Detection of Listeria monocytogenes in Food Using the Proofman-LMTIA Assay.

Microbial factors, including bacteria, viruses, and other pathogens, are significant contributors to foodborne illnesses, posing serious food safety risks due to their potential for rapid growth and contamination. Listeria monocytogenes is one of the most common types of foodborne bacteria that can cause serious foodborne diseases or even fatalities. In this study, a novel nucleic acid amplification method called Proofman-LMTIA was employed to detect Listeria monocytogenes contamination in food. This method combines proofreading enzyme-mediated probe cleavage with ladder-shape melting temperature isothermal amplification. A positive recombinant plasmid was used as a control to ensure the accuracy of the detection results, and primers and Proofman probes were specifically designed for the LMTIA. Genomic DNA was extracted, the reaction temperature was optimized, and the primers' specificity was verified using foodborne pathogens like Staphylococcus aureus, Escherichia coli O157:H7, and Salmonella. The sensitivity was assessed by testing serial dilutions of genomic DNA, and the method's applicability was confirmed by detecting artificially contaminated fresh pork. The established LMTIA method exhibited both high specificity and sensitivity. At the optimal reaction temperature of 63 °C, the primers specifically identified Listeria monocytogenes contamination in pork at a concentration of 8.0 ± 0.7 colony-forming units (CFUs) per 25 g. Furthermore, the Proofman-LMTIA method was applied to test Listeria monocytogenes DNA in 30 food samples purchased from a Chinese retail market, and reassuringly, all results indicated no contamination. Proofman-LMTIA can serve as a reliable and rapid method for detecting Listeria monocytogenes in food, contributing to public health by safeguarding consumers from foodborne illnesses, and strengthening food safety regulations.

Hydrogen cyanide catalytic hydrolysis mechanism by Al-doped graphene: A density functional theory study.

CONTEXT: The hydrogen cyanide (HCN) hydrolysis reaction mechanism over Al-doped graphene was investigated through the density functional theory method. HCN preferentially adsorbed vertically on the Al top site to form a stable adsorption configuration. H2O preferentially adsorbed parallel on the Al top site to form a stable adsorption configuration. The competitive adsorption of HCN and H2O weakened the adsorption stability of each molecule over Al-doped graphene. The break of C-N and H-O bonds was the key process in the preferential fracture pathway of the C-H bond. The break of C-N and C-H bonds was the key process in the preferential fracture pathway of the H-O bond. HCN played the role of bridge in the joint adsorption process. H atom transfer and C-N bond cleavage promoted the generation of CO and NH3. The change in the order of H atom transfer determined the reaction energy barrier. NH2CHO was more likely to act as an intermediate to promote the hydrolysis process. METHODS: The calculation work was achieved from the Dmol3 program in Material Studio 2017 using the GGA/PBE method with DNP basis, including the geometric structure and reaction pathway optimization, and adsorption energy calculation. All calculations were performed using a spin-polarized set and the TS method was used for DFT-D correction.

Study on detection of soybean components in edible oil with ladder-shape melting temperature isothermal amplification (LMTIA) assay.

A ladder-shape melting temperature isothermal amplification (LMTIA) assay was established and used to detect soybean components in edible oils. LMTIA primers were designed with the sequence of the internal transcribed spacer (ITS) gene as the target, the reaction temperatures were optimized, the sensitivity was determined, and the suitability of the DNA extraction method for edible oil was assessed, with H2O and genomic DNA (gDNA) from corn, rapeseed, cottonseed, sesame, chili, chicken, pork, beef, and mutton as negative controls to test the false positives of the LMTIA assay. The established LMTIA assay gave a sensitivity of 1 pg at an optimal temperature of 57 °C. The Edible Oil DNA Extraction Kit was suitable for the LMTIA assay to detect soybean components in refined plant oil. No false positives occurred from all negative controls. This study successfully established the LMTIA assay for the detection of soybean ITS genes in edible oils, which could be used to detect soybean components in edible oils.

Design of Nanostraws in Amine-Functionalized MCM-41 for Improved Adsorption Capacity in Carbon Capture.

Polymeric amine encapsulation in high surface area MCM-41 particles for CO2 capture is well established but has the drawback of leaching out the water-soluble polymer upon exposure to aqueous environments. Alternatively, chemical (covalent) grafting amine functional groups from an alkoxysilane such as 3-aminopropyltriethoxysilane (APTES) on MCM-41 offer better stability against this drawback. However, the diffusional restriction exhibited by the narrow uniform MCM-41 pores (2-4 nm) may impede amine functionalization of the available silanol groups within the inner mesoporous core. This leads to incomplete amine functionalization and could reduce the CO2 adsorption capacity in such materials. Our concept to improve access to the MCM-41 interior is based on the incorporation of nanostraws with larger inner diameter (15-30 nm) to create a hierarchical porosity and enhance the molecular transport of APTES. Halloysite nanotubes (HNT) are used as tubular straws that are integrated into the MCM-41 matrix using an aerosol-assisted synthesis method. Characterization results show that the intrinsic structure of MCM-41 remains unaltered after the incorporation of the nanostraws and amine functionalization. At an optimal APTES loading of 0.5 g (X = 2.0), the amine-functionalized composite of MCM-41 with straws (APTES/M40H) has a 20% higher adsorption capacity than the amine-modified MCM-41 (APTES/MCM-41) adsorbent. Furthermore, the CO2 adsorption capacity APTES/M40H doubles that of APTES/MCM-41 when normalized based on the composition of MCM-41 in the composite particle with straws. The facile integration of nanostraws in MCM-41 leading to hierarchical porosities could be effective toward the mitigation of diffusional restriction in porous materials with potential for other catalytic and adsorption technologies.

Development of a Ladder-Shape Melting Temperature Isothermal Amplification Assay for Detection of Duck Adulteration in Beef.

ABSTRACT: Ladder-shape melting temperature isothermal amplification (LMTIA) is a newly developed technology, and the objective of this study was to establish its effectiveness for detection of duck adulteration in beef. LMTIA primers were designed with the prolactin receptor gene of Anas platyrhynchos as the target. The LMTIA reaction system was optimized, and its performance was compared with that of the loop-mediated isothermal amplification (LAMP) assay in terms of specificity, sensitivity, and limit of detection (LOD). Our results showed that the LMTIA assay was able to specifically detect 10 ng of genomic DNAs (gDNAs) of A. platyrhynchos, without detecting 10 ng of gDNAs of Bos taurus, Sus scrofa, Gallus gallus, Capra hircus, Felis catus, and Canis lupus familiaris. The sensitivity of the LMTIA assay was 1 ng of gDNAs of A. platyrhynchos; it was able to detect duck adulteration in beef with a 0.1% LOD. Although the LAMP assay could not clearly distinguish A. platyrhynchos from G. gallus, it had a sensitivity of 10 ng of gDNAs of A. platyrhynchos and a LOD of 1% duck adulteration in beef. This study may help facilitate the surveillance of commercial adulteration of beef with duck meat.

Development of a ladder-shape melting temperature isothermal amplification (LMTIA) assay for detection of African swine fever virus (ASFV).

BACKGROUND: Due to the unavailability of an effective vaccine or antiviral drug against the African swine fever virus (ASFV), rapid diagnosis methods are needed to prevent highly contagious African swine fever. OBJECTIVES: The objective of this study was to establish the ladder-shape melting temperature isothermal amplification (LMTIA) assay for the detection of ASFV. METHODS: LMTIA primers were designed with the p72 gene of ASFV as the target, and plasmid pUC57 was used to clone the gene. The LMTIA reaction system was optimized with the plasmid as the positive control, and the performance of the LMTIA assay was compared with that of the commercial real-time polymerase chain reaction (PCR) kit in terms of sensitivity and detection rate using 200 serum samples. RESULTS: Our results showed that the LMTIA assay could detect the 104 dilution of DNA extracted from the positive reference serum sample, which was the same as that of the commercial real-time PCR kit. The coincidence rate between the two assays was 100%. CONCLUSIONS: The LMTIA assay had high sensitivity, good detection, and simple operation. Thus, it is suitable for facilitating preliminary and cost-effective surveillance for the prevention and control of ASFV.

Inflammatory epithelial cytokines after in vitro respiratory syncytial viral infection are associated with reduced lung function.

Respiratory syncytial virus (RSV) infections in early life predispose children with cystic fibrosis (CF) to more severe lung function decline in later life. The mechanisms explaining the associations between RSV and progression of CF lung disease are not clear. In this study, a human bronchial epithelial cell line and primary human nasal epithelial cells (PNECs) from individuals with CF and healthy control donors were infected with RSV. Real-time PCR, plaque assay, cytokine detection, immunofluorescence and Western blot analyses were performed. RSV is replicated to a higher degree in CF epithelial cells as compared to control cells; however, no defects in innate immune pathways were identified in CF cells. Rather, primary p.Phe508del cystic fibrosis transmembrane conductance regulator PNECs produced more cytokines after RSV infection than control cells. Moreover, interleukin-8 and tumour necrosis factor-α production post RSV negatively correlated with lung function (% predicted forced expiratory volume in 1 s) in the individuals who donated the cells. These data suggest that CF epithelium has a dysfunctional response to RSV allowing for enhanced viral replication and an exaggerated inflammatory response that ultimately may predispose to greater airway inflammation and reduced lung function.