Reporter bacteriophage T7NLC utilizes a novel NanoLuc::CBM fusion for the ultrasensitive detection of Escherichia coli in water.

Rapid detection of bacteria responsible for foodborne diseases is a growing necessity for public health. Reporter bacteriophages (phages) are robust biorecognition elements uniquely suited for the rapid and sensitive detection of bacterial species. The advantages of phages include their host specificity, ability to distinguish viable and non-viable cells, low cost, and ease of genetic engineering. Upon infection with reporter phages, target bacteria express reporter enzymes encoded within the phage genome. In this study, the T7 coliphage was genetically engineered to express the newly developed luceriferase, NanoLuc (NLuc), as an indicator of bacterial contamination. While several genetic approaches were employed to optimize reporter enzyme expression, the novel achievement of this work was the successful fusion of the NanoLuc reporter to a carbohydrate binding module (CBM) with specificity to crystalline cellulose. This novel chimeric reporter (nluc::cbm) bestows the specific and irreversible immobilization of NanoLuc onto a low-cost, widely available crystalline cellulosic substrate. We have shown the possibility of detecting the immobilized fusion protein in a filter plate which resulted from a single CFU of E. coli. We then demonstrated that microcrystalline cellulose can be used to concentrate the fusion reporter from 100 mL water samples allowing a limit of detection of \u3c10 CFU mL-1 E. coli in 3 hours. Therefore, we conclude that our phage-based detection assay displays significant aptitude as a proof-of-concept drinking water diagnostic assay for the low-cost, rapid and sensitive detection of E. coli. Additional improvements in the capture efficiency of the phage-based fusion reporter should allow a limit of detection of \u3c10 CFU per 100 mL

A phage-based assay for the rapid, quantitative, and single CFU visualization of E. coli (ECOR #13) in drinking water

Drinking water standards in the United States mandate a zero tolerance of generic E. coli in 100mL of water. The presence of E. coli in drinking water indicates that favorable environmental conditions exist that could have resulted in pathogen contamination. Therefore, the rapid and specifc enumeration of E. coli in contaminated drinking water is critical to mitigate signifcant risks to public health. To meet this challenge, we developed a bacteriophage-based membrane fltration assay that employs novel fusion reporter enzymes to fully quantify E. coli in less than half the time required for traditional enrichment assays. A luciferase and an alkaline phosphatase, both specifcally engineered for increased enzymatic activity, were selected as reporter probes due to their strong signal, small size, and low background. The genes for the reporter enzymes were fused to genes for carbohydrate binding modules specifc to cellulose. These constructs were then inserted into the E. coli-specifc phage T7 which were used to infect E. coli trapped on a cellulose flter. During the infection, the reporters were expressed and released from the bacterial cells following the lytic infection cycle. The binding modules facilitated the immobilization of the reporter probes on the cellulose flter in proximity to the lysed cells. Following substrate addition, the location and quantifcation of E. coli cells could then be determined visually or using bioluminescence imaging for the alkaline phosphatase and luciferase reporters, respectively. As a result, a detection assay capable of quantitatively detecting E. coli in drinking water with similar results to established methods, but less than half the assay time was developed

Determination of zearalenone and its metabolites in endometrial cancer by coupled separation techniques

This study presents a selective method of isolation of zearalenone (ZON) and its metabolite, α-zearalenol (α-ZOL), in neoplastically changed human tissue by accelerated solvent and ultrasonic extractions using a mixture of acetonitrile/water (84/16% v/v) as the extraction solvent. Extraction effectiveness was determined through the selection of parameters (composition of the solvent mixture, temperature, pressure, number of cycles) with tissue contamination at the level of nanograms per gram. The produced acetonitrile/water extracts were purified, and analytes were enriched in columns packed with homemade molecularly imprinted polymers. Purified extracts were determined by liquid chromatography (LC) coupled with different detection systems (diode array detection - DAD and mass spectrometry - MS) involving the Ascentis RP-Amide as a stationary phase and gradient elution. The combination of UE-MISPE-LC (ultrasonic extraction - molecularly imprinted solid-phase extraction - liquid chromatography) produced high (R ≈ 95–98%) and repeatable (RSD < 3%) recovery values for ZON and α-ZOL

A bacteriophage detection tool for viability assessment of Salmonella cells

Available online 7 September 2013Salmonellosis, one of the most common food and water-borne diseases, has a major global health and economic impact. Salmonella cells present high infection rates, persistence over inauspicious conditions and the potential to preserve virulence in dormant states when cells are viable but non-culturable (VBNC). These facts are challenging for current detection methods. Culture methods lack the capacity to detect VBNC cells, while biomolecular methods (e.g. DNA- or protein-based) hardly distinguish between dead innocuous cells and their viable lethal counterparts. This work presents and validates a novel bacteriophage (phage)-based microbial detection tool to detect and assess Salmonella viability. Salmonella Enteritidis cells in a VBNC physiological state were evaluated by cell culture, flow-cytometry and epifluorescence microscopy, and further assayed with a biosensor platform. Free PVP-SE1 phages in solution showed the ability to recognize VBNC cells, with no lysis induction, in contrast to the minor recognition of heat-killed cells. This ability was confirmed for immobilized phages on gold surfaces, where the phage detection signal follows the same trend of the concentration of viable plus VBNC cells in the sample. The phage probe was then tested in a magnetoresistive biosensor platform allowing the quantitative detection and discrimination of viable and VBNC cells from dead cells, with high sensitivity. Signals arising from 3 to 4 cells per sensor were recorded. In comparison to a polyclonal antibody that does not distinguish viable from dead cells, the phage selectivity in cell recognition minimizes false-negative and false-positive results often associated with most detection methods

Formulation and Search of Assembly Sequence Design Spaces for Efficient Use of Assembly Plant Resources for New Products

Efficient procedures for generation of feasible assembly sequences and effective utilization of available assembly plant resources can greatly reduce the development time and cost of platforms for new product family members. This article presents a method to generate feasible assembly sequences and an approach to select an assembly process that reduces the existing plant modification cost. Assembly sequence design space is combinatorial in nature. Mathematical models to solve the effects of constraints on these spaces and algorithms to efficiently enumerate feasible spaces are explored in this research. Algorithms to search the feasible space to identify assembly process that can reduce the modification cost of the existing assembly plant can help increase utilization of existing resources. A software application that implements the method and algorithms has been developed. The algorithms use the concept of recursive partitioning of set of components to generate assembly sequence space. The assembly processes are then evaluated to determine the process that maximizes resource utilization for new platforms. The application of the proposed approach is demonstrated using automotive underbody front structure family.Yeshttps://us.sagepub.com/en-us/nam/manuscript-submission-guideline

TRENDS Trends and opportunities in food pathogen detection

Despite the recent advances in food pathogen detection, there still exist many challenges and opportunities to improve the current technology. Techniques such as immunomagnetic separation (IMS) and polymerase chain reaction (PCR) have paved the way for rapid and sensitive detection of foodborne pathogens, and advances in nanobiotechnology have allowed for miniaturization of devices. Collaborations between workers in the fields of engineering, nanotechnology and food science have introduced new lab-on-a-chip technologies permitting development of portable, hand-held biosensors for food pathogen detection. This report highlights examples within the current state of the art, and emphasizes areas in which further research is needed. In 1999 it was estimated that foodborne pathogens were responsible for 76 million illnesses annually, resulting in 5,000 deaths [1]. This report identified Salmonella, Listeria and Toxoplasma as the major causative agents, being responsible for 1,500 of the reported deaths. Data published in 2006 by the CDC suggested that infections due to Yersinia, Shigella, Listeria, Campylobacter, Escherichia coli O157:H7 and Salmonella have decreased dramatically, while infections due to Vibrio have increased [2]. A more recent report indicated similar findings, with a decrease in Yersinia, Shigella, Listeria and Campylobacter cases, and again a significant increase in Vibrio infections [3]. The declining rates of infection due to Listeria monocytogenes and E. coli O157:H7 are likely a result of increased awareness. The FDA, USDA and EU have all implemented a zero-tolerance rule for L. monocytogenes in ready-to-ea

Development of fluorescent nanoparticle-labeled lateral flow assay for the detection of nucleic acids

The rapid, specific and sensitive detection of nucleic acids is of utmost importance for the identification of infectious agents, diagnosis and treatment of genetic diseases, and the detection of pathogens related to human health and safety. Here we report the development of a simple and sensitive nucleic acid sequence-based and Ru(bpy)(3)(2+)-doped silica nanoparticle-labeled lateral flow assay which achieves low limit of detection by using fluorescencent nanoparticles. The detection of the synthetic nucleic acid sequences representative of Trypanosoma mRNA, the causative agent for African sleeping sickness, was utilized to demonstrate this assay. The 30 nm spherical Ru(bpy)(3)(2+)-doped silica nanoparticles were prepared in aqueous medium by a novel method recently reported. The nanoparticles were modified by 3-glycidoxypropyl trimeth oxysilane in order to conjugate to amine-capped oligonucleotide reporter probes. The fluorescent intensities of the fluorescent assays were quantified on a mictrotiter plate reader using a custom holder. The experimental results showed that the lateral flow fluorescent assay developed was more sensitive compared with the traditional colloidal gold test strips. The limit of detection for the fluorescent lateral flow assay developed is approximately 0.066 fmols as compared to approximately 15 fmols for the colloidal gold. The limit of detection can further be reduced about one order of magnitude when ``dipstick'' format was used

A Novel Method for the Preparation of Ru(Bpy)32+-Doped Silica Nanoparticles

A novel method for the preparation of spherical Ru(bpy)(2+)(3) -doped silica nanoparticles with adjustable diameters and narrow particle size distribution with high yield was developed. Unlike the most commonly used Stober method which uses a reverse microemulsion system (oil-based, NH4OH as a catalyst, pH 11-12), the proposed method synthesizes the nanoparticles in a water-based system (pH 9-10) using L-lysine as a catalyst in the presence of the dye. Both the size of the spherical dye-doped nanoparticles and the Ru(bpy)(2+)(3) content which is incorporated in silica nanoparticles are precisely controlled by the multiple-step addition of tetraethyl orthosilicate and Ru(bpy)(2+)(3) solution, respectively. (C) 2012 Elsevier B.V. All rights reserved