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University of Wyoming

UW Technologies Available for Licensing

 

Licensed - Technology Disclosure: 05-019, 05-099, 06-019 Fast, Economical, Accurate Diagnostic for Pathogens In Convenient, One-Tube Assay - Licensed 


Each year, foodborne contamination with E. coli O157:H7 results in approximately 62,500 illnesses, requires 1,843 hospitalizations, and causes 52 deaths. There are many methods used to detect E. coli O157:H7 in food and water. These detection systems include cultural techniques, immunological procedures, and nucleic acid-based assays.

However, most of these tests require a combination of long incubation times to grow the bacteria to high enough levels for detection, meaning that test results are not known for several days. In the time interval required to obtain a test result, contaminated food may have already been disseminated to retail stores and purchased by consumers. Also, these tests are expensive are often quite complex, requiring intensive training for laboratory personnel. Therefore, there remains a need for very sensitive, rapid, and simple methods capable of detecting E. coli O157:H7.

Reporter bacteriophages represent a novel and sensitive alternative to conventional methods for the detection of bacteria within food. A bacteriophage (phage) is a virus that specifically infects bacteria, and a reporter gene is a segment of DNA that encodes for a protein that is easily measurable (i.e. a fluorescent protein or an enzyme). In this method, a bacteriophage is modified to carry a reporter gene. The reporter gene is introduced into a target bacterium via the bacteriophage during its normal infection cycle. Once the reporter gene has been introduced to the bacterium, it is expressed (i.e. the protein is produced), thereby allowing bacterial cells to be rapidly identified. Recently, researchers in the food microbiology laboratory in the College of Agriculture’s Department of Animal Science have developed a novel assay, known as the Phast Swab, to detect E. coli O157:H7 that is based on the use of reporter phages.

To demonstrate the effectiveness of the Phast Swab for E. coli O157:H7, researchers at the University of Wyoming developed such an assay, using a reporter phage carrying the E. coli β-galactosidase gene, and the substrate CPRG. The Phast Swab integrates a sampling method, a reporter bacteriophage, immunomagnetic separation, and bacterial enrichment in one easy to use device (Figure 1).  The Phast Swab is a simple to use device. The bottom of the device contains bacterial growth media and specific immunomagnetic (IMS) beads. The swab (or dropper if sampling liquid) is removed, the surface to be tested is swabbed, and the swab is returned to the device, followed by an 8 hour enrichment. Following enrichment, the IMS beads (with target bacteria attached) are concentrated, and the growth media is removed. Following a wash step, the reporter phage is mixed with the target bacteria (this is accomplished directly in the device) and the Phast Swab is incubated at 37oC for 1.5 hours. Finally, the cap of the Phast Swab is broken, releasing the beta-galactosidase substrate into the bottom of the device, where it reacts with any beta-galactosidase present. A positive test is indicated by the development of a red color, while in a negative test, the color remains yellow (Figure 2).      

                             

                                                Figure 1.  The Phast Swab

 

  

                Figure 2.  Detection of Escherichia coli O157:H7 using the Phast Swab.  The bottom of the device contains growth media (tryptic soy broth supplemented with 0.2 M glucose) and E. coli O157:H7 specific immunomagnetic (IMS) beads (Figure 2 #1).  The swab is removed, the surface to be tested is swabbed, and the swab is returned to the device, followed by an 8 hour enrichment.  Following enrichment, the IMS beads (with E. coli O157:H7 cells attached) are concentrated, and the growth media is removed (Figure 2 #2).  Following a wash step, the reporter phage is added, and the Phast Swab is incubated at 37oC for 1.5 hours (Figure 2 #3). Finally, the cap of the Phast Swab is broken, releasing the beta-galactosidase (CRPG) substrate into the bottom of the device (Figure 2 #4), where it will react with any beta-galactosidase present. A positive test is indicated by the development of a red color, while in a negative test, the color remains yellow (Figure 2 #5).

 

The nature of this detection method is such that it effectively detects E. coli O157:H7 in a simple and rapid manner, and also distinguishes between viable, and non-viable cells, since phages can only grow within living bacteria. In addition, the fact that a color reaction is used to evaluate the test result eliminates the need for instrumentation to read the test, making test interpretation simple and cheap. However, the versatility of the assay is such that, other substrates for β-galactosidase (fluorescent, luminescent) could be employed in the one tube assay, allowing for more sensitive detection of the target pathogen in settings (laboratories) where instrumentation to detect the fluorescent or luminescent signal is present. For example, we have modified the Phast Swab to be used in conjunction with hand held luminometers, which are already widely used in the food industry for hygiene monitoring. 

This technology is also broadly applicable to the detection of other bacterial foodborne pathogens, since another reporter phage with a different host range could be employed to detect other foodborne pathogens (i.e. Salmonella or Listeria), in the same one-tube (Phast Swab) format described here (see below). Also, since there are numerous colorimetric substrates for β-galactosidase, it will be possible to multiplex the assay to allow simultaneous detection of multiple pathogens.

The reporter bacteriophage technology described here is based on bacteriophage T4, which belongs to a super family of bacteriophages (the T even family) that infect many diverse bacteria, including pathogens that are the cause of food spoilage and human and animal illness. Therefore, it is possible to create multiple detection assays that specifically detect different bacterial pathogens, based on a single platform—the T4 bacteriophage.

The bacteriophages within the T even family are genetically related to one another, with varying degrees of homology. One area of highly conserved homology within these bacteriophages lies in the regions upstream and downstream (gene 36 upstream and gene t downstream) of the genes that encode the tail fibers required for bacterial host recognition. This makes it possible to amplify the tail fiber genes from bacteriophages in the T even family using the polymerase chain reaction (PCR), and a single set of PCR primers. Herein lies the strategy for the creation of a reporter phage template. The T4 phage used in these studies carry amber mutations in its tail genes. This mutation will make the phage unable to grow in wildtype strains (the phage will only grow in an amber suppressor strain). A reporter gene (beta-galactosidase) will be stably incorporated into the bacteriophage T4. This will form the basis of the reporter phage template. To produce reporter phages capable of detecting the bacteria discussed above, the host range of the reporter phage template will be altered via homologous recombination between the tail fiber negative reporter phage and a plasmid carrying tail fibers amplified from a T even phage that infects the bacterial host of interest. The reporter phage will develop functional tail fibers via marker rescue of the amber mutation, enabling the reporter phage to infect, and detect the bacteria of interest.

Once the reporter phages have been developed, they will form the basis of Phast Swabs that can detect different bacteria.

The advantages of this system are numerous when compared to conventional techniques. First, as previously stated, the reporter bacteriophages used in this technology are capable of distinguishing between viable and non-viable bacteria (bacteriophages can only grow in viable bacteria), which is a major advantage over conventional PCR assays and ELISA techniques. Second, the labor required is greatly reduced compared to those other methods because this technique is self contained in one testing device. Third, the platform used in this technology, T4 bacteriophage with an integrated beta-galactosidase gene, allows for colorimetric detection of the signal making the test “instrument-less.” Fourth, this technique is highly cost effective because multiple assays can be produced from a single bacteriophage. The assays can be produced based on a standardized platform, without the need to completely genetically and phenotypically characterize new bacteriophages for every new test. Finally, this technology eliminates many of the traditional reporter bacteriophage creation steps, uses a lytic bacteriophage (that cannot transfer virulence genes to the host), does not incorporate antibiotic resistance genes into the bacteriophage chromosome, and allows for the creation of multiple detection assays within a matter of weeks or months, rather than years using conventional methods. (read College of Agriculture article)

If you would like to learn more about this novel method for rapid and simple detection of bacterial pathogens and how your company may apply it in commercial situations, please contact the director of the University of Wyoming Research Products Center, Davona Douglass. We would be pleased to share further details.