H7 In Milk Research Articles

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.

Engineering of a Chimeric Template Triggers RNase H-Based Isothermal Amplification Approach for Sensitive Detection of Pathogen RNA.

RNA-based detection of pathogenic organisms is an emerging field of research that is crucial for disease diagnosis and environmental and food safety. By rationally engineering an RNA-DNA tandem (RDT) structural template, we proposed a novel RNase H-based isothermal exponential amplification (RH-IEA) reaction to rapidly identify long-stranded RNA. In this strategy, the rigid and compact RDT template selectively recognized the target RNA and formed a stable hybrid with it. Upon site-specific cleavage of RNase H, the 3' overhang of the target RNA was cut off, and a free hydroxyl end at the hydrolysis site was generated to trigger an exponential amplification reaction (EXPAR). This method maintained the high efficiency and rapid amplification kinetics of EXPAR. As a result, the RH-IEA strategy was able to sensitively and specifically detect the characteristic sequence of Escherichia coli O157:H7 RNA, with a detection sensitivity of 1 fg/μL. Besides, the RDT template can be used as an RNA protector to prevent specific segments of the target RNA from being degraded by RNase enzymes, allowing the sample to be stored at room temperature for a long time. With this advantage, the practicality of RH-IEA will be more flexible than the reverse transcription polymerase chain reaction. It was successfully applied in the identification of E. coli O157:H7 in milk with a minimum detection concentration of 1.0 × 102 CFU/mL. Therefore, the RH-IEA method will serve as a powerful tool for detecting long-stranded RNA and will also shed light on the pathogen detection in food safety and molecular diagnosis.

An esterase activity-based biosensor for rapid and sensitive detection of viable Escherichia coli O157:H7 in milk

Escherichia coli O157:H7 is harmful to humans by producing toxins that cause thrombotic thrombocytopenic purpura and haemolytic uraemic syndrome. Real-time recombinase-aided amplification (rRAA) can generate real-time fluorescence signals to achieve sensitive detection of pathogens within 20 min but cannot distinguish dead bacteria from viable bacteria, leading to false-positive results. Thiazole orange monoazide (TOMA) is an activity-labile nucleic acid-modifying compound, permeating into bacterial cells and, in dead cells, covalently linking to genomic DNA to suppress DNA amplification. In viable cells, however, TOMA is cleaved by active esterase and so inhibition of DNA amplification would not occur. A rRAA assay integrated with TOMA was successfully developed potentially enabling rapid and sensitive identification of viable E. coli O157:H7 in milk. The limit of detection was less than 10 cfu mL−1 in both pure culture and skimmed milk. Moreover, this assay could resist interference from non-target bacteria and high concentrations of dead bacteria.

Multivalent aptamer meshed open pore membrane and signal amplification for high-flux and ultra sensitive whole cell detection of E. coli O157:H7 in complex food matrices

We develop a multivalent aptamer meshed open pore membrane with a signal amplification method as a simple and robust platform for sensitive, robust, and high-flux detection of E. coli O157:H7 whole cells in food samples. Nylon open pore membranes are surface modified by multi-handled poly(amidoamine) dendrimers as templates that are subsequently functionalized with multiple branches of capturing RCA (cRCA). The resulting cRCA networks are composed of tandem repeating aptamers that can specifically recognize and capture E. coli O157:H7 cells. The captured cells are then conjugated with signaling RCA (sRCA) to amplify the fluorescent detection signal. Our results demonstrate that the cRCA-based aptamer meshwork significantly enhances the surface-capturing performance of E. coli O157:H7 cells while achieving high-fluxes. Moreover, the membrane surface modification using dendrimers renders the resulting membrane nonfouling thus significantly reducing the background noises by approximately 96 times while the sRCA signal intensification enhances the fluorescent detection signals by approximately 27 times. We show that the membranes exhibit excellent performances in detecting E.coli O157:H7 in milk and orange juice samples with limit of detection of 10 cells per 250 mL with excellent specificity, offering a robust and practical approach for rapid and sensitive detection of foodborne bacteria.

Contamination of Escherichia coli O157:H7 in Milk and Its Dairy Products in Depok, Cianjur, Sukabumi and Bandung

Escherichia coli O157:H7 is known as a foodborne pathogen that can infect humans as well as animals. Dairy cows are the main reservoir of E. coli O157:H7 strains. These bacteria have the potential to pollute milk through direct contact with cattle and the dairy farming environment. Bacteria can survive during the production process and dairy products such as cheese. The presence of E. coli O157:H7 in dairy products has not been widely reported in Indonesia. The purpose of this study was to examine E. coli O157:H7 in the fresh milk from livestock and dairy products obtained from supermarkets and traditional markets in Depok, Cianjur, Sukabumi, and Bandung. A conventional method for E. coli isolation and testing techniques and serotyping tests on O157 and H7 was used in the study. The results showed that 26 isolates (23.85%) of a total of 109 fresh milk samples were positive for E. coli. They were identified as E. coli O157:H7 were 17 (65.38%) of 26 E. coli isolates. In dairy products, E. coli O157:H7 was not detected. This indicates that biosafety and biosecurity in the of dairy cows need to be to prevent E. coli O157:H7 contamination in milk and their livestock environment.

Photoelectrochemical Sensors with Near-Infrared-Responsive Reduced Graphene Oxide and MoS2 for Quantification of Escherichia Coli O157:H7.

The photoelectric response is crucial for photocatalysis, having applications in solar cells and photoelectrochemical (PEC) sensors. In this study, we demonstrate improvements in the near-infrared (NIR)-light-driven PEC response via synergism between reduced graphene oxide (rGO) and MoS2. Intriguingly, rGO modulates the morphology of MoS2, facilitating carrier generation and migration, improving the PEC performance of the resultant rGO-MoS2 sheets (GMS), and yielding an approximately 8-fold increase in the photocurrent compared to that of the pure MoS2. Based on these findings, a NIR-responsive PEC immunosensing platform for the "turn-on" analysis of Escherichia coli O157:H7 on 980 nm light irradiation is reported. Specifically, the device is a three-dimensional magnetic screen-printed paper-based electrode assembled on a home-made PEC cell, and it enables integrated separation and detection. Using a sandwich-type immunocomplex bridged by E. coli O157:H7 and a GMS PEC probe, the immunosensing platform detected E. coli O157:H7 between 5.0 and 5.0 × 106 CFU mL-1, having an extremely low detection limit of 2.0 CFU mL-1. Further, the assay enables the direct analysis of E. coli O157:H7 in milk without the need for pretreatment. Our findings suggest directions for the development of NIR-responsive paper-based PEC materials for portable biomolecule sensing.

Application of a novel lytic phage vB_EcoM_SQ17 for the biocontrol of Enterohemorrhagic Escherichia coli O157:H7 and Enterotoxigenic E. coli in food matrices.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 and Enterotoxigenic E. coli (ETEC) are important foodborne pathogens, causing serious food poisoning outbreaks worldwide. Bacteriophages, as novel antibacterial agents, have been increasingly exploited to control foodborne pathogens. In this study, a novel broad-host range lytic phage vB_EcoM_SQ17 (SQ17), was isolated, characterized, and evaluated for its potential to control bacterial counts in vitro and in three different food matrices (milk, raw beef, and fresh lettuce). Phage SQ17 was capable of infecting EHEC O157:H7, ETEC, and other E. coli strains. Morphology, one-step growth, and stability assay showed that phage SQ17 belongs to the Caudovirales order, Myoviridae family, and Mosigvirus genus. It has a short latent period of 10 min, a burst size of 71 PFU/infected cell, high stability between pH 4 to 12 as well as thermostability between 30°C and 60°C for 60 min. Genome sequencing analysis revealed that the genome of SQ17 does not contain any genes associated with antibiotic resistance, toxins, lysogeny, or virulence factors, indicating the potential safe application of phage SQ17 in the food industry. In Luria-Bertani (LB) medium, phage SQ17 significantly decreased the viable counts of EHEC O157:H7 by more than 2.40 log CFU/ml (p < 0.05) after 6 h of incubation at 37°C. Phage SQ17 showed great potential to be applied for biocontrol of EHEC O157:H7 in milk and raw beef. In fresh lettuce, treatment with SQ17 also resulted in significant reduction of viable cell counts of EHEC O157:H7 and ETEC at both 4°C and 25°C. Our results demonstrate that SQ17 is a good candidate for application as an EHEC O157:H7 and ETEC biocontrol agent in the processing stages of food production and food preservation.

Polydopamine nanospheres-assisted direct PCR for rapid detection of Escherichia coli O157:H7

Polymerase chain reaction (PCR) is one of the most common methods for rapid monitoring of foodborne pathogens; however, it requires purified nucleic acid as a template. Conventional nucleic acid purification is a time-consuming and laborious process. To overcome this, we developed polydopamine nanospheres (PDA NPs)-assisted direct PCR for detecting Escherichia coli O157:H7 (E. coli O157:H7). PDA NPs significantly enhanced PCR efficiency because of their strong interaction with PCR reagents, including polymerase and primers, thereby enabling regulation of the PCR performance. The optimal concentration and diameter for PDA NPs were 0.10 μg/μL and 504 nm, respectively. The PDA NPs-assisted direct PCR exhibited high sensitivity in E.coli O157:H7 detection. The detection limit of PDA NPs-assisted direct PCR was 6.7 × 104 CFU/mL, which was 10-fold lower than that of direct PCR (6.7 × 105 CFU/mL). Moreover, the sensor demonstrated excellent selectivity against E. coli O157:H7, with a negative reaction to eight other common pathogens. Most importantly, the PDA NPs-assisted direct PCR detected the order of 104−5 CFU/mL E.coli O157:H7 in milk, beef, and watermelon samples. No cultural enrichment was required, with the whole process taking <3 h. Therefore, PDA NPs-assisted direct PCR has tremendous potential in the rapid and sensitive detection of pathogens.

I2/I−-mediated fluorescence quenching of an Ag+-doped gold nanocluster-based immunoassay for sensitive detection of Escherichia coli O157:H7 in milk

Escherichia coli O157:H7 is a type of hazardous bacteria in the field of food safety. A sensitive and effective method is urgently needed to detect it, avoiding enormous harm for the human health. In this study, we synthesized stable Ag+-doped gold nanoclusters (Ag-AuNC) with a fluorescence intensity 4.8 times stronger than that of AuNC. It was further demonstrated that Ag0 existing in the AuNC core and a fraction of Ag+ anchored on the AuNC shell eliminated the surface defects and improved the luminescent properties of AuNC. A combination of I2 and I- was used to quench fluorescence-enhanced Ag-AuNC, which was first applied in ELISA for detecting E. coli O157:H7 to improve the sensitivity. In the presence of E. coli O157:H7, the biotinylated anti-E. coli O157:H7 mAb and streptavidin-alkaline phosphatase would be immobilized and catalyze l-ascorbic acid 2-phosphate sesquimagnesium salt hydrate to produce ascorbic acid. After addition of KIO3, I2/I- were generated. The I2 could trigger oxidative etching of Ag-AuNC and I- could combine with Ag+ to decrease the Ag+ concentration of Ag-AuNC, which resulted in fluorescence quenching of Ag-AuNC. Under optimal conditions, the linear range of I2/I--mediated fluorescence quenching of Ag-AuNC-based immunoassay for detecting E. coli O157:H7 was 3.3 × 103 to 106 cfu/mL, with a detection limit of 9.2 × 102 cfu/mL, 10.7-fold lower than that of the traditional ELISA. The proposed immunoassay exhibits excellent sensitivity, specificity, recovery, and accuracy, which is useful for quantitative detection of E. coli O157:H7 in food safety.

A novel colorimetric sensor using aptamers to enhance peroxidase-like property of gold nanoclusters for detection of Escherichia coli O157:H7 in milk

Escherichia coli O157:H7 is a pathogenic foodborne bacterium of public health concern. A simple, rapid, and environmentally friendly method was developed to synthesise papain-modified gold nanoclusters (papain@AuNCs). Following this, aptamers-modified papain@AuNCs (aptamers@papain@AuNCs) were constructed by reacting Au–S covalent bonds between thiolated aptamers and the papain@AuNCs, enhancing the peroxidase activity. E. coli O157:H7 could be captured through the aptamer@papain@AuNCs, significantly improving the catalytic activity toward the peroxidase substrate 3,3′,5,5′-tetramethylbenzidine, and the blue products could be generated. Papain was used for the first time as a template for the synthesis of AuNCs of peroxidase-like activity for detection. The quantitative detection for E. coli O157:H7 was 39 cfu mL−1 in pure culture. The detection limit of E. coli O157:H7 was 5.6 × 102, 5 × 102 and 4.9 × 102 cfu mL−1 in ultra-high temperature sterilised, pasteurised, and raw milk samples, respectively, after 2 h pre-incubation, indicating that the developed sensor has potential for pathogen detection in foods.

Real-time recombinase-aided amplification with improved propidium monoazide for the rapid detection of viable Escherichia coli O157:H7 in milk

Escherichia coli O157:H7, the causative agent of thrombotic thrombocytopenic purpura and hemolytic uremic syndrome in humans, generates a effective harm to community health because of its high pathogenicity. A real-time recombinase-aided amplification (rRAA) is an emerging method for nucleic acid detection. However, genomic DNA of bacteria could exist in food and the environment for a long time after death and could be amplified by rRAA assay, resulting in false-positive signal; thus, developing a fast and sensitive method is necessary to detect viable foodborne pathogens in food products. In our research, rRAA assay coupled with an enhanced nucleic acid binding dye named improved propidium monoazide (PMAxx) was established and applied in viable E. coli O157:H7 identification in skim milk. The PMAxx could eliminate interference from dead bacteria by permeating impaired membranes and covalently linking to DNA to prevent DNA amplification. The PMAxx-rRAA assay was performed with high sensitivity and good specificity. The PMAxx-rRAA assay could detect as low as 5.4 × 100 cfu/mL of viable E. coli O157:H7 in pure culture, and 7.9 × 100 cfu/mL of viable E. coli O157:H7 in skim milk. In addition, the PMAxx-rRAA assay was performed in the presence of a high concentration of dead bacteria or nontarget bacteria in skim milk to verify the capacity to resist interference from dead bacteria and nontarget bacteria. Therefore, the established PMAxx-rRAA assay is a valuable tool for the identification of viable E. coli O157:H7 in complex food matrix.

Electrochemical Biosensing Interface Based on Carbon Dots-Fe3O4 Nanomaterial for the Determination of Escherichia coli O157:H7.

Escherichia coli (E. coli) O157:H7 can cause many food safety incidents, which seriously affect human health and economic development. Therefore, the sensitive, accurate, and rapid determination of E. coli O157:H7 is of great significance for preventing the outbreak and spread of foodborne diseases. In this study, a carbon dots-Fe3O4 nanomaterial (CDs-Fe3O4)-based sensitive electrochemical biosensor for E. coli O157:H7 detection was developed. The CDs have good electrical conductivity, and the surface of carbon dots contains abundant carboxyl groups, which can be used to immobilize probe DNA. Meanwhile, the CDs can be used as a reducing agent to prepare CDs-Fe3O4 nanomaterial. The Fe3O4 nanomaterial can improve the performance of the electrochemical biosensor; it also can realize the recovery of CDs-Fe3O4 due to its magnetism. As expected, the electrochemical biosensor has excellent specificity of E. coli O157:H7 among other bacteria. The electrochemical biosensor also exhibited good performance for detecting E. coli O157:H7 with the detection range of 10–108 CFU/ml, and the detection limit of this electrochemical biosensor was 6.88 CFU/ml (3S/N). Furthermore, this electrochemical biosensor was successfully used for monitoring E. coli O157:H7 in milk and water samples, indicating that this electrochemical biosensor has good application prospect. More importantly, this research can provide a new idea for the detection of other bacteria and viruses.

Poly-l-lysine-functionalized magnetic beads combined with polymerase chain reaction for the detection of Staphylococcus aureus and Escherichia coli O157:H7 in milk

Rapid and credible detection of pathogens is essential to prevent and control outbreaks of foodborne diseases. In this study, a poly-l-lysine-functionalized magnetic beads (PLL-MB) strategy combined with a PCR assay was established to detect Staphylococcus aureus. We also detected Escherichia coli O157:H7 to further verify the strategy for gram-negative bacteria detection. Poly-l-lysine has strong positive charges because of its amino groups, which can conjugate with the carboxyl of carboxyl magnetic beads. Furthermore, it can be used to combine with bacteria through electrostatic adsorption. Under optimum conditions, the developed PLL-MB complexes showed 90% capture efficiency in phosphate-buffered saline and 85% capture efficiency in milk for S. aureus detection. The limit of detection of the PLL-MB-PCR assay was 102 cfu/mL (1.8 × 102 cfu/mL for S. aureus and 7 × 102 cfu/mL for E. coli O157:H7) in phosphate-buffered saline and milk samples. The whole assay can be performed within 4 h. The proposed strategy showed a lower limit of detection when compared with the conventional PCR assay without enrichment. In addition, this method exhibited the advantages of a high-efficient, cost-efficient, and simple operation, indicating its potential applications in foodborne pathogen detection.

A detection method of Escherichia coli O157:H7 based on immunomagnetic separation and aptamers-gold nanoparticle probe quenching Rhodamine B's fluorescence: Escherichia coli O157:H7 detection method based on IMS and Apt-AuNPs probe quenching Rho B' s fluorescence.

This research aimed to detect Escherichia coli O157:H7 in milk based on immunomagnetic probe separation technology and quenching effect of gold nanoparticles to Rhodamine B. Streptavidin-modified magnetic beads (MBs) were combined with biotin-modified antibodies to capture E. coli O157:H7 specifically. Gold nanoparticle (AuNPs) was incubated with sulfhydryl-modified aptamers (SH-Aptamers) to obtain the Aptamers-AuNPs probe. After magnetic beads captured target bacteria and formed a sandwich structure with the gold nanoprobe, Rhodamine B was added into complex to obtain fluorescent signal changes. Our results demonstrated that the established method could detect E. coli O157:H7 in the range of 101-107CFU/mL, and the limit of detection (LOD) was 0.35CFU/mL in TBST buffer (pH = 7.4). In milk simulation samples, the LOD of this method was 1.03CFU/mL. Our research provides a promising approach on the detection of E. coli O157:H7.

A novel smartphone-based colorimetric aptasensor for on-site detection of Escherichia coli O157:H7 in milk

Effective testing tools for Escherichia coli O157:H7 can prevent outbreaks of foodborne illness. In this paper, a smartphone-based colorimetric aptasensor was developed using functionalized gold nanoparticles (GNP) and multi-walled carbon nanotubes (MWCNT) for monitoring E. coli O157:H7 in milk. The maximum absorption peak of GNP bonded with aptamer (Apt) generated evident transformation from 518 to 524 nm. The excess GNP-Apt was removed by functionalized MWCNT magnetized with carbonyl iron powder (CIP) and hybridized with a DNA probe, whereas the GNP-Apt immobilized on E. coli O157:H7 remained in the system. In the presence of a high-salt solution, the GNP-Apt that captured E. coli O157:H7 remained red, but the free GNP-Apt aggregated and appeared blue. The chromogenic results were analyzed by a smartphone-based colorimetric device that was fabricated using acrylic plates, a light-emitting diode, and a mobile power pack. To our knowledge, this was the first attempt to use a smartphone-based colorimetric aptasensor employing the capture of GNP-Apt coupled with separation of MWCNT@CIP probe to detect E. coli O157:H7. The aptasensor exhibited good reproducibility and no cross-reaction for other bacteria. A concentration of 8.43 × 103 cfu/mL of E. coli O157:H7 could be tested in pure culture, and 5.24 × 102 cfu/mL of E. coli O157:H7 could be detected in artificially contaminated milk after 1 h of incubation. Therefore, the smartphone-based colorimetric aptasensor was an efficient tool for the detection of E. coli O157:H7 in milk.

An aptamer-exonuclease III (Exo III)–assisted amplification-based lateral flow assay for sensitive detection of Escherichia coli O157:H7 in milk

Escherichia coli O157:H7 (E. coli O157:H7), one of the most widespread foodborne pathogens, can cause a series of diseases and even lead to death. In this study, a highly sensitive method was developed by combining aptamer-exonuclease III (Exo III)-assisted amplification with lateral flow assay (LFA) based on gold nanoparticles (AuNP). The compound of single-stranded (ss) DNA-anti-E. coli O157:H7 aptamer (ssDNA-aptamer) was formed by hybridization between designed target ssDNA and aptamer. When E. coli O157:H7 was present, target bacteria were bound with the aptamer, and the free target ssDNA was hybridized with the probes of the designed hairpin (HP) structure. Exo III digests the 3' double-stranded blunt end of the complex and releases the enzyme product. Because the remaining sequence of the HP of the designed enzyme product was the same as the target ssDNA sequence, the target ssDNA could be amplified. Finally, the enhanced target ssDNA was combined with AuNP-LFA to achieve visual detection of E. coli O157:H7. The quantitative ability of this platform for E. coli O157:H7 was 7.6 × 101 cfu/mL in pure culture, and the detection limit in milk was 8.35 × 102 cfu/mL. This LFA was highly specific to E. coli O157:H7, and the time for detection of E. coli O157:H7 in milk was 4 h. Hence, this system has important application prospects in the detection of pathogenic bacteria in dairy products.

Isolation, characterization and application of an alkaline resistant virulent bacteriophage JN01 against Escherichia coli O157:H7 in milk and beef

Bacteriophage is a new biological control tool for the prevention and treatment of foodborne pathogens. In this study, an alkali-resistant phage against Escherichia coli (E. coli), named JN01, was isolated and characterized from wastewater. It exhibited lytic activities against 16 of 20 E. coli strains, 11 of which were more than three multi-drug resistant strains. The biological characteristics of JN01 showed that the latent period and lysis period was 15 min and 85 min, respectively. The burst size of JN01 was about 134 PFU per infected cell. JN01 was relatively stable under stressed conditions such as temperatures (30°C–70 °C) and pH (4–13). The genome of JN01 is 88,360-bp double-stranded DNA molecules, with a total of 130 open reading frames (ORFs) and 38.76% of G + C content. Importantly, no known virulence-associated, antibiotic and lysogeny-related genes were identified in the genome of JN01. Morphological and phylogenetic analyses confirmed that JN01 belongs to the Myoviridae. Viable cell counts of E. coli O157: H7 were reduced in milk and raw beef samples when treated with JN01 at 4 °C. The results indicated that JN01 could be a natural potential biological control agent for E. coli O157: H7 in foods.

Cell-based fluorescent microsphere incorporated with carbon dots as a sensitive immunosensor for the rapid detection of Escherichia coli O157 in milk

The rapid and early detection of foodborne pathogens in contaminated food is important for ensuring food safety and quality. In this study, a highly sensitive fluorescent immunosensor was developed to detect Escherichia coli O157:H7 in milk, by using microspheres labeled with carbon dots (CDs). The CDs-microspheres were prepared with Staphylococcus aureus cells as the carrier to incorporate CDs particles. Characterization of the microsphere revealed strong intensity, good stability and high uniformity in fluorescence. With Staphylococcal Protein A (SPA) on the surface of S. aureus cells, the microsphere could be easily coupled with various antibodies (e.g., immunoglobulin G). In combination with the immunomagnetic beads technique, a CDs-microsphere immunosensor was established for the specific detection of E. coli O157:H7 in milk. The limit of detection for E. coli O157:H7 is 2.4 × 102 colony-forming unit (CFU)/mL, comparable to that of real-time PCR methods. Milk samples spiked with E. coli O157:H7 at concentrations from 2.4 × 102 to 2.4 × 107 CFU/mL could be detected within 30 min. The coefficients of variation of the intra-assay tests were less than 10%, indicating a good repeatability. Moreover, the method was able to detect trace amounts of E. coli O157:H7 (<10 CFU) in real milk samples, with a 100% (10/10) accuracy after bacterial enrichment. This CDs-microsphere immunosensor shows considerable potential as a rapid and sensitive tool to detect pathogens in milk and other foods.

A Novel Real-Time Loop-Mediated Isothermal Amplification Combined with Immunomagnetic Beads Separation and Ethidium Bromide Monoazide Treatment for Rapid and Ultrasensitive Detection of Viable Escherichia coli O157:H7 in Milk

In this study, a real-time loop-mediated isothermal amplification (Rti-LAMP) combined with immunomagnetic beads separation (IMS) and ethidium bromide monoazide (EMA) treatment was developed for the rapid and ultrasensitive detection of viable Escherichia coli (E. coli) O157:H7 in milk without enrichment. Polyclonal antibodies against E. coli O157:H7 flagellum and specific immunomagnetic beads (IMBs) were prepared. The capture efficiency of target bacteria was more than 95% by adding 50 μL of prepared IMBs into 1 mL of E. coli sample for incubation at 37 °C for 20 min. Then, Gly-HCl buffer (0.2 M, pH 2.2) was a key to the effective eluting and separating of bacteria from IMB-bacterial complexes. The eluted bacteria were resuspended with 20 μL TZ lysing solution and boiled at 99.5 °C for 10 min, as a DNA template of Rti-LAMP. The development of IMS-Rti-LAMP assay could specifically detect viable E. coli O157:H7 as low as 2.7 CFU/mL in pure culture. Furthermore, the optimum volume for detection of E. coli O157:H7 in milk was 1 mL of the sample by IMS-Rti-LAMP assay. Before Gly-HCl elution, 4 μg/mL of EMA was used to treat bacterial cells which significantly inhibited DNA amplification derived from dead cells in Rti-LAMP. The limit of detection (LOD) of viable E. coli O157:H7 in milk was 6 CFU/mL by the IMS-EMA-Rti-LAMP, and the whole assay could be completed in 4 h. The development of IMS-EMA-Rti-LAMP assay was a rapid, ultrasensitive, and specific detection of viable E. coli O157:H7 in milk without enrichment.

Naked-eye based point-of-care detection of E.coli O157: H7 by a signal-amplified microfluidic aptasensor

Fast and sensitive detection of E.coli O157: H7 is significantly essential for clinical management as well as for transmission prevention during disease outbreaks. Though many types of detection strategies have been implemented for measuring E.coli O157: H7, most of them still rely on complex instruments or tedious/laborious setups, which restrict their applications in resource-limited scenarios. Herein, we introduce an eye-based microfluidic aptasensor (EA-Sensor) for fast detection of E.coli O157: H7 without the assist of any instruments. We demonstrate the perfect coupling of aptamer sensing, hybridization chain reaction (HCR)-amplification and a distance-based visualized readout to quantitatively determine the pathogen concentration. We first used gel-electrophoresis assay to evaluate the system and the results proved that E.coli O157: H7 was well recognized by the aptamer and HCR could increase the signal by about 100 folds. In addition, the Aptamer specificity and signal-amplification ability were verified on the EA-Sensor for sensing E.coli O157: H7 by naked eyes. Furthermore, we demonstrated that E.coli O157: H7 in milk could be accurately and conveniently measured with good performance. With the benefits of operation integration and strategy integration, our EA-Sensor shows advantages of high specificity, easy operation, efficient amplification and visualized readout, which offers a favorable point-of-care tool for E.coli O157: H7 or other pathogen detection in resource-constrained settings.