Structural basis of directional switching by the bacterial flagellum.

The bacterial flagellum is a macromolecular protein complex that harvests energy from uni-directional ion flow across the inner membrane to power bacterial swimming via rotation of the flagellar filament. Rotation is bi-directional, with binding of a cytoplasmic chemotactic response regulator controlling reversal, though the structural and mechanistic bases for rotational switching are not well understood. Here we present cryoelectron microscopy structures of intact Salmonella flagellar basal bodies (3.2-5.5 Å), including the cytoplasmic C-ring complexes required for power transmission, in both counter-clockwise and clockwise rotational conformations. These reveal 180° movements of both the N- and C-terminal domains of the FliG protein, which, when combined with a high-resolution cryoelectron microscopy structure of the MotA5B2 stator, show that the stator shifts from the outside to the inside of the C-ring. This enables rotational switching and reveals how uni-directional ion flow across the inner membrane is used to accomplish bi-directional rotation of the flagellum.

Combining deep learning and fluorescence imaging to automatically identify fecal contamination on meat carcasses.

Food safety and foodborne diseases are significant global public health concerns. Meat and poultry carcasses can be contaminated by pathogens like E. coli and salmonella, by contact with animal fecal matter and ingesta during slaughter and processing. Since fecal matter and ingesta can host these pathogens, detection, and excision of contaminated regions on meat surfaces is crucial. Fluorescence imaging has proven its potential for the detection of fecal residue but requires expertise to interpret. In order to be used by meat cutters without special training, automated detection is needed. This study used fluorescence imaging and deep learning algorithms to automatically detect and segment areas of fecal matter in carcass images using EfficientNet-B0 to determine which meat surface images showed fecal contamination and then U-Net to precisely segment the areas of contamination. The EfficientNet-B0 model achieved a 97.32% accuracy (precision 97.66%, recall 97.06%, specificity 97.59%, F-score 97.35%) for discriminating clean and contaminated areas on carcasses. U-Net segmented areas with fecal residue with an intersection over union (IoU) score of 89.34% (precision 92.95%, recall 95.84%, specificity 99.79%, F-score 94.37%, and AUC 99.54%). These results demonstrate that the combination of deep learning and fluorescence imaging techniques can improve food safety assurance by allowing the industry to use CSI-D fluorescence imaging to train employees in trimming carcasses as part of their Hazard Analysis Critical Control Point zero-tolerance plan.

Thermal inactivation of Salmonella during hard and soft cookies baking process.

This study validated a simulated commercial baking processes for hard and soft cookies to control Salmonella, and determined D- and z-values of 7-serotype Salmonella (Newport, Senftenberg, Tennessee, Typhimurium, and three isolates from dry pet food) cocktail in cookie doughs. Cookie doughs were prepared using flour mist-inoculated with the Salmonella cocktail. Hard and soft cookies were baked at 185 °C for 16 min and 165.6 °C for 22 min, respectively, followed by 30 min of ambient air cooling. D-values of the cocktail in cookie doughs were determined using thermal-death-time disks. Studies were designed as randomized complete blocks with three replications as blocks (α = 0.05). Salmonella populations decreased by > 5 log CFU/g in hard and soft cookies at 11.5 and 20.5 min of baking, respectively. Salmonella was not detected in hard cookies at the end of baking (as determined by enrichment), whereas in soft cookies, 0.6 log CFU/g Salmonella was present at the end of baking and cooling. Salmonella D-values in hard cookie dough at 60, 65 and 70 °C were 59.6, 28.1 and 11.9 min, respectively; while in soft cookie dough they were 62.3, 28.6 and 14.4 min, respectively. The Salmonella z-values in hard and soft cookie doughs were 14.5 and 15.8 °C, respectively.

Rapid identification of pathogens by using surface-enhanced Raman spectroscopy and multi-scale convolutional neural network.

Salmonella is a prevalent pathogen causing serious morbidity and mortality worldwide. There are over 2600 serovars of Salmonella. Among them, Salmonella Enteritidis, Salmonella Typhimurium, and Salmonella Paratyphi were reported to be the most common foodborne pathogenic serovars in the EU and China. In order to provide a more efficient approach to detect and distinguish these serovars, a new analytical method was developed by combining surface-enhanced Raman spectroscopy (SERS) with multi-scale convolutional neural network (CNN). We prepared 34-nm gold nanoparticles (AuNPs) as the label-free Raman substrate, measured 1854 SERS spectra of these three Salmonella serovars, and then proposed a multi-scale CNN model with three parallel CNNs to achieve multi-dimensional extraction of SERS spectral features. We observed the impact of the number of iterations and training samples on the recognition accuracy by changing the ratio of the number of the training and testing sets. By comparing the calculated data with experimental one, it was shown that our model could reach recognition accuracy more than 97%. These results indicate that it was not only feasible to combine SERS spectroscopy with multi-scale CNN for Salmonella serotype identification, but also for other pathogen species and serovar identifications.

Ohmic heating increases inactivation and morphological changes of Salmonella sp. and the formation of bioactive compounds in infant formula.

The effect of ohmic heating (OH) (50, 55, and 60 °C, 6 V/cm) on the inactivation kinetics (Weibull model) and morphological changes (scanning electron microscopy and flow cytometry) of Salmonella spp. in infant formula (IF) was evaluated. In addition, thermal load indicators (hydroxymethylfurfural and whey protein nitrogen index, HMF, and WPNI) and bioactive compounds (DPPH, total phenolics, ACE, α-amylase, and α-glucosidase inhibitory activities) were also studied. OH presented a more intense inactivation rate than conventional heating, resulting in a reduction of about 5 log CFU per mL at 60 °C in only 2.91 min, being also noted a greater cell membrane deformation, higher formation of bioactive compounds, and lower values for the thermal load parameters. Overall, OH contributed to retaining the nutritional value and improve food safety in IF processing.

Thermal inactivation kinetics of Salmonella and Enterococcus faecium NRRL B-2354 on dried basil leaves.

The enhanced heat resistance of Salmonella developed at low water activity makes it a serious challenge to eliminate them during thermal processing. The objectives of this research are to (i) investigate the effect of water activity on thermal inactivation of Salmonella cocktail (Agona, Tennessee, Mbandaka, Montevideo, and Reading) in dried basil leaves, and (ii) evaluate Enterococcus faecium NRRL B-2354 as an appropriate surrogate for Salmonella in dried basil leaves. Dried basil leaves, inoculated with a Salmonella cocktail and E. faecium separately, were equilibrated to different water activities (aw: 0.40, 0.55, and 0.70) in a humidity-controlled chamber. The basil samples were packed (1.6 ± 0.1 g) in aluminum pouches and thermally treated at 70, 75, and 80 °C using a dry heating method for 0-180 min to obtain the thermal death curve. The microbial survival data was fit using two primary models (Log-linear and Weibull model). Results from AICc showed that the log-linear model fits well for thermal inactivation of both microorganisms. As the aw decreases from 0.70 to 0.40 at 75 °C, the D-value increases from 3.30 to 9.14 min for Salmonella and 6.53 to 14.07 min for E. faecium. Based on the AICc values, the modified Bigelow model fits the D-values better than the response surface model for both the microorganisms. The kill ratio of surrogate to pathogen ranged from 1.4 to 2.8, indicating that it is a conservative surrogate for Salmonella for performing validation of the thermal pasteurization process. The identification of suitable surrogate and development of modified Bigelow model will help the spice industry in developing the thermal processes for improving the safety of basil leaves.

Salmonella survival after exposure to heat in a model meat juice system.

The effect of heat against eleven Salmonella strains in model meat juices was examined. Juices from beef, lamb and goat were made from either the fatty layer (FL), muscle (M) or a mixture of both (FLM). The pH of each FLM sample was altered to match the pH of PBS and vice versa to determine the pH effect on the survival of Salmonella against the effect of heat. Salmonella were exposed to either gradual heating to 70 °C in FLM, M and FL or heat shock at 70 °C for 5 min in FLM. Fat, fatty acid profile and iron content of the juices were determined. Gradual heat treatment significantly (p ≤ 0.05) reduced Salmonella as compared to the untreated controls (~1.92-7.61 log CFU ml-1) while heat shock significantly (p ≤ 0.05) reduced Salmonella as compared to the untreated controls (~5.80-7.36 log CFU ml-1). Survival of Salmonella was higher in lamb juices than other juices. The fat content in lamb FL (3.25%) was significantly higher (p ≤ 0.05) than beef (1.30%) and goat FL (1.42%). Iron content in lamb FLM (~127 mg kg-1) was significantly (p ≤ 0.05) lower than beef (~233 mg kg-1) and goat FLM (~210 mg kg-1). The omega 6 and linoleic acid content in goat FLM (~36.0% and ~34.4%) was significantly higher (p ≤ 0.05) than beef (~29.1% and ~27.1%). Fat, fatty acids and iron may differentially protect Salmonella against the effect of heat in these juices.

Mobilization of Tn1721-like structure harboring blaCTX-M-27 between P1-like bacteriophage in Salmonella and plasmids in Escherichia coli in China.

The aim of this study was to explore the characteristics of blaCTX-M-27 carriage and mobilization in Salmonella and Escherichia coli isolates from food-producing animals in China. A total of 2280 E. coli and 229 Salmonella isolates collected from food animals from June 2003 to September 2014 were screened for the presence of blaCTX-M-27 gene. The blaCTX-M-27-positive isolates were typed and plasmid DNA sequenced to determine the genetic context of blaCTX-M-27 and plasmid types present. Bacterial fitness was evaluated by growth curve and plasmid stability in vitro. CTX-M-27-positive E. coli (18, 0.79 %) and Salmonella (34, 14.85 %) were detected. PFGE profiles of CTX-M-27-positive strains revealed a wide variety of genotypes and S. Indiana was the most prevalent serotype. Replicon typing, S1-PFGE and hybridization of CTX-M-27-carrying plasmids confirmed that blaCTX-M-27 gene was located on IncFII (12/18), IncN (4/18), and non-typeable (2/18) plasmids in E. coli and on P1-like bacteriophage (21/34), IncP (4/34), IncFIB (4/34), IncN (2/34), IncHI2 (2/34), and IncA/C (1/34) plasmids in Salmonella. Comparison and analysis of gene context of blaCTX-M-27 in P1-like bacteriophage and plasmids revealed they shared the same structure and contained an identical genetic context with the Tn1721-like structure ΔISEcp1B-blaCTX-M-27-IS903D-iroN-Δmap-Tn1721. In addition, plasmid stability tests indicated that the blaCTX-M-27 P1-like bacteriophage were more stable than plasmids in the absence of cefotaxime selective pressure. These results demonstrate that Tn1721-like transposons harboring CTX-M-27 could be mobilized between different plasmids in E. coli and P1-like bacteriophage disseminated among Salmonella.

Designing an Outer Membrane Protein (Omp-W) Based Vaccine for Immunization against Vibrio and Salmonella: An in silico Approach.

BACKGROUND: Cholera triggered by Vibrio cholerae remains the main reason for morbidity and mortality all over the world. In addition, salmonellosis is regarded as an infectious disease that makes it essential for the identification and detection of Salmonella. With a beta-barrel structure consisting of eight non-parallel beta strands, OmpW family is widely distributed among gram-negative bacteria. Moreover, OmpW isolated from S. typhimurium and Vibrio cholerae can be used in vaccine design. METHODS: Topology prediction was determined. T-cell and B-cell epitopes were selected from exposed areas, and sequence conservancy was evaluated. The remaining loops and inaccessible residues were removed to prepare OmpW-1. High antigenicity peptides were detected to replace inappropriate residues to obtain OmpW-2. Physicochemical properties were assessed, and antigenicity, hydrophobicity, flexibility, and accessibility were compared to the native Omp-W structure. Low score areas were removed from the designed structure for preparing the OmpW-3. To construct OmpW-4, TTFrC was used as T-CD4+ cell-stimulating factor and CTB as adjuvant to the end of the C-terminal of this sequence, which can increase the antigenicity and sequence density. The sequences were re-analyzed to delete the unfavorable residues. Besides, the solubility of the mature OmpW and the designed structure were predicted while overexpressed in E. coli. RESULTS: The designed vaccine is a stable protein that has immune cells recognizing epitopes and is considered as an antigen. The construct can be overexpressed in an E. coli. CONCLUSION: The multi-epitope vaccine is a suitable stimulator for the immune system and would be a candidate for experimental research. Recent patents describe numerous inventions related to the clinical facets of vaccine peptide against human infectious disease.

Cell blocking: An enhancement of foodborne pathogen detection by fluorescent signals of recombinant yeasts.

Cell blocking (CB) technique has been widely applied in many studies since the last century. In our research, this technique was mostly used to study the enhancement of the vacuolar response-based system that could detect Shigella sp. and Salmonella sp. investigated in previous studies. The recombinant yeast cells were blocked by mixing with agarose gel on a 96-wells plate, then storing this plate in -80 °C before using. The optimal conditions for the new system, such as agarose concentration, maximum storage time, were also established. Finally, the efficiency of the vacuolar response-based system was improved, and this system could be used as a portable detector for the foodborne pathogen.

In vivo assessment of pathogens toxicity on Daphnia magna using fluorescent dye staining.

Daphnia has been widely used as an indicator species in aquatic biomonitoring for decades. Traditional toxicity assays based on lethality take a long time to assess, and the effect mode of contaminants is not clear. Because of the translucency of the Daphnia body and the application of fluorescent probes in cell staining, different intoxicated parts can be visualized. In this study, a double-staining method using two fluorescent dyes, Calcein AM (cell-permeant dye) and Propidium Iodide (cell-impermeant dye), was carried out on Daphnia magna exposed to six pathogens: Salmonella spp. (four strains) and Shigella spp. (two strains). The results showed that those bacteria caused different infections on daphnia depending on the age of this organism and bacterial concentrations. In detail, S. dublin and S. sonnei are the most harmful to Daphnia when they cause damage at smaller concentrations at the younger stage (3 weeks old). Interestingly, older Daphnia can give responses to nearly 10 CFU/ml to less than 100 CFU/ml of some bacteria strains. In another experiment, S. sonnei disturbed Daphnia after just 10 min of exposure, and Daphnia adapted to S. choleraesuis, S. typhi, and S. flexneri at the early stage (3 weeks old) after 1 h of exposure. Moreover, the damaged areas of the daphnia body were directly observed via a microscope, contributing to the understanding and the prediction of toxicity mechanisms.

A Hybrid Eukaryotic-Prokaryotic Nanoplatform with Photothermal Modality for Enhanced Antitumor Vaccination.

Cytomembrane-derived nanoplatforms are an effective biomimetic strategy in cancer therapy. To improve their functionality and expandability for enhanced vaccination, a eukaryotic-prokaryotic vesicle (EPV) nanoplatform is designed and constructed by fusing melanoma cytomembrane vesicles (CMVs) and attenuated Salmonella outer membrane vesicles (OMVs). Inheriting the virtues of the parent components, the EPV integrates melanoma antigens with natural adjuvants for robust immunotherapy and can be readily functionalized with complementary therapeutics. In vivo prophylactic testing reveals that the EPV nanoformulation can be utilized as a prevention vaccine to stimulate the immune system and trigger the antitumor immune response, combating tumorigenesis. In the melanoma model, the poly(lactic-co-glycolic acid)-indocyanine green (ICG) moiety (PI)-implanted EPV (PI@EPV) in conjunction with localized photothermal therapy with durable immune inhibition shows synergetic antitumor effects as a therapeutic vaccine. The eukaryotic-prokaryotic fusion strategy provides new perspectives for the design of tumor-immunogenic, self-adjuvanting, and expandable vaccine platforms.

Functional protein shells fabricated from the self-assembling protein sheets of prokaryotic organelles.

Fabricating protein compartments from protein units is challenging and limited by the use of external stimuli and crosslinkers. Here we explore the fabrication of all-protein compartments using self-assembled proteins of prokaryotic organelles. These proteins have intrinsic interacting domains which are ionic in nature, and spontaneously self-assemble into sheets when over-expressed. Using a one-step approach, we maneuvered the formation of the protein shells from the sheets without any external stimuli or crosslinker. The spontaneous self-assembly of the native protein sheets into protein shells not only preserves the native functional properties of the protein but also enhances their thermal stability compared to the sheets. We further demonstrate that these compartments can encapsulate macromolecular enzymes and, more interestingly, permit the free exchange of small molecules and substrates through their intrinsic conduit channels. The porous nature of the shell housing active enzymes and allowing movement of small molecules makes them suitable as active bioreactors. Furthermore, to extend the tunability of these protein-compartments with respect to stability, enzyme-encapsulation, and permeability, we fabricated three different compartments using three different sheet proteins, PduA/B/B' and compared their properties. Interestingly we find that all three protein shells show similar behaviour with respect to an encapsulated diol-dehydratase enzyme and vitamin B12, which are native to the Pdu BMC system. Furthermore, for the non-native enzyme CytC, the small molecule R6G dye, doxorubicin, NR and curcumin they behave diversely. Insights from this analysis will allow us to design and develop sheet protein based synthetic active bioreactors requiring meticulous, compartmentalization in process optimization.

Combination of attenuated Salmonella carrying PD-1 siRNA with nifuroxazide for colon cancer therapy.

Colon cancer is a member of malignant tumors in the digestive system. Traditional treatment strategies are ineffective and improving the treatment of colon cancer is an urgent need. Targeting programmed cell death-1 (PD-1) by monoclonal antibodies has shown some therapeutic effectiveness and has advantages. Additionally, the Stat3 inhibitor nifuroxazide was employed to promote the antitumor activity. Here, we hypothesized that combining nifuroxazide with PD-1 small interfering RNA carried by attenuated Salmonella would exert a synergistic antitumor effect on colon cancer. Indeed, treatment with this combination effectively inhibited the development of colon cancer in mice and improved the survival rate. These two novel anticancer agents worked synergistically to elicit potent antitumor immunity and achieve improved therapeutic efficacy. The underlying mechanisms are mainly involved with immune regulation and cell apoptosis. This study provides a previous framework for combining this Stat3 inhibitor with RNAi designed to block immune checkpoint signaling for cancer therapy.

Artificial neural network-assisted Fourier transform infrared spectroscopy for differentiation of Salmonella serogroups and its application on epidemiological tracing of Salmonella Bovismorbificans outbreak isolates from fresh sprouts.

Salmonellae represent one of the most common bacterial infection reagents in both humans and animals. For detection and epidemiological elucidation of Salmonella infections, determination of Salmonella serotypes and differentiation between different Salmonella isolates is crucial. In the first part of this study, Artificial Neural Network (ANN)-assisted Fourier transform infrared (FTIR) spectroscopy was used to establish a method for subtyping Salmonella isolates according to their serogroups. For this, 290 Salmonella strains from 35 different serogroups were used to establish an ANN for differentiation between infrared spectra of 10 different Salmonella serogroups (B, C1, C2-C3, D1/D2, E1, E4, F, G, H, O:55) vs. the remaining serogroups. In the final ANN, sensitivity values ranged between 90 and 100% for most of the 10 serogroups under investigation. In the second part of this study, ANN-assisted FTIR spectroscopy was applied for epidemiological distinction of Salmonella Bovismorbificans outbreak isolates from fresh sprouts vs. isolates from other sources. Four Salmonella Bovismorbificans isolates from human and food origin in the context of a Southern German outbreak were successfully discriminated from other S. Bovismorbificans isolates from various sources. ANN-assisted FTIR spectroscopy is thus an effective tool for discrimination of Salmonella isolates at or even below serogroup level.

Influence of autolyzed whole yeast and yeast components on broiler chickens challenged with salmonella lipopolysaccharide.

The objective of this study was to assess the effect of dietary yeast products on broiler chickens challenged with salmonella lipopolysaccharide (LPS). The chicks were divided into 8 treatments with 6 replicates and 9 birds per replicate. The treatments consisted of a positive control (PC) [without supplementation and not challenged]; negative control (NC) [without supplementation but challenged]; whole yeast and challenged; yeast cell wall and challenged; yeast glucan and challenged; yeast mannan and challenged; zinc bacitracin and challenged; and Salinomycin and challenged. Whole yeast or Yeast cell wall was included at 2.0 g/kg diet. Yeast glucan or mannan was added at 0.20 g/kg diet. Zinc bacitracin (ZNB) and Salinomycin (SAL) was included at 50 and 60 ppm, respectively. Dietary treatments had no effect (P > 0.05) on feed intake (FI) at day 10. Supplementation with yeast and its derivatives improved (P < 0.05) body weight gain (BWG) and feed conversion ratio (FCR) on day 10. On days 24 and 35, LPS challenge declined FI, BWG, FCR, and flock uniformity (day 28) in the NC group compared to the PC group. Yeast products and antibiotics improved (P < 0.05) FI, BWG, FCR, and flock uniformity in LPS-challenged birds. On day 24, spleen weight increased while bursa weight decreased in the NC group relative to the PC group; this effect was reversed (P < 0.05) by feeding all yeasts and antibiotics. On day 24, application of all the dietary treatments ameliorated the changes observed in white blood cell, lymphocyte and monocyte counts as well as albumin and immunoglobulin G of NC birds. On day 35, all yeasts additives, ZNB and SAL improved (P < 0.05) the meat yield of broilers challenged with LPS. In conclusion, supplementation of diets with yeast and its derivatives can ameliorate the negative effects of salmonella LPS challenge on broiler chicks, thus improving the performance, flock uniformity, and meat yield.

Evaluation of Salmonella bongori derived biosurfactants and its extracellular protein separation by SDS-PAGE using petridishes: A simply modified approach.

Presently, through the preliminary screening assays, the Salmonella bongori BH11 was found to be an effective biosurfactants (BSFs) producer. The secreted BSFs were extracted using methanol: chloroform and characterized through FTIR, TLC, HPLC and GCMS analyses. Further, the extracellular protein was extracted (TCA/acetone method), estimated (Lowry's method) and separated (standard and modified SDS-PAGE). Through the obtained characteristic FTIR peaks (1107.09cm-1), its content was presumed to be glycolipids and as rhamnose/rhamnolipids through the TLC-Rf value. GCMS revealed 6 compounds, in which Toluene (32%) and 5-(2-Thienyl) pentanoic acid (23%) are the major ones. The crude BSFs exhibited preponderant antibacterial effects on Staphylococcus aureus and Serratia marcescens. Also, it inhibited the biofilm formation of S. aureus, Pseudomonas aeruginosa, P. fluorescens and S. marcescens. Considerably, 76% mortality of IV instar larvae of Culex quinquefasciatus was recorded from BSFs, when compared to SDS. The presently followed protein separation technique using two petridishes might attract the attention of the researchers, as it would emerge as a standard procedure in future. This is the first report on the screening of BSFs from Salmonella bongori that showed antagonistic property, larvicidal potentials and the presently followed modified SDS-PAGE protein separation technique is a simple, reliable and cost effective one.

Architecture of the Bacterial Flagellar Distal Rod and Hook of Salmonella.

The bacterial flagellum is a large molecular complex composed of thousands of protein subunits for motility. The filamentous part of the flagellum, which is called the axial structure, consists of the filament, the hook, and the rods, with other minor components-the cap protein and the hook associated proteins. They share a common basic architecture of subunit arrangement, but each part shows quite distinct mechanical properties to achieve its specific function. The distal rod and the hook are helical assemblies of a single protein, FlgG and FlgE, respectively. They show a significant sequence similarity but have distinct mechanical characteristics. The rod is a rigid, straight cylinder, whereas the hook is a curved tube with high bending flexibility. Here, we report a structural model of the rod constructed by using the crystal structure of a core fragment of FlgG with a density map obtained previously by electron cryomicroscopy. Our structural model suggests that a segment called L-stretch plays a key role in achieving the distinct mechanical properties of the rod using a structurally similar component protein to that of the hook.

Relationships of Water Activity and Moisture Content to the Thermal Inactivation Kinetics of Salmonella in Low-Moisture Foods.

HIGHLIGHTS: Water affects thermal inactivation kinetics of Salmonella in low-moisture foods. Water activity and moisture content are both feasible predictors of heat resistance. Sorption state of food materials may affect Salmonella heat resistance.

Sensitivity Enhancement of Nucleic Acid Lateral Flow Assays through a Physical-Chemical Coupling Method: Dissoluble Saline Barriers.

Nucleic acid lateral flow assays (NALFAs) have attracted much attention due to their rapid, robust, simple, and cost-effective features. However, the current NALFAs are still limited by low sensitivity because of the poor understanding and control of the underlying complex flow and reaction processes. Although enormous efforts have been devoted to enhancing detection sensitivity of NALFAs, developing simple NALFAs with high sensitivity remains difficult. Thus, we proposed a novel physical-chemical coupling method using dissoluble saline barriers and developed the corresponding mathematical model to better understand the underlying processes to enhance the NALFA sensitivity. Through optimizing the design parameters (e.g., saline barriers patterns, volume, and concentrations) experimentally and numerically, we achieved the highest 10-fold sensitivity enhancement for detection of nucleic acids (including HBV, Staphylococcus aureus, and salmonella as model targets) using this method. The physical-chemical coupling method offers a facile strategy for developing highly sensitive NALFAs.