagnostic Platform for Emetic Bacillus cereus Based on RPA-Assisted CRISPR/Cas12a.

Emetic Bacillus cereus (B. cereus), which can cause emetic food poisoning and in some cases even fulminant liver failure and death, has aroused widespread concern. Herein, a universal and naked-eye diagnostic platform for emetic B. cereus based on recombinase polymerase amplification (RPA)-assisted CRISPR/Cas12a was developed by targeting the cereulide synthetase biosynthetic gene (cesB). The diagnostic platform enabled one-pot detection by adding components at the bottom and cap of the tube separately. The visual limit of detection of RPA-CRISPR/Cas12a for gDNA and cells of emetic B. cereus was 10-2 ng µL-1 and 102 CFU mL-1, respectively. Meanwhile, it maintained the same sensitivity in the rice, milk, and cooked meat samples even if the gDNA was extracted by simple boiling. The whole detection process can be finished within 40 min, and the single cell of emetic B. cereus was able to be recognized through enrichment for 2-5 h. The good specificity, high sensitivity, rapidity, and simplicity of the RPA-assisted CRISPR/Cas12a diagnostic platform made it serve as a potential tool for the on-site detection of emetic B. cereus in food matrices. In addition, the RPA-assisted CRISPR/Cas12a assay is the first application in emetic B. cereus detection.

Highly selective monoclonal antibody-based lateral flow immunoassay for visual and sensitive determination of conazole fungicides propiconazole in vegetables.

The conazole fungicide propiconazole is frequently found in vegetables although usage is not allowed. To overcome the high-cost and time-consuming labour requirements of instrumental methods, we developed a simple and visual lateral flow immunoassay for the sensitive determination of propiconazole. A hapten was carefully designed to raise a monoclonal antibody against propiconazole. Bal b/c mice were immunised with the hapten-carrier protein conjugate and a specific monoclonal antibody (mAb) was produced. Based on this mAb, a sensitive immunochromatographic strip assay (ICA) was established for rapid screening of propiconazole in vegetable samples. After optimisation of analytical parameters, the ICA strip showed a detection limit of 0.13 ng g-1 and a linear range from 0.5 to 80 ng g-1 using a strip reader. The assay also can be read by the naked eye with a visual limit of detection of 80 ng g-1. The recoveries for spiked vegetable samples by ICA ranged from 85.2% to 114.9%, with a coefficient of variation less than 11.7%. The assay time is within 45 min for a single sample including the sample pre-treatment. For spiked and blind samples, the detection capability of ICA was equivalent to liquid chromatography-mass spectrometry.