H7 In Milk Samples Research Articles

Sensitive Detection of Escherichia coli O157:H7 Using Allosteric Probe and Hairpin Switches-Based Isothermal Transcription Amplification.

Pathogens pose a serious threat to public and population health, leading to serious outbreak and spread of diseases irrespective of the region. The capability to directly, sensitively, and specifically detect viable pathogens in low numbers in food and clinical samples is very desirable but remains a challenge. In this work, we present a novel assay of a combination of an aptamer-based allosteric probe and hairpin switch-controlled T7 RNA polymerase-based isothermal transcription amplification, which enables rapid, ultrasensitive, label-free detection of direct pathogens. It can detect Escherichia coli as low as 73.2 CFU/mL. Moreover, with the usage of the proposed assay, sensitive quantification of E. coliO157:H7 in milk samples has been achieved, showing significant potential as a simple and sensitive tool to quantify pathogens in milk and other foods.

Label-free and sensitive fluorescent detection of Escherichia coli O157: H7 in milk based on cascade strand displacement amplification and G-quadruplex-thioflavin T

The development of a simple, rapid, sensitive, and accurate method for detecting Escherichia coli O157:H7 in complex matrices is critical for ensuring food safety. In this study, a fluorescent biosensor employing aptamer (Apt) -functionalized magnetic beads and cascade strand displacement amplification (SDA) was devised for the detection of E. coli O157:H7 in milk samples. E. coli O157:H7 is captured by the aptamer, causing the complementary DNA (cDNA) to not bind to the aptamer and to be released into the supernatant. Following magnetic separation, the cDNA present in the supernatant initiates the opening of the hairpin (HP) structures. This enables primers to attach to the opened HP, triggering the initial SDA process, which generates a substantial quantity of double-stranded DNA (dsDNA) and recycles cDNA to open more HP structures. The dsDNA activates a second SDA, producing single-stranded DNA (ssDNA) for template binding and instigating a third SDA process. Triple SDA leads to the production of numerous G-quadruplex sequences and the formation of a G-quadruplex structure in the presence of K+. The inclusion of ThT into the G-quadruplex significantly enhances fluorescence. Under optimal conditions, this approach can detect E. coli O157:H7 at levels as low as 101 CFU/mL in pure cultures within 2.5 h, with a broad linear detection range of 101–106 CFU/mL. This method exhibits remarkable specificity towards the target bacteria over non-target bacteria. Furthermore, the technique has been effectively utilized for the detection of E. coli O157:H7 in milk samples. Notably, the method involves only two main steps, identification and amplification, significantly reducing contamination risks and minimizing operational steps.

An engineered antimicrobial peptide as an alternative bioreceptor for the detection of pathogenic Escherichia coli O157:H7

Escherichia coli O157:H7, which can cause acute infections even in extremely small numbers, is the most common pathogen responsible for foodborne illnesses. Both the development of antimicrobial agents and establishment of a sensitive and rapid detection method are essential to prevent foodborne illnesses. In this study, three antimicrobial peptides (AMPs) were derived from magainin 1 and clavanin A through peptide engineering. Subsequently, AMP 3, which showed the highest antimicrobial activity against E. coli O157:H7 in antibacterial activity assays, was screened as a potential candidate bioreceptor. To achieve sensitive and selective detection, a hydrophilic tannic acid (TA)-polyethyleneimine (PEI) hydrogel was modified on a gold electrode, and AMP 3 was immobilized to develop an AMP-based electrochemical sensor. The electrochemical performance of the Au/TA-PEI/AMP 3-based electrochemical biosensor was monitored using cyclic voltammetry and differential pulse voltammetry. The limit of detection of the Au/TA-PEI/AMP 3 biosensor was determined to be 3.4 colony-forming units (CFU)/mL, with a linear detection range of 101–105 CFU/mL. In addition, the biosensor was applied to quantitatively detect E. coli O157:H7 in milk samples, achieving excellent recovery rates ranging from 89.1 to 122.0 %. Overall, the developed AMP-based biosensor exhibits a good selectivity and sensitivity in the detection of E. coli O157:H7; these features render it a potential tool for detecting bacteria that pose a threat to public health and safety.

G-triplex/hemin DNAzyme mediated colorimetric aptasensor for Escherichia coli O157:H7 detection based on exonuclease III-assisted amplification and aptamers-functionalized magnetic beads

Escherichia coli O157: H7 (E. coli O157: H7) is one of the most common foodborne pathogens and is widespread in food and the environment. Thus, it is significant for rapidly detecting E. coli O157: H7. In this study, a colorimetric aptasensor based on aptamer-functionalized magnetic beads, exonuclease III (Exo III), and G-triplex/hemin was proposed for the detection of E. coli O157: H7. The functional hairpin HP was designed in the system, which includes two parts of a stem containing the G-triplex sequence and a tail complementary to cDNA. E. coli O157: H7 competed to bind the aptamer (Apt) in the Apt-cDNA complex to obtain cDNA. The cDNA then bound to the tail of HP to trigger Exo III digestion and release the single-stranded DNA containing the G-triplex sequence. G-triplex/hemin DNAzyme could catalyze TMB to produce visible color changes and detectable absorbance signals in the presence of H2O2. Based on the optimal conditions, E. coli O157: H7 could be detected down to 1.3 × 103 CFU/mL, with a wide linear range from 1.3 × 103 to 1.3 × 107 CFU/mL. This method had a distinguished ability to non-target bacteria, which showed good specificity. In addition, the system was successfully applied to detect E. coli O157: H7 in milk samples.

Sensitive fluorescence ELISA with streptavidin scaffolded DNA tetrads for the detection of Escherichia coli O157:H7

Escherichia coli O157:H7 poses a threat to humans. Traditional ELISA is not a sensitive method for the detection of E. coli O157:H7. Here, an efficient method was designed for improving the load capacity of alkaline phosphatase (ALP) with streptavidin scaffolded DNA tetrad (SS-DNAt). With more ALP, more ascorbic acid 2-phosphate was catalyzed to ascorbic acid that was used to synthesize fluorescence poly adenine-thymine-templated copper nanoclusters. Based on SS-DNAt, fluorescence ELISA was successfully proposed for improving the sensitivity for detection of E. coli O157:H7 in milk samples. The method showed a linear range of 104 to 106 cfu/mL. The limit of detection of fluorescence ELISA was 3.75 × 103 cfu/mL and 6.16-fold better than that of traditional ELISA. The recovery of the fluorescence ELISA was 86.7 to 93.6% with the coefficient of variation of 5.6 to 10.5% in milk. This method could be used to detect hazardous material in food.

Prevalence and Susceptibility Profile of E. Coli O157:H7 Isolated from Raw Milk in Kohat, Pakistan

Diseases development due to food is a major threat to consumers; especially in countries where sanitation facilities are poor. E. coli O157:H7 strain causes major outbreaks among foodborne diseases. The current work investigated the prevalence of E. coli O157:H7 and their antibiogram profile in raw milk samples. A total of 180 samples were collected from milk shops in Kohat, Pakistan. The isolates were initially identified on colony morphology on different Media and later through biochemical tests. Isolates were also screened to differentiate E. coli O157:H7 from other fecal E. coli via sorbitol MaCconkey agar. Out of 180 samples 134 (74.4%) milk samples were positive for E. coli and 46 (25.6%) milk samples were negative. Antibiotic susceptibility test was performed by using antibiotics of different classes. Most of the isolates showed susceptibility to ciprofloxacin (82.5%) (80.4%), gentamicin (82.5%) (80.5%) and Oxytetracycline (85.5%) (85.6%) respectively and slightly resistant to Tetracycline (55.8%) (48.2%), Imepenam (52.6%) (42.8%), Ceftriaxone (45.5%) (40.6%), Trimethoprim (54.4%) (36.6%), Chloramphenicol (38.6%) (30.5%), and shows resistance to Amikacin (25.5%) (18.4%) and Cloxacillin (10.5%) (4.5%) respectively. The high frequency of E. coli O157:H7 in milk samples indicate poor hygiene during milking, transportation and milk storage. The awareness against spreading of this pathogen is necessary among stakeholders at different levels of milk handling. Keywords: E. coli O157:H7; Antibiotic resistance, Milk

Sandwich immunoassay based on antimicrobial peptide-mediated nanocomposite pair for determination of Escherichia coli O157:H7 using personal glucose meter as readout.

A sandwich immunoassay was developed for determination of E. coli O157:H7. This is based on an antimicrobial peptide-mediated nanocomposite pair and uses a personal glucose meter as signal readout. The antimicrobial peptides, magainins I, and cecropin P1 were employed as recognition molecules for the nanocomposite pair, respectively. With a one-step process, copper phosphate nanocomposites embedded by magainins I and Fe3O4 were used as "capturing" probes for bacterial magnetic isolation, and calcium phosphate nanocomplexes composed of cecropin P1 and invertase were used as signal tags. After magnetic separation, the invertase of the signal tags hydrolyzed sucrose to glucose, thereby converting E. coli O157:H7 levels to glucose levels. This lattercan be quantified by a personal glucose meter. Under optimal conditions, the concentration of E. coli O157:H7 can be determined in a linear range of 10 to 107CFU·mL-1 with a detection limit of 10CFU·mL-1. The method was successfully applied to the determination of E. coli O157:H7 in milk samples. Graphical abstract Schematic representation of sandwich immunoassay for E. coli O157:H7. One-pot synthetic of Fe3O4-magainins I nanocomposites (MMP) were used for magnetic capture. Cecropin P1-invertase nanocomposites (PIP) were used as signal tags. A personal glucose meter was used as readout to determine the target.

A quantitative foam immunoassay for detection of Escherichia coli O157:H7 based on bimetallic nanocatalyst‑gold platinum

Inspired by the very popular demonstration, Elephant's Toothpaste, we developed a cost-effective, portable foam immunoassay for quantitative detection of Escherichia coli O157:H7 (E. coli O157:H7) by measuring foam volume changes using a low-cost ruler. This quantitative immunoassay for E. coli O157:H7 is built by integrating a catalyzed foam-generation reaction with pathogen recognition component. E. coli O157:H7 was enriched and captured by monoclonal antibody to E. coli O157:H7 (mAb1) modified Fe3O4 magnetic nanoparticles (MNP). The peroxidase-like nanozymes gold and platinum nanoparticles (Au@Pt NP) were loaded on silica nanoparticle (SiO2 NP), forming Au@Pt/SiO2 NP. The Au@Pt/SiO2 NP were functionalized by antibody against E. coli O157:H7 (mAb2-Au@Pt/SiO2 NP) and used as signal labels. In the final sandwich immune complex, Au@Pt NP catalyzes the hydrolysis of hydrogen peroxide (H2O2) to produce oxygen (O2). In the presence of sodium dodecyl sulfate (SDS), O2 is trapped by SDS forming thick foam. The foam rises in an acrylic tube and produces sensitive height readout. There is a linear relationship between the foam height and the logarithm of E. coli O157:H7 concentration. Under the optimum conditions, the detection limit was 2.16 × 102 CFU·mL−1 (3σ) in the range of 1.19 × 103–1.19 × 107 CFU·mL−1. This method demonstrates good practical application potential during detection of E. coli O157:H7 in milk samples. This new signaling strategy opens up a new way for portable, simple and sensitive on-site analysis in various environments.

An electrochemical immunobiosensor for ultrasensitive detection of Escherichia coli O157:H7 using CdS quantum dots-encapsulated metal-organic frameworks as signal-amplifying tags

We report here cadmium sulfide quantum dots (CdS QDs)-encapsulated metal-organic frameworks as signal-amplifying tags for ultrasensitive electrochemical detection of Escherichia coli O157:H7 (E. coli O157:H7). CdS QDs were encapsulated in zeolitic imidazolate framework-8 (ZIF-8) to form CdS@ZIF-8 muti-core-shell particles by in situ growth of ZIF-8 in the presence of CdS QDs. To specifically recognize E. coli O157:H7 cells, CdS@ZIF-8 particles were coated with polyethyleneimine to introduce amino groups on their surfaces, followed by surface modification of anti-E. coli O157:H7 antibody. A sandwich-type electrochemical immunobiosensor for the detection of E. coli O157:H7 was fabricated using CdS@ZIF-8 particles as signal tags. Cd(II) ions were released from CdS@ZIF-8 tags by HCl leaching, enabling the detection of E. coli O157:H7 by differential pulse voltammetry. Under the optimized conditions, the linear range of the biosensor is from 10 to 108 colony forming units (CFU) per mL for E. coli O157:H7 detection, with the detection limit of 3 CFU mL−1 (S/N = 3). The sensitivity of the biosensor for E. coli O157:H7 detection using CdS@ZIF-8 particles as signal tags is 16 times that of a biosensor using CdS QDs as signal tags, because the number of CdS QDs labeled to each bacterial cell increases greatly resulting from a great number of CdS QDs encapsulated in each CdS@ZIF-8 label. This method was successfully used to detect E. coli O157:H7 in milk samples.

Multicolour digital image analysis system for identification of bacteria and concurrent assessment of their respiratory activity

To develop a rapid and simple multicolour digital image analysis system for simultaneous identification of bacteria and assessment of their metabolic activity. We developed an image analyser capable of distinguishing triple-stained bacterial cells. Bacteria were stained with a nucleic acid stain, a fluorescent antibody and a fluorescent metabolic indicator for enumeration, species identification and assessment of metabolic activity. This multicolour image analyser was used to simultaneously identify Escherichia coli O157:H7 in milk samples and assess their respiratory activity. The images of the triple-stained bacteria were captured using a combination of blue light and u.v. excitation and an epifluorescence microscope and were processed by our image analyser. We found a good correlation between the counts of actively respiring (r = 0.93) and total (r = 0.94) E. coli O157:H7 measured by digital image analysis and visual observation. The multicolour digital image analysis system described here was able to quantify active pathogenic micro-organisms within 2 h. This multicolour image analysis allows the rapid and simultaneous quantification of bacteria, identification of species and assessment of metabolic activity.