Rapid Detection of Escherichia coli O157:H7 by Using Green Fluorescent Protein-Labeled PP01 Bacteriophage

Abstract

A previously isolated T-even-type PP01 bacteriophage was used to detect its host cell, Escherichia coli O157:H7. The phage small outer capsid (SOC) protein was used as a platform to present a marker protein, green fluorescent protein (GFP), on the phage capsid. The DNA fragment around soc was amplified by PCR and sequenced. The gene alignment of soc and its upstream region was g56-soc.2-soc.1-soc, which is the same as that for T2 phage. GFP was introduced into the C-and N-terminal regions of SOC to produce recombinant phages PP01-GFP/SOC and PP01-SOC/GFP, respectively. Fusion of GFP to SOC did not change the host range of PP01. On the contrary, the binding affinity of the recombinant phages to the host cell increased. However, the stability of the recombinant phages in alkaline solution decreased. Adsorption of the GFP-labeled PP01 phages to the E. coli cell surface enabled visualization of cells under a fluorescence microscope. GFP-labeled PP01 phage was not only adsorbed on culturable E. coli cells but also on viable but nonculturable or pasteurized cells. The coexistence of insensitive E. coli K-12 (W3110) cells did not influence the specificity and affinity of GFP-labeled PP01 adsorption on E. coli O157:H7. After a 10-min incubation with GFP-labeled PP01 phage at a multiplicity of infection of 1,000 at 4°C, E. coli O157:H7 cells could be visualized by fluorescence microscopy. The GFP-labeled PP01 phage could be a rapid and sensitive tool for E. coli O157:H7 detection.

Luminescence based enzyme-labeled phage (Phazyme) assays for rapid detection of Shiga toxin producingEscherichia coliserogroups

“…20 Similarly, several researchers combined the reporter phage concept and labeling of phages to create genetically modified labeled phages, which displayed the green fluorescent reporter protein on the capsid surface. 21,22 This technique allowed the detection of viable but non-culturable bacteria (VBNC) within one hour, if sufficient levels of bacteria were present. 21,22 Phages have also been labeled with quantum dots and used to detect bacteria.…”

“…21,22 This technique allowed the detection of viable but non-culturable bacteria (VBNC) within one hour, if sufficient levels of bacteria were present. 21,22 Phages have also been labeled with quantum dots and used to detect bacteria. 23 One disadvantage of the above studies is the fact that they all required expensive instrumentation to detect the fluorescently phage-based methods have been developed to effect rapid detection of bacteria.…”

“…The binding properties of bacteriophages (phages) have often been exploited to create rapid bacterial detection assays. [18][19][20][21] The purpose of this study was to develop fieldbased diagnostic assays with the ability to detect five main STEC serogroups: O26, O103, O111, O145 and O157. To achieve this, luminescent assays, based on enzyme-labeled phages (Phazymes), were developed by combining the Phazymes with a sampling device (swab), STEC specific immunomagnetic (IMS) beads, bacterial enrichment broth and a luminescent substrate to form an integrated assay that detected STEC in food and water samples within 9 hours.…”

Development and Prospects of Dedicated Tracers for the Molecular Imaging of Bacterial Infections

“…in sewage water. 169,170 Bacteriophages have been fluorescently labeled with fluorescent nucleic acid dyes SYBR gold and SYBR green I, after which they were used to target and label bacteria. 171,172 By combining immuno-magnetic isolation by antibody-coated magnetic beads with staining by bacteriophages, Goodridge et al could reach very low detection limits (10 1 -10 2 CFU/mL) of bacterial pathogens in food samples.…”

Application of gold-labeled antibody biosensor in simultaneous determination of total aflatoxins using artificial neural network

“…The measurement of antibody or antigen concentrations based on biospecific recognition interactions such as biosensors has been considered as a major analytical method and used in environmental and biochemical studies. This method has generated much interest due to its cost-effectiveness, sensitivity and specificity [15][16][17][18][19]. Gold nanoparticles (GNPs) have recently attracted significant attention due to their non-toxic nature and excellent biological compatibility [20,21].…”