High-resolution melting real-time PCR assays for subtyping of five diarrheagenic Escherichia coli by a single well in milk.

Diarrheagenic Escherichia coli (DEC) is a kind of foodborne pathogen that poses a significant threat to both food safety and human health. To address the current challenges of high prevalence and difficult subtyping of DEC, this study developed a method that combined multiplex polymerase chain reaction (PCR) with high resolution melting (HRM) analysis for subtyping 5 kinds of DEC. The target genes are amplified by multiplex PCR in a single well, and HRM curve analysis was applied for distinct amplicons based on different melting temperature (Tm) values. The method enables discrimination of different DEC types based on characteristic peaks and distinct Tm values in the thermal melting curve. The assay exhibited 100% sensitivity and 100% specificity with a detection limit of 0.5-1 ng/µL. The results showed that different DNA concentrations did not influence the subtyping results, demonstrating this method owed high reliability and stability. In addition, the method was also used for the detection and subtyping of DEC in milk. This method streamlines operational procedures, shorts the detection time, and offers a novel tool for subtyping DEC.

Epidemiological and Whole Genome Sequencing Analysis of Restaurant Salmonella Enteritidis Outbreak Associated with an Infected Food Handler in Jiangxi Province, China, 2023.

In China, Salmonella is one of the most frequent causes of bacterial gastroenteritis, and food handlers in restaurants as an important contaminated source were rarely reported. In May 2023, an outbreak of Salmonella enterica serovar Enteritidis infection in a restaurant in Jiangxi Province, China, was investigated. Cases were interviewed. Stool samples from cases, anal swabs from restaurant employees, suspicious raw food materials, and semifinished food were collected and examined. Pulsed-field gel electrophoresis (PFGE) and whole genome sequencing (WGS) were performed to determine the relatedness of the pathogen isolates. Antimicrobial resistance genes and virulence genes of isolates were analyzed by WGS. The antimicrobial profile of the isolates was detected by broth microdilution, which involved 20 different antibiotics. Among the 31 patrons, 26 showed gastrointestinal symptoms. Five Salmonella Enteritidis strains were isolated from patients (2), semifinished food (2), and food handler (1). The results of PFGE and single-nucleotide polymorphism showed that these five isolates were identical clones. These findings demonstrated that this outbreak was a restaurant Salmonella Enteritidis outbreak associated with an infected food handler. The rates of resistance to nalidixic acid and colistin and intermediate resistance to ciprofloxacin were 100%, 80%, and 100%, respectively. These outbreak isolates harbored point mutation gyrA p.D87G. The cause of inconsistency between the genotype and phenotype of resistance was deeply discussed. A total of 107 virulence genes were found in each isolate, with many being associated with Salmonella pathogenicity island (SPI)-1 and SPI-2. As an overlooked contamination source, infected food handlers can easily cause large-scale outbreaks. This outbreak highlighted that the government should enhance the training and supervision of food hygiene and safety for food handlers to prevent foodborne outbreaks.

Promoted Mid-Infrared Photodetection of PbSe Film by Iodine Sensitization Based on Chemical Bath Deposition.

In recent years, lead selenide (PbSe) has gained considerable attention for its potential applications in optoelectronic devices. However, there are still some challenges in realizing mid-infrared detection applications with single PbSe film at room temperature. In this paper, we use a chemical bath deposition method to deposit PbSe thin films by varying deposition time. The effects of the deposition time on the structure, morphology, and optical absorption of the deposited PbSe films were investigated by x-ray diffraction, scanning electron microscopy, and infrared spectrometer. In addition, in order to activate the mid-infrared detection capability of PbSe, we explored its application in infrared photodetection by improving its crystalline quality and photoconductivity and reducing tge noise and high dark current of PbSe thin films through subsequent iodine treatment. The iodine sensitization PbSe film showed superior photoelectric properties compared to the untreated sample, which exhibited the maximum of responsiveness, which is 30.27 A/W at 808 nm, and activated its detection ability in the mid-infrared (5000 nm) by introducing PbI2, increasing the barrier height of the crystallite boundary and carrier lifetimes. This facile synthesis strategy and the sensitization treatment process provide a potential experimental scheme for the simple, rapid, low-cost, and efficient fabrication of large-area infrared PbSe devices.

Epitaxial Topological Insulator Bi2Te3 for Fast Visible to Mid-Infrared Heterojunction Photodetector by Graphene As Charge Collection Medium.

Three dimensional topological insulators have a thriving application prospect in broadband photodetectors due to the possessed topological quantum states. Herein, a large area and uniform topological insulator bismuth telluride (Bi2Te3) layer with high crystalline quality is directly epitaxial grown on GaAs(111)B wafer using a molecular beam epitaxy process, ensuring efficient out-of-plane carriers transportation due to reduced interface defects influence. By tiling monolayer graphene (Gr) on the as-prepared Bi2Te3 layer, a Gr/Bi2Te3/GaAs heterojunction array prototype was further fabricated, and our photodetector array exhibited the capability of sensing ultrabroad photodetection wavebands from visible (405 nm) to mid-infrared (4.5 µm) with a high specific detectivity (D*) up to 1012 Jones and a fast response speed at about microseconds at room temperature. The enhanced device performance can be attributed to enhanced light-matter interaction at the high-quality heterointerface of Bi2Te3/GaAs and improved carrier collection efficiency through graphene as a charge collection medium, indicating an application prospect of topological insulator Bi2Te3 for fast-speed broadband photodetection up to a mid-infrared waveband. This work demonstrated the potential of integrated topological quantum materials with a conventional functional substrate to fabricate the next generation of broadband photodetection devices for uncooled focal plane array or infrared communication systems in future.

Immuno-HCR based on contact quenching and fluorescence resonance energy transfer for sensitive and low background detection of Escherichia coli O157:H7.

Escherichia coli O157:H7 makes a major threat to human health. Aiming to detect Escherichia coli O157:H7 sensitively, hybridization chain reaction signal amplified immunoassay (immuno-HCR) based on contact quenching (CQ) and fluorescence resonance energy transfer (FRET) was developed. The background of the new designed HCR hairpins (CQ-FRET hairpins) was reduced by contact-quenching fluorescein (FAM) and breaking FRET from donor (FAM) to acceptor (Cy5). The F/F0 ratio of CQ-FRET hairpins (37.02) was obviously higher than that of two other common HCR fluorescent hairpins (CQ hairpins, 21.45; FRET hairpins, 4.61). The limit of detection of the assay was 3.5 × 101 CFU/mL and obviously lower than that of CQ hairpins based immuno-HCR (3.28 × 103 CFU/mL) and FRET hairpins based immuno-HCR (6.49 × 104 CFU/mL). The proposed low fluorescent background immuno-HCR with high sensitivity which was verified in contaminated milk samples could be potentially used in the detection of various pathogens.

Molecular cloning, expression, and functional analysis of the copper amine oxidase gene in the endophytic fungus Shiraia sp. Slf14 from Huperzia serrata.

Huperzine A (HupA) is a drug used for the treatment of Alzheimer's disease. However, the biosynthesis of this medicinally important compound is not well understood. The HupA biosynthetic pathway is thought to be initiated by the decarboxylation of lysine to form cadaverine, which is then converted to 5-aminopentanal by copper amine oxidase (CAO). In this study, we cloned and expressed an SsCAO gene from a HupA-producing endophytic fungus, Shiraia sp. Slf14. Analysis of the deduced protein amino acid sequence showed that it contained the Asp catalytic base, conserved motif Asn-Tyr-Asp/Glu, and three copper-binding histidines. The cDNA of SsCAO was amplified and expressed in Escherichia coli BL21(DE3), from which a 76 kDa protein was obtained. The activity of this enzyme was tested, which provided more information about the SsCAO gene in the endophytic fungus. Gas Chromatograph-Mass Spectrometry (GC-MS) revealed that this SsCAO could accept cadaverine as a substrate to produce 5-aminopentanal, the precursor of HupA. Phylogenetic tree analysis indicated that the SsCAO from Shiraia sp. Slf14 was closely related to Stemphylium lycopersici CAO. This is the first report on the cloning and expression of a CAO gene from HupA-producing endophytic fungi. Functional characterization of this enzyme provides new insights into the biosynthesis of the HupA an anti-Alzheimer's drug.

[Molecular cloning, expression and characterization of lysine decarboxylase gene of endophytic fungus Shiraia sp. Slf14 from Huperzia serrata].

Objective: Huperzine A (HupA) was approved as a drug for the treatment of Alzheimer's disease. The HupA biosynthetic pathway was started from lysine decarboxylase (LDC), which catalyzes lysine to cadaverine. In this study, we cloned and expressed an LDC gene from a HupA-producing endophytic fungus, and tested LDC activities. Methods: An endophytic fungus Shiraia sp. Slf14 from Huperzia serrata was used. LDC gene was obtained by RT-PCR, and cloned into pET-22b(+) and pET-32a(+) vectors to construct recombinant plasmids pET- 22b-LDC and pET-32a-LDC. These two recombinant plasmids were transformed into E. coli BL21, cultured for 8 h at 24 °C, 200 r/min with 1×10­3 mol/L IPTG into medium to express the LDC proteins, respectively. LDC proteins were purified by Ni2+ affinity chromatography. Catalytic activities were measured by Thin Layer Chromatography. At last, the physicochemical properties and structures of these two LDCs were obtained by bioinformatics software. Results: LDC and Trx-LDC were expressed in E. coli BL21 successfully. SDS-PAGE analysis shows that the molecular weight of LDC and Trx-LDC were 24.4 kDa and 42.7 kDa respectively, which are consistent with bioinformatics analysis. In addition, TLC analysis reveals that both LDC and Trx-LDC had catalytic abilities. Conclusion: This work can provide fundamental data for enriching LDC molecular information and reveal the HupA biosynthetic pathway in endophytic fungi.