Taisong Fang

Efficient oral delivery of RNA to the target site is a long-standing issue for nucleic acid–based therapy. Herein, we adopted a vesicle-to-vesicle transfer strategy and established an efficient approach to encapsulate and stabilize RNA for targeted oral delivery. The amphiphilic specifically acylated epigallocatechin directly performed encapsulation of RNA and generated nanovesicles in high efficiency without assistance of additional materials. The RNA encapsulated in the nanovesicles was efficiently transferred to outer membrane vesicles (OMVs) derived from Escherichia coli Nissle 1917 probiotic through membrane fusion with simple operation. The derived hybrid vesicles (HVs) were further anchored with bilirubin and Lys-Asp-Glu-Leu grafted hyaluronic acid (HA-BR-KDEL) ligand for sequential cellular and intracellular targeting to the endoplasmic reticulum of inflammatory cells in inflamed intestinal tract. Oral delivery of HVs@HA-BR-KDEL notably alleviated colitis symptoms in mice and contributed to the restoration of intestinal homeostasis. The tea polyphenol hybrid OMV strategy holds great promise for oral gene-mediated treatment.

Background: Shiga toxin-producing Escherichia coli (STEC) is a significant cause of foodborne illness causing various gastrointestinal diseases including hemolytic uremic syndrome (HUS), the most severe form, which can lead to kidney failure or even death. Objective: Here, we report the development of recombinase aided amplification (RAA)-exo-probe assays targeting the stx1 and stx2 genes for the rapid detection of STEC in food samples. Methods: Primers and exo-probes were designed and optimized for the detection of stx1 and stx2 using RAA technology. The optimal STEC RAA-exo-probe assays were then tested for specificity and sensitivity, and validated in both spiked and real food samples. Results: These assays were found to be 100% specific to STEC strains and were also highly sensitive with a detection limit of 1.6 × 103 CFU/mL or 32 copies/reaction. Importantly, the assays were able to successfully detect STEC in spiked and real food samples (beef, mutton, and pork), with a detection limit as low as 0.35 CFU/25g in beef samples after an overnight enrichment step. Conclusions: Overall, the RAA assay reactions completed within ∼20 min and were less dependent on expensive equipment, suggesting they can be easily adopted for in-field testing requiring only a fluorescent reader. Highlights: As such, we have developed two rapid, sensitive, and specific assays that can be used for the routine monitoring of STEC contamination in food samples, particularly in the field or in poorly equipped labs.

Escherichia coli O157:H7, being the cause of hemorrhagic colitis in humans, is recognized as one of the most dangerous and widespread foodborne pathogens. A highly specific, sensitive, and rapid E. coli O157:H7 detection method needs to be developed since the traditional detection methods are complex, costly, and time-consuming. In this study, a recombinase aided amplification (RAA) assisted CRISPR/Cas12a (RAA-CRISPR/Cas12a) fluorescence platform for specific, sensitive, and rapid nucleic acid detection of E. coli O157:H7 was introduced. First, the feasibility (components of CRISPR/Cas12a system) of the developed method was evaluated. Then a total of 34 bacterial strains were used for the specificity test, and gradient dilutions of extracted DNA and bacterial solutions of E. coli O157:H7 were prepared for the sensitivity test. Third, a real-time PCR assay for detection of the specific wzy gene of E. coli O157:H7 (FDA’s Bacteriological Analytical Manual) was used for sensitivity comparison. Finally, analysis of RAA-CRISPR/Cas12a detection in spiked and 93 real ground beef samples was carried out. The developed RAA-CRISPR/Cas12a method showed high specificity, and the detection could be completed within 30 min (after 4 h enrichment in spiked ground beef samples). The limit of detection (LOD) of bacterial concentrations and genomic DNA was 5.4 × 10² CFU/mL and 7.5 × 10⁻⁴ ng/μL, respectively, which exhibited higher sensitivity than the RAA-gel electrophoresis and RT-PCR methods. Furthermore, it was shown that E. coli O157:H7 in ground beef samples could be positively detected after 4 h enrichment when the initial bacterial inoculum was 9.0 CFU/25 g. The detection results of the RAA-CRISPR/Cas12a method were 100% consistent with those of the RT-PCR and traditional culture-based methods while screening the E. coli O157:H7 from 93 local collected ground beef samples. The developed RAA-CRISPR/Cas12a method showed high specificity, high sensitivity, and rapid positive detection of E. coli O157:H7 from ground beef samples. The RAA-CRISPR/Cas12a system proposed in this study provided an alternative molecular tool for quick, specific, sensitive, and accurate detection of E. coli O157:H7 in foods.