Food Science Packaging – Gary S. Sayler, Steven A. Ripp, Bruce Applegate, Purdue Research Foundation, University of Tennessee Research Foundation
Abstract for “Bioluminescent biosensor device”
“Disclosed is a method and device for detecting bacteria. It relies on the recognition and infection by one or more strains of bacteria with bacteriophage genetically altered to cause production an inducer molecule in bacteria following phage infection. The inducer molecule is released by the infected bacteria and is detected using genetically modified bacterial bioreporter cell lines that emit bioluminescence when stimulated by the inducer. Autoamplification allows detection of low levels without the need for sample enrichment. Methods for detecting select bacteria and kits for detecting selected bacteria are also disclosed.
Background for “Bioluminescent biosensor device”
“1.1 Field of The Invention.”
“The invention relates to methods and devices that detect targeted microorganisms, such as bacteria, by inducing bioluminescence within bioreporter cell cells. Infecting target bacteria with genetically engineered bioreporter cell-derived bacteriophage is possible. An inducer stimulates the bioreporter cells to produce light. The bacteriophage infects the target bacteria and produces the inducer.
“1.2 Description of Related Art”
“The current technology is focusing on the development biologically-based detection methods. This has led to efforts to identify specific microbial pathogens. There have been many methods to determine the presence of microbial contaminants over the years. Typically, culture methods were used in the past, but they were slow and inefficient. Bioreporter technology has allowed for the use of genetically engineered bacteria and bacteriophage in order to identify toxic chemicals, as well as specific species of bacteria.
Bioreporters are genetically engineered organisms that detect specific compounds. They do this by inducing expression of a detectable product gene in the bioreporter cells using a promoter that is heterologous to the target compound. As used herein, bioluminescent bioreporters are genetically engineered bacteria that have genes that, when expressed, result in bioluminescence. The bioreporter cells respond to the presence of a particular compound by emitting light. The lux gene is a popular gene that can be used for this purpose. The lux genes can be expressed under the right conditions and bioluminescence can be detected using a variety optical methods. Many constructs found in bioluminescent bioreporter species are derived from Vibrio fischeri, a bioluminescent marine bacteria (King and al. 1990).
“Sayler et al. “Sayler et al. A variety of lux-based bacterial bioreporters has been used to detect and monitor naphthalene (Heitzer et al., 1994), BTEX (benzene, toluene, ethylbenzene, and xylene) (Applegate et al., 1998), polychlorinated biphenyls (PCBs) (Layton et al., 1998), 2,4-dichlorophenoxyacetic acid (2,4-D) (Hay et al,. 2000, ammonia (Simpson and al. 2001), and the food-salivating indicator chemical?phenylamine (Rippet al. 2000a).
“Genetic constructs that impart bioluminescence to bacterial Bioreporter Cells have in general used a lux gene cassette derived form the marine bacterium Vibrio Fischeri (Engebrecht et al. 1983). As used herein, ?cassette? A recombinant DNA construct is one that is made from a combination of inserted DNA sequences and a vector. The complete lux cassette is composed of five genes (i.e. luxA, B. C., D, and E. These genes are responsible for producing bioluminescence. luxC. and D code for an aldehyde.
“The light response of bioluminescent bioreporters can be measured using optical transducers, such as photomultiplier tube, photodiodes or microchannel plates, and charge-coupled devices. It is necessary to transfer the bioluminescent signal from the transducer. This requires fiber optic cables, lenses, or liquid light guides. These instruments are not suitable for field use. This results in a bulky, heavy instrument that is attached to power and optic cables. Ripp et. al. described field release experiments as an example. A bioluminescent bioreporter was developed for the detection and monitoring of naphthalene in soil. Multiplexed photomultiplier tubes were used to detect bioluminescent signals. These cables were expensive, fragile and cumbersome to use.
“Battery-operated hand-held photomultiplier devices that can be interfaced with a computer laptop have been described. They are used in conjunction bioreporters to analyze groundwater for hydrocarbon contamination (Ripp, 1999a). Special bioluminescent integrated circuits for bioreporter integrated systems (BBICs), have been described (Simpson et. al. 2001). These self-contained units can detect environmental contaminants like naphthalene or BTEX by simply exposing them to these compounds (Ripp et. al. 1999b). These devices use bioluminescent bioreporters, which are genetically modified bacteria bioreporters. They respond to certain chemicals by producing visible light.
Another area of interest is the detection of pathogenic organisms. This is in contrast to chemical agents. For human safety, pathogens like those that cause human and animal disease, foodbome diseases and those used to wage biological warfare are very important. The need for advanced detection systems is further reinforced by the constant appearance of new strains.
“Microbial contamination of fresh fruits or vegetables is a growing concern in the food industry. This is due to increased consumption of these products and recognition of new foodborne pathogens like Escherychia coli o157:H7 and Campylobacter jejuni (Tauxe 1992). While federal agencies have recommended safe food handling practices to minimize risk, they are still unable to provide rapid and accurate methods of detecting pathogens in production, processing and distribution systems. Particular concern when monitoring food safety is the identification of bacteria that causes the majority food-related deaths in America, such as Salmonella, Escherychiacoli O157.H7, Escherychia monocytogenes, Escherychia pneumoniaes, and Campylobacter.
Bioluminescent methods are used in the food industry to detect bacterial contamination. One technology that relies on the detection of ATP is based on the biochemical requirements of bacteria to use ATP for energy production. This is essential for growth and survival. The ATP detection method does not distinguish between bacterial species and does not distinguish pathogens from non-pathogenic ones (Vanne et al. 1996).
“Several reports have reported bioluminescent detections of target bacteria using bacteriophage infection. These procedures have identified a select number of pathogens, as shown in Table 1.
“TABLE 1\nBioluminescence detection of bacterial pathogens by\nbacteriophage containing a luxAB insert.\nPathogen Bacteriophage Detection Limit Test Source Reference\nEnterobacteriaceae Unspecified 10 cells/g/cm2 Surface and meat Kodikara\ncarcass swabs et al.,\n1991\nEscherichia coli ? Charon 100 cells/ml Milk Ulitzer and\nspecies Kuhn,\n1987\nEscherichia coli ? V10 Not determined Pure culture Waddell\nO157:H7 and\nPoppe,\n1999\nListeria A511 10 cells/g Cheese, pudding, Loessner\nmonocytogenes cabbage et al.,\n1996\nSalmonella P22 10 cfu/ml Eggs Chen and\nspecies Griffiths,\n1996\nSalmonella P22 100 cells/ml Pure culture Stewart et\ntyphimurium al., 1989\nStaphylococcus Unspecified 1000 cfu/ml Pure culture Pagotto et\naureus al., 1996”
“In all these cases, the bacteriaiophage contained only an incomplete lux gene (i.e. luxAB. Although useful for the detection of certain pathogenic species, there are several drawbacks to this technique. For detection of the bioluminescent response, it is necessary to have an exogenous source for the aldehyde substrate required for the luciferase reaction if only the luxAB genes were used. This can cause problems when attempting to detect the bioluminescent response. Additional problems arise when conditions like the amount of inducer used may need to be changed. This can be especially problematic if you use the methods in environments such as farms, where the environment is not conducive to running the tests and the end-user may not have the necessary training.
“An additional problem with bacterial detection is the fact that pathogens can often be found in low levels. Existing bioluminescent techniques may not be able to detect such low levels of pathogens. There are many non-bioluminescent methods that can be used to detect low levels of bacteria. These methods include amplification steps such as sample pre-enrichment to increase pathogen concentrations to detectable levels or DNA-based polymerase chains reaction (PCR). These amplification steps have a disadvantage: they require extensive user training, and costly instrumentation.
“There is a need to develop methods and devices that can detect specific bacteria, especially pathogens, and do so selectively, accurately, quickly, and with high sensitivity. To monitor a wide range of common pathogens such as those that are implicated in food safety, food processing, hospital environments, and biological warfare, new devices are required.
“The present invention addresses some of the deficiencies in the methods and devices presently employed in detecting individual species of bacteria, by providing a novel internally amplified bioluminescent bacteriophage/bioreporter system. The disclosed devices allow for rapid and sensitive detections of specific pathogens using a fully integrated, easy-to-use system that requires no more than sample addition. A signal amplifier mechanism is integrated into the design to increase the device’s sensitivity. Two cooperating elements are included in the invention, i.e. The invention includes two cooperating elements, i.e. biosensor and bioreporter element that work together in a unique two-step process. The invention’s biosensor elements are demonstrated by genetically modified bacteria, while the bioreporter element may be any one of many genetically modified cell line. The biosensor bacteriophage infects a selected pathogen (e.g., bacteria) and causes an inducer to cause the bioreporter cells line to express the Lux gene cassette. This results in amplified bioluminescence which is easily detectable.
“In certain embodiments, the invention uses a bacteriophage that has been genetically modified to have a luxI-gene. The luxI gene encodes the protein product, acyl heteroserine latone synthetase. This enzyme performs a condensation reaction between cell metabolites that results in the production acyl en homoserine (AHL) lactone. The genetically modified bacteria is introduced to a selected target bacterium. The phage luxI gene of the bacterium is transcribed into the bacterium. This results in the expression of the LuxI protein in infected cells. The target cell produces AHL molecules that diffuse into the surrounding medium.
“Infected bacteria is caused by the presence of bioreporter cell, which have been genetically engineered to produce light when stimulated with AHL. The bioreporter cells don’t produce any light if the AHL inducer is absent. The bioreporter cell’s ability to absorb AHL molecules from the environment after phage infection causes the production of bioluminescent protein. This is because the bioreporter cells are genetically engineered with a lux gene cassette (luxR+luxI+luxCDABE), which is responsive to AHL. AHL is an autoinducer which positively regulates lux operon. The AHL complex stimulates the lux genes of the bioreporter cells, which results in the production light.
“The invention’s unique feature is the amplifying of bioluminescence by the presence of lux-modified bioreporter cell. The induction of the light-producing genes in one bioreporter cells results in the production not only of light-producing protein but also AHL molecules. These AHL molecules diffuse from the light-producing bioreporter cell and induce the expression of the lux gene in nearby bioreporter cell. Intense bioluminescence is caused by this cascade effect that involves multiple bioreporter cell neighbors. Infection of a target bacteria results in a chain reaction that produces bioluminescence in many bioreporter cell. This allows for detection of extremely low levels of target bacteria. This innovative integrative method allows rapid detection of pathogens without the need for sample enrichment.
“The bacteriophage/bioreporter system employs a luxI-integrated bacteriophage that infects only a particular bacterium. The invention involves the first selection of a target bacterium. Next, the identification of a bacteriophage that is specific for the target bacterium is made and then genetic engineering the bacteriophage with the luxI gene. You can use the specificity of phage infections to detect, monitor and identify certain species of bacteria.
“In most situations in real life, the target bacteria is in a natural environment. This can often be in the presence of microbes or other contaminants. The bacteriophage/bioreporter system addresses this problem by directing the luxI bacteriophage against specific strains of bacteria. You can choose from many commonly recognized pathogens to target bacteria. Several types of bacteria are often linked to food contamination. These include Salmonella, Escherychia coli, O157:H7, Listeria moncytogenes and enterobacteriaceae.
Many other bacteria can be detected with the methods and devices described, provided that an appropriate infectious phage is identified or engineered. In practice, the first step in identifying a pathogen is to identify a pathogen-specific bacteria. There are many. For example, bacteriophage m13 which infects E. coli is one of them. Additional examples of bacteriophage which specifically infect pathogenic bacteria species are found in Table 4.
“An important consideration is the identification of a species-specific bacteriophage that harbors the luxI cassette, and the ability to penetrate the bacterial cells so that the luxI gene can be expressed in the target bacterium. E.coli can be infected by bacteriophage, M13. coli.”
“Some embodiments of this invention allow simultaneous contact with a sample using multiple bacteriophage-biosensors. Each of these biosensors specifically recognizes a specific bacterium and is kept in a separate compartment. Multiple target cells may be detected simultaneously in this manner. The bacteriophage/bioreporter elements can be integrated onto a chip surface to provide a convenient, easily handled device.”
“Some embodiments include multi-component packaged kit kits that contain both sensor and detector elements to detect select strains. These kits can be used to detect bacterial presence in samples. They contain one or more types genetically engineered bacteriaophages, each of which is designed to infect a specific bacterium and, upon infecting, cause the bacterium to express an inducer molecule. The bacteriophage may contain a luxI genetic product that results in the formation of AHL, which is an inducer of lux genes in bioluminescent cells. The kits contain a number of genetically engineered bacteria bioreporter cells that are capable of producing bioluminescence when they are stimulated with the inducer. Particular embodiments of the bioreporter bacteria include a luxR?luxpro/luxI/luxCDABE gene that is stimulated with AHL to produce light. Instructions for use and optionally a device to measure the generated light may be included in kits. This could include an integrated circuit that detects a bioluminescent signal. An integrated circuit could include a photodetector and low-noise electronics (e.g. On-chip wireless communication system, biocompatible housing and semi-permeable membrane that covers the bioreporter area.”
The disclosed methods and devices are based on biologically-based sensor technology, which can be easily adapted to quality control programs and pathogen detection. This system is extremely easy to use because the necessary elements, biosensors and autoamplifying biologicalluminescent bioreporters, are integrated into the detector device’s compartments. The system is simple to use. Simply contact a sample with the detector device’s sample chamber and allow the device to process it and send the results. This technology is a new way to detect, monitor, and prevent biological contamination.
“4.1 Vibrio Fischeri Bioluminescence”
“Genetically engineered bioluminescent bioreporter cell cells are used in the invention. These cells are capable of producing light due to the incorporation of lux genes. They are responsible for bioluminescence produced by the marine bacterium Vibrio Fischeri. FIG. FIG. 1. This is a schematic representation illustrating the lux genes. It shows positive regulation by the luxI- and luxR gene product. The luciferase gene (luxAB), encodes proteins that generate bioluminescence, while the synthetase and reductase(luxC), transferase/luxD genes code for proteins required for the production of an aldehyde substrate. FIG. FIG. The luxR, luxI gene regulatory elements are represented by small circles. Acyl-homoserine (AHL), synthetase is the expressed product of luxI gene. Its product, acyl-homoserine lactone [AHL], acts as an inducer for the bioluminescent response. AHL (represented by black circles) forms a complex that is bound to the expression product of the LuxR gene, i.e. The LuxR transcriptional regulator is represented by white circles. This complex (black and wavy circles) binds with the promoter of the luxI (black box). Inducing transcription is done in the direction indicated by the lower arrow of luxI or luxCDABE. These codes code for proteins involved in biochemical reactions that produce 490 nm of light. This mechanism of positive regulation by the LuxR?AHL complex of the lux operon occurs in both native Vibrio fischeri as well as in the recombinantly added lux gene cassette in bioreporter cells.
Autoinduction is a mechanism that amplifies light generation in Vibrio cells and bioreporter cells. AHL is produced by the luxI gene. This causes AHL to diffuse into the extracellular environment and induce luxI and luxCDABE transcription in nearby bioluminescent cells. This cascade effect eventually generates intense bioluminescence. Autoinduction is a mechanism that increases the production of light by engaging many cells. This invention uses autoinduction to increase the production of light using genetically engineered lux -based bioluminescent bioreporter cell.
“4.2 Bioluminescent Bioreporter System”
“As shown in FIG. “As seen in FIG. 2, the invention uses two elements, i.e. A biosensor and a Bioreporter are the two elements of the invention. The biosensor serves two purposes. A luxI-integrated bacteriophage is an example of a biosensor that infects a specific strain of bacteria. The biosensor DNA infects the target bacterial cell and causes it to produce the gene products encoded by biosensor DNA. The luxI-integrated bacteriophage case shows that the infected bacterium generates the luxI gene product acylhomoserine lactone synthetase. This leads to the ultimate production of AHL.
“A second element of the bacteriophage/bioreporter system is the bioreporter cell. The bioreporter serves two purposes. It responds to the biosensor signal and amplifies that signal to allow multiple bioreporters to be responsive to the signal from infected targets cells. A bioreporter is a bacterial line that has been genetically engineered so it can produce light when stimulated by the target cell signal. AHL is the signal used in systems that use luxI bacteriophage.
“FIG. 2 shows a bacteriophage/bioreporter system in which the bacteriophage biosensor incorporates a luxI construct and the bacterial bioreporter cell incorporates the lux R+I+CDABE constructs. By including the entire luxCDABE gene cassette, the bioreporter element can produce light without the need to add exogenous chemicals. This is because the luxI-luxR genes positively regulate the lux operon in Vibrio as well as in genetically engineered bioreporter cell cells. (FIG. 1).”
“Using the autoinduction mechanism Vibrio fischeri to amplify the bioluminescent signal, invention is achieved through diffusion and uptake by multiple bioreporter cell bioreporters. Nearby cells absorb the AHL molecules released by one cell from the medium. This AHL binds with the luxR binding points in neighboring cells. This causes lux gene transcription to be initiated from the promotor (Plux), and more AHL production by these cells. The number of LuxR binding episodes increases with increasing AHL concentrations. Bioluminescence is intensified when multiple bioluminescing cell are involved.
“4.3 Materials and Methods to Build Bacteriophage Bacterial Cell Lines
Table 2 lists “Plasmids” and “Bacteria strains that are suitable for the practice of the invention.”
“TABLE 2\nPlasmids and bacterial strains utilized in bioluminescent bioreporter and luxI\nbacteriophage construction strategies.\nRelevant genotype/characteristics Reference\nPlasmids\nPCR? ?II 3.9kb cloning vector to PCR products with three? A Invitrogen\noverhangs, ApR, KmR Carlsbad, CA.\npUTK214 pUT/mini-Tn5KmNX ,ApR, KmR Applegate et\nal., 1998\npUTK222 pUT/mini-Tn5KNX-lux containing the promoterless lux Hay et al.,\ngene cassette with unique NotI-XbaI cloning sites for 2000\npromoter insertion, ApR, KmR\nBacterial Strains\nE. coli SV17- ?pir, recA, thi, pro, hsdR?M+, RP4:2-Tc:Mu:Km DeLorenzo et\n1(?pir) Tn7TpRSmR; mobilizing strain for pUT mini-Tn5 al., 1990,\nderivatives 1993\nE. coli INVIF? Strain used in combination with TA Cloning Vector, pCR? ?II Invitrogen\nF? ?80lacZ? ?M15 ? (lacZYA argF)U169 recA1 Carlsbad CAnendA1 HsdR17(rK?, mK+) phoA?E44? ?thi-1 gvrA96\nrelA1\nP. Fluorescens 5R Naphthalene metabolizing Strain, harboring the archetypal Sanseverino etnNAH plasmid pKA1 (93)
“4.3.1 Bioluminescent Bioreporter Line Responsive To AHL Inducer Molecules.”
“Only a single bioluminescent bioreporter cell line needs to be constructed since its function, to respond to AHL molecules, remains the same regardless of the bacteriophage/pathogen system with which it is coupled. These methods can be used to construct several bioluminescent cell lines for chemical sensing (Table 3).
“TABLE 3\nWhole cell bioluminescent reporters constructed utilizing\nthe MiniTn5NXlux transposon.\nBioluminescent\nReporter lux fusion Reference\nPseudomonas putida chromosomal-based tod-lux fusion for the Applegate et\nTVA8 detection of toluene al., 1998\nRalstonia eutropha tfd-lux to detect the herbicide 2,4- Hay et al., 2000\nJMP134-32 dichlorophenoxyacetic acid (2,4-D)\nPseudomonas putida Ferric uptake regulatory (fur) responsive Bright et al.,\nFeLux-1 promoter fused to lux to determine the 2000\nbioavailability of Fe in aqueous systems”
“The bioluminescent bioreporter is constructed by a promoterless luxCDABE genetic cassette in a MiniTn5 transposon named MiniTn5NXlux. (Applegate et al. 1998). This construct has a unique NotI/XbaI Cloning Site that allows for direct insert of promoter fragments. MiniTN5 transposon that contains luxR and an associated promoter element (hereafter luxR?luxproluxI) can be constructed by amplifying luxR and divergent promoter with appropriate primers containing base modification. These restriction sites allow for directional cloning. 3). Touchdown PCR is used to amplify the fragment to accept the primer modifications and decrease spurious products (Don et. al., 1991).
“The MiniTn5/luxR/luxpro transposon was transformed into E. coli SV17-1(pir) and biparentally mated to Pseudomonas Fluorescens 5R. P. fluorescens5R is the best strain for this purpose, as it produces the highest levels light (King and al. 1990). Transconjugants can be selected using minimal media with 50 mg/L kanamycin and salicylate to provide a single carbon and energy source. The E.coli donor strain harbors the archetypal NAH DNA plasmid, pKA1, which permits the strain to use naphthalene or salicylate as carbon and/or energy sources. Salicylate metabolism allows for the isolation of the recombinant Pseudomonas. Transconjugants, which are not inserted into the chromosome, are tested for their ability to increase the number of genetic reporter genes. The plasmid is extremely stable and can be mobilized into other strains, if needed.
“4.3.2 LuxI Bacteriophage”
“A variety of bacteriophage can be genetically incorporated into the luxI gene to detect unique pathogenic species. Table 4 lists examples of pathogens and associated phage. These examples were chosen because they have been extensively used for epidemiological typing of the specific pathogen. Based on phage adsorption coefficients, latency times, and lysis time in pure culture studies, temperate and virulent phages are more likely to produce higher AHL concentrations upon host infection. Carriere and colleagues have conducted studies to support this conclusion. (1997) using luxAB reporter phage to Mycobacterium tuberculosis. It was shown that rapid cell lysis with virulent phage caused a rapid decrease in light output, while the temperate reporter phage produced longer light responses due to an accumulation of luciferase proteins in the host. L. monocytogenes temperate (A511) and virulent (A118) phages have been sequenced. They can be used in the homologous recombination process described below to create luxI integrated Phage.
“TABLE 4nBacteriophages and their corresponding host pathogensnHost Paragon ReferencenVirulent PhagenKH1 Escherichiacoli O157:H7 Kudva, 1999nE79Pseudomonas Aeruginosa Hayashi 1981nFelixO-1 Salmonella spp. Stewart et al., 1998\nTwort Staphylococcus aureus Loessner et al., 1998\n?4 Campylobacter spp. Frost et al., 1999\nA511 Listeria monocytogenes Loessner et al., 1996\nTemperate Phage\n?V10 Escherichia coli O157:H7 Khakhria et al., 1990\nG101 Pseudomonas aeruginosa Miller et al., 1974\nP22 Salmonella spp. Chen and Griffiths 1996n??11 Staphylococcus Aureus Stewart et.al., 1985n??C Campylobacter species. Bokkenheuser et al., 1979\nA118 Listeria monocytogenes Loessner et al., 2000; van der Mee-\nMarquet et al., 1997”
Primers may be used to amplify the luxI gene of V. fischeri using standard PCR techniques. The 5? The primer contains stop codons in the three reading frames for the luxI start codon, ribosomal binding sites and a primer to prevent frame shifting that could lead to fusion proteins. To ensure optimal expression in the target organism, ribosomal binding site are modified. The resulting fragments are cloned in the TA cloning vector PCR. 2. According to the manufacturer’s instructions. To verify the orientation and size of fragments in transformants with inserts, they are subject to restriction analysis.”
“Strains with inserts in the correct orientation (lacproluxI), are screened to produce the diffusible AHL Signal by testing the supernatant’s induction activities using an AHL-responsive, bioluminescent reporter strain. After growing E.coli cultures with the correct inserts, the assay is performed by centrifugation followed by growth to an optical density (1.0) at 546nm. By adding aliquots of the reporter strain to the supernatant, the supernatant can be tested. For verification, clones that produce functional AHL will be sequenced.
“4.4 Bioluminescent Response.”
“The bioluminescent light response, regardless of whether it is bacterial or bacteriophage, is usually measured using optical transducers like photomultiplier tubes or photodiodes. Additional equipment is required to transfer the bioluminescent signal from the transducer. This includes fiber optic cables, lenses or liquid light guides. The result is often a bulky, heavy instrument that can’t be used in the field. use. Azur Corporation (Carlsbad Calif.) has among other things developed hand-held, battery-operated photomultiplier units that can directly be interfaced with a laptop.
“?Field-friendly? “Field-friendly? The bioluminescent integrated circuits (BBICs), which are bioluminescent and bioreporter, have a 5 mm2 surface. They consist of two main components: photodetectors to capture bioluminescent signals on-chip and signal processors for managing the storage and management of bioluminescence information. Remote frequency (RF) transmitters may be added to the overall circuit design for wireless data relay. All elements required are contained within the BBIC. This allows for the BBIC’s operational capabilities to be realized simply by exposing it to the test sample.
“4.5 Use of Bioluminescent Pathogen Detection Systems”
“4.5.1 Bioreporter Line Cell Line”
“Detection limits and response times, saturation kinetics, and basal expression levels lux (Winson, 1998) were observed in bioreporter cells lines using standardized bioavailability tests (Heitzer, et. al. 1992). The bioreporter cells are grown in yeast extract-peptone-glucose (YEPG) medium to exponential phase (OD546=0.35) whereupon 100 ?l aliquots are transferred to 96-well microtiter plates. Microtiter wells are incubated with Acyl homoserine Lactone (AHL), at concentrations of 0.01 to 1000ppm. Light readings are continuously taken using a scintillator counter for 24 hours. Vials containing no AHL are used to determine the background levels of bioluminescence caused by basal expression.
“Plotting background-corrected Bioluminescence against time produces standard curves that indicate detection limits and response times. For the analysis of AHL concentrations, standard HPLC techniques can be used (Winson and al. 1998). After baseline measurements have been made, microtiter plates are used to perform tests using the bioreporter and varying levels of luxI bacteriophage or associated pathogen. These tests are done in similar formats in order to determine detection limits, response time, saturation kinetics and background induction.
“Measurements can also be taken with an Azur DeltaTox (Carlsbad. Calif.) photomultiplier device. The Azur photomultiplier, a battery-operated handheld unit, interfaces directly with a laptop computer. This makes it ideal for field measurements. monitoring. To identify potential effects on bioreporter responses, parameters such as pH and temperature are closely monitored. To account for AHL molecules intrinsic to the organism, a negative control is made up of samples that are free of bacteriophage.
An integrated circuit photodetector can also be used to analyze samples. A test bed with integrated circuits to measure the replicate amount of induced bioreporter-bioluminescence. This analysis uses integrated circuits that are connected to a flowcell system, through which the desired substance passes. The integrated circuit records the bioluminescent reactions and then downloads them to a computer interface.
“4.5. “4.5.
“The physiological status of bacteria can influence the degree bioreporter reaction, since luminescence requires active reproduction of the bacteriophage (i.e. acyl-homoserine latone synthetase) in the pathogen. Analyses using log-phase cells could underestimate the field conditions sensitivity of bioreporter systems. Studies are done with strains of interest in a variety of physiological conditions, including starvation and disinfectant treatment with chlorine. Starvation can be induced by extracting log-phase cells and then rinsing the cells three times. Finally, these cells are stored in minimal salts media. To provide a wide range in metabolic states, stored cells are tested after 0 hr to 1 hr and 1 hr to 3 d, 3 days, and 7 days (Morita 1982). Similar log-phase suspensions that have been washed are also treated with a variety of levels of chlorine (0,0.5, 1, 2 and 3 mg/L) for two minutes to create a gradient in active cells (Boulos, et al. 1999).
“Samples of the different starvation times are evaluated for bioreporter responses. Several types of cell counts are performed, including 1) total direct count using acridine orange staining (Hobbie and al. 1977); 2) viable cells using LIVE/DEAD (Molecular Probes Eugene, Oreg. ), and 3) respiring cells using 5-cyano-2.3-diotyl Tetrazoloium (5CTC). Bioreporter tests may utilize an Azur Deltatox photomultiplier unit following standard procedures defined during baseline studies. AO counts are one of the many cell counts. They provide an estimate of total cells, and should not change with starvation or chlorine treatment. CTC counts are a measure of active respiration in cells that have been exposed to the fluorescent formazan dye (CTC dye) and cells that have active electron transport activity. CTC response (both as a function of the number and fluorescence per cells) responds rapidly to carbon source availability (Cook & Garland, 1997) and stress such a chlorine treatment (Boulos, 1999).
The LIVE/DEAD Baclight Kit contains two nucleic acid-binding stain stains that can be used to distinguish between viable and dead cells. SYTO 9, which is a fluorescent green dye, passes through all cells. Propidium iodide can penetrate cells only with damaged membranes and stain them red. Dual staining gives separate estimates of dead (red) and live cells, using membrane integrity to distinguish them. LIVE/DEAD counts have shown less response to stress than CTC count estimates (Boulos and al. 1999; Braux and al. 1997). Analyzing samples simultaneously with the different methods allows for a direct assessment of the impact that respiration and viability play on the bioreporter’s response. It is crucial to determine the extent to which viable cells, but not actively breathing, respond to the biosensor assay. If viable cells respond weakly to the biosensor assay, it is possible that potentially virulent cells are not detected.
“4.5.3 The Effect of the Sample Matrix on Bioluminescence
The bioreporter’s response may be affected by the sample matrix, which is particulate matter that includes microorganisms. Particulate matter can bind to cells and prevent infection of the bacteria, which could lead to non-specific phage binding and/or general quenching. This could decrease the detection limit of a bioreporter. Non-specific infections, though unlikely, can lead to false positives.
“In certain embodiments of the invention it may be desirable for the bioreporter cell to be immobilized in a stabilizing matrix. Without adverse effects on viability, alginate has been used successfully to encapsulate cells. As long as alginate-encased cells are moist, it is possible to sustain long-term viability (weeks or months). Lyngberg et. al., 1999 reported that latex copolymers are also useful in immobilizing E. Coli as well as maintaining viability. Other matrices include carrageenan, acrylic vinyl acetate copolymer, polyvinyl chloride polymer, sol-gel, agar, agarose, micromachined nanoporous membranes, polydimethylsiloxane (PDMS), polyacrylamide, polyurethane/polycarbomyl sulfonate, or polyvinyl alcohol. You may also use electrophoretic deposition.
“4.5.4 Shelf-Life Assessment and Bioreporter Lyophilization”
“Lyophilization (freeze drying) is a significant advantage when using microorganisms to report on the environment. This allows for long-term storage, ranging from months to years. There is little risk of them losing viability. It is desirable that the bacteriophage/bioreporter system be placed in a physiological state amenable to long-term storage such that the end-user can simply revive a pellet of cells whenever measurements are required. These components are lyophilized and resuscitated at different intervals to determine the shelf life of the bioluminescent bioreporters. Bioluminescence is then measured in accordance with the above.
“5.0 EXAMPLES”
“5.1 Example 1”
“Amplification of Bioluminescent Sign in Bioreporter cells”
The detection scheme used to quantify pathogenic targets is based on the ability of AHL molecules induce bioluminescence so that it can be correlated and correlated with the original number AHL-producing targets in the sample. This technique is based on the same principles as quantitative PCR, except that the initial AHL concentrations (instead of nucleic acids) allow differential detection of the exponential rise in signal, i.e. bioluminescence in reporter cells (Heid and al., 1996).
“Amplification of bioluminescent responses of bioreporter cell occurs through the autoinduction mechanism V. fischeri AHL is released to the extracellular environment after target cell infection with the biosensor (i.e. a luxI bacteriophage). Light production is stimulated by the neighboring bioluminescent bioreporter cell’s uptake of AHL. This AHL stimulates the production of more light. This cascade effect results in intense bioluminescent light because of the involvement of multiple binding episodes within multiple bioluminescent cell types.
“FIG. “FIG. V. fischeri overnight culture was diluted to 0.01 OD546. Standard dilutions for N-(3-oxohexanoyl] homoserine lactone (Quorum Sciences were prepared by resuspending 213 mg in 1mL acidified ethyl alcohol. This gave rise to a 100mM stock, followed by dilutions with acidified ethyl alcohol. After placing 100 mL of N- (3-oxohexanoyl] homoserine lactone/ethyl-acetate solution in shell vials, the assays were carried out. The test vials were then filled with one mL of V. fischeri (prepared in the same manner). The vials were shaken at 140 RPM. Light measurements were taken at both time zero and 30-minute intervals with a Zylux portable lumenometer. The data were plotted in photons per second against time. This was the average of three replicates.
“A threshold line is used in data analysis to distinguish between samples (FIG. 4). This line indicates the photons per second at a given time when the bioluminescent bioreporter has been autoinduced. The curve characteristics on the graph determine this value. Autoinduction occurs when the curve’s slope increases rapidly. To determine the threshold line for the assay, the autoinduction of the control specimen is used. After determining the threshold value, the sample data can be analyzed to determine the time when autoinduction occurred. Autoinduction occurs sooner if the inducer concentration is higher. You can use this in either a quantitative or qualitative format, depending on your application.
The results showed that homoserine lactone concentrations are more important than light production. 4) All samples achieved the same light levels. However, it is only the beginning of the geometric increase of light that allows quantification of inducers. These results show that homoserine-lactone molecules increase the lux-based, bioluminescent signal in quantifiable ways.
“In the bacteriophage/bioreporter system, the target bacterium is infected by a specific bacteriophage carrying the luxI gene. The bacterium becomes capable of producing the inducer, acylhomoserine lactone, (AHL) after infection. The higher the number of phage infections, the more AHL is produced and the shorter time it takes for the light to be produced in bioluminescent bioreporter cell cells. To measure the amount or number of bacteria infected with the luxIbacteriophage, the time difference between the samples and the control is used.
“5.2 Example 2”
“Exemplary strains of LuxI Bacteriophage
“Because the genomes A511 and A118 of L. monocytogenes phages A511 have been characterized, luxI incorporation in these bacteriophage can be achieved through homologous recombination. The A511:luxAB phage was described by Loessner et. al. (1996). This is the basic strategy. The luxI construct is described in section 4.3.2. It contains appropriate ribosome binding site for L. monocytogenes. A set of primers containing flanking sequences at the 3? End of the cps gene at phages A511 and 118. The product is amplified, and then inserted into the Phage by Recombination. Bacteria are screened and enhanced essentially according to Loessner and colleagues (1996). Supernatants from primary lysates are tested for their ability inducing the lux genes of the reporter strain, since AHL production is the preferred phenotype. RFLP analysis is used to verify that the phage containing luxI has been selected.
For uncharacterized phage genes, the transposon mutagenesis technique of Waddel & Poppe, 1999 can be used to generate luxI phage constructs. The MiniTn5 transposon contains a promoterless gene for luxI that is used to mutagenize the phage. The MiniTn5luxI transposon can be constructed by inserting the appropriate luxI previously constructed into the unique cloning location of pUTK214. (Applegate and al., 1998). The construct is converted into E. coli SV17-1 (pir). The restriction fragment analysis is used to screen transformants for inserts. Once MiniTn5luxI transposons have been identified, they can be used to mutagenize (via biparental marriage) the appropriate phages for that specific application. The screening of phages is the same as described by Waddell and Poppe (1999), with the exception that kanamycin is used to select. The bioluminescent reporter strain is used to screen Phage that show the KmR genotype for production of acylhomoserine lactone. The detection assays are performed on phage that produce significant amounts of AHL.
“The M13 bacteriaiophage is an example of a system that can detect various pathogens. luxI genes can also be modified to be expressed by the following organisms: E. coli o157:H7 L. monocytogenes Salmonella spp. Campylobacter spp. B. anthracis B. thuringiensis B. thuringiensis B. subtilus.”
“5.3 Example 3”
“Detection and Treatment of Bacterial Pathogens In Food”
Salmonella, Listeria monocytogenes and Escherichiacoli O157-H7 are four pathogens that are particularly concerning to the food industry. Recent estimates show that these four types of bacteria are responsible for more than 95% of all food-related death in the U.S. (Mead and al. 1999). Cantaloupe (Riess and al. 1990), alfalfa seeds (Mahon and al. 1997), tomatoes (Hedberg und al. 1994) as well as watermelon (del Rosario and al. 1995). Salmonella-related illnesses have been linked to cantaloupe. A wide range of fresh vegetables have been found to contain salmonella, including artichoke and cabbage, celery and eggplant as well as lettuce mustard cress and parsley (Sumner & Peters, 1997).
E. coli O157.H7 is an emerging human pathogen that was first linked to outbreaks of food illness (e.g., fast food hamburgers). It can cause death in children and adults, especially if it produces enterohemorrhagic toxic toxins. Although cattle appear to be the primary reservoir, E.coli O157H7 has been linked with outbreaks in cantaloupe and broccoli (Sumner & Peters 1997). Cubed watermelon and cubed melon can be used to grow the organism (del Rosario & Beuchat 1995). It has also been isolated from celery, cilantro, coriander, and cabbage.
“Campylobacter, an emerging pathogen that causes acute stomachitis and Guillan-Barre syndrome (an acute neurological disorder), has been recognized as a common antecedent. Most often, illness is associated with contaminated poultry or raw milk. However, Campylobacter can also be found in raw fruits and vegetables (Bean et al. 1990; Harris et. al. 1986).
“The invention can be used to detect food-borne pathogens, taking into account the nature of the sample being tested. Sample matrix, which is particulate material that results from the rinsing and blending of vegetable matter, can affect the bioreporter’s response. Particulate material can bind to target cells, block infection by the bacteria, and/or cause general quenching in the light signal from the bioreporter cell. To test the effect of the sample matrix on the bioreporter, samples from tomato and lettuce may be tested. Lettuce, tomato, and other plant types are used as examples. They are both commonly eaten fresh produce that has been contaminated with some or all of the pathogens important to the food industry. The two crops are also distinct types of plant material (i.e. leafy vegetables versus fruit), with potentially unique matrix characteristics.
“Surface washings are made of lettuce by shaking aseptically-cut strips in sterile tris buffer for 2 hr. Then, homogenized samples can be obtained using a stomacher mixer (Donegan et. al., 1991; Jacques and Morris 1995). Tomatoes can be surface rinsed using gentle hand rub and placed in sterile bags with sterile buffer. The stomacher blender homogenizes them (Zhuang, et al. 1995). Others use mixed samples of tomato and lettuce. Log-phase cells can be introduced at levels that are well within the detection limits established by baseline studies. Results with additional vegetable matter are also compared to controls.
“Other tests provide baseline information about the effectiveness of the biosensor for detecting contamination in plant production, processing and distribution systems. Plant material is given specific amounts of pathogens either during or shortly after the plant’s growth cycle. The survival of the pathogen can be compared using the biosensor to monitor its survival up until harvest and after storage. This is then compared with the results obtained by reverse transcriptase – quantitative PCR (Heid, 1996) as described below.
“Tomatoes and lettuce are examples of how the invention can be used to detect foodborne pathogens. The typical production cycle of lettuce (cv Waldmann?s Green) takes 28 days in controlled environmental chambers (Wheeler and al., 1994). The production cycle of tomato (cv. Reimann Phillip) is grown in controlled environment chambers for 90 days. However, ripe fruits can be harvested as early as day 60 (Mackowiak and al., 1999). The plants are inoculated for pathogens on the day 60-aged fruit, and they are then removed from the plant after 1, 7, 14 and 21 days or at other appropriate intervals.
“Reverse transcriptase quantitative PCR (RTQPCR)” is a method that can be used to determine the copy number of DNA in a sample of nucleic acid extracts (Heid et. al. 1996). This method uses either PCR or RNA to target amplification and kinetic detection of PCR product at each cycle of the ABI 7700 Sequence Detection System in Foster City, Calif. The specific target molecule can be detected in a single PCR reaction. This involves primers that flank the area of interest, a dual-labeled diagnostic probe with a fluorescent reporter dye as well as a quencher. The 5?-nuclease activity (Taq) cleaves perfectly annealed probes. The result is an increase in reporter signal. Laser-induced fluorometry (LCD) and charge coupled devices are used to detect the fluorescent signal produced by PCR products labeled. This instrumentation and method are compatible with multiple reporter chemicals.
“The TaqMan? The assay (Applied Biosystems Foster City, Calif.), can detect 5 copies of the target against a background of 500 ng non-target DNA/RNA. This is 5 copies per sample and has a dynamic range of five orders of magnitude (i.e. 5 to 106 copies). RTQPCR is currently the most sensitive method to quantify and detect molecular targets in pathogen detection. It is therefore the best method to evaluate biosensor sensitivity.
“Primer-directed PCR amplification combined with probe hybridization has a higher specificity than membrane-bound nucleic acids hybridization. It allows for the quantitative detection of DNA and RNA. RT-PCR can be used to quantify mRNA or rRNA. This allows us to identify potential pathogens in agricultural products and determine their relative proportions. Both mRNA and rRNA are related to the physiological state of the cell/community; rRNA generally increases with growth rate.
Two primer-probe detection systems are used to detect Salmonella in QPCR. They target different molecular markers that are specific for Salmonella. The TaqMan? The TaqMan? in raw meat products (Chen et al., 1997; Kimura et al., 1999). The second primer set amplifies either a 173 bp (or 107 bp) product from a Salmonella typhimurium-specific region of the phase 1-flagellin filament gene. Its application has been demonstrated in the quantitative detection in soil systems of viable, but non-culturable S. Typhimurium populations.
QPCR detects Listeria monocytogenes by targeting the hemolysin-A (hlyA), transcript, which is unique to Listeria. Norton and Batt (1999) developed the hlyA detection method. It allows for the quantitative detection and quantification of viable Listeria populations using a 210-bp segment from the transcribed hlyA genes as a target.
“Escherichiacoli O157H7 detection for QPCR uses the 5? Oberst et al. developed a nuclease test. (Oberst and al., 1998) which targets the intimin-protein encoded by eaeA. The eaeA test generates a 631 bp section of the gene that contains target sequence, which may be detected using one of four probe sequences.
“Campylobacter Jejuni detection uses the nestedPCR approach of Winters and al. (1998, 2000) In a modified 5 Nuclease assay. Initial PCR amplification produces a 159-bp product, which can be used as a template for a second one using the original forward primer and a complementary reverse primer to a region within the 3. End of the first amplification product. The nested product produces a C. jejuni product with a 122-bp C. product, but no C. coli.”
“5.4 Example 4”
Summary for “Bioluminescent biosensor device”
“1.1 Field of The Invention.”
“The invention relates to methods and devices that detect targeted microorganisms, such as bacteria, by inducing bioluminescence within bioreporter cell cells. Infecting target bacteria with genetically engineered bioreporter cell-derived bacteriophage is possible. An inducer stimulates the bioreporter cells to produce light. The bacteriophage infects the target bacteria and produces the inducer.
“1.2 Description of Related Art”
“The current technology is focusing on the development biologically-based detection methods. This has led to efforts to identify specific microbial pathogens. There have been many methods to determine the presence of microbial contaminants over the years. Typically, culture methods were used in the past, but they were slow and inefficient. Bioreporter technology has allowed for the use of genetically engineered bacteria and bacteriophage in order to identify toxic chemicals, as well as specific species of bacteria.
Bioreporters are genetically engineered organisms that detect specific compounds. They do this by inducing expression of a detectable product gene in the bioreporter cells using a promoter that is heterologous to the target compound. As used herein, bioluminescent bioreporters are genetically engineered bacteria that have genes that, when expressed, result in bioluminescence. The bioreporter cells respond to the presence of a particular compound by emitting light. The lux gene is a popular gene that can be used for this purpose. The lux genes can be expressed under the right conditions and bioluminescence can be detected using a variety optical methods. Many constructs found in bioluminescent bioreporter species are derived from Vibrio fischeri, a bioluminescent marine bacteria (King and al. 1990).
“Sayler et al. “Sayler et al. A variety of lux-based bacterial bioreporters has been used to detect and monitor naphthalene (Heitzer et al., 1994), BTEX (benzene, toluene, ethylbenzene, and xylene) (Applegate et al., 1998), polychlorinated biphenyls (PCBs) (Layton et al., 1998), 2,4-dichlorophenoxyacetic acid (2,4-D) (Hay et al,. 2000, ammonia (Simpson and al. 2001), and the food-salivating indicator chemical?phenylamine (Rippet al. 2000a).
“Genetic constructs that impart bioluminescence to bacterial Bioreporter Cells have in general used a lux gene cassette derived form the marine bacterium Vibrio Fischeri (Engebrecht et al. 1983). As used herein, ?cassette? A recombinant DNA construct is one that is made from a combination of inserted DNA sequences and a vector. The complete lux cassette is composed of five genes (i.e. luxA, B. C., D, and E. These genes are responsible for producing bioluminescence. luxC. and D code for an aldehyde.
“The light response of bioluminescent bioreporters can be measured using optical transducers, such as photomultiplier tube, photodiodes or microchannel plates, and charge-coupled devices. It is necessary to transfer the bioluminescent signal from the transducer. This requires fiber optic cables, lenses, or liquid light guides. These instruments are not suitable for field use. This results in a bulky, heavy instrument that is attached to power and optic cables. Ripp et. al. described field release experiments as an example. A bioluminescent bioreporter was developed for the detection and monitoring of naphthalene in soil. Multiplexed photomultiplier tubes were used to detect bioluminescent signals. These cables were expensive, fragile and cumbersome to use.
“Battery-operated hand-held photomultiplier devices that can be interfaced with a computer laptop have been described. They are used in conjunction bioreporters to analyze groundwater for hydrocarbon contamination (Ripp, 1999a). Special bioluminescent integrated circuits for bioreporter integrated systems (BBICs), have been described (Simpson et. al. 2001). These self-contained units can detect environmental contaminants like naphthalene or BTEX by simply exposing them to these compounds (Ripp et. al. 1999b). These devices use bioluminescent bioreporters, which are genetically modified bacteria bioreporters. They respond to certain chemicals by producing visible light.
Another area of interest is the detection of pathogenic organisms. This is in contrast to chemical agents. For human safety, pathogens like those that cause human and animal disease, foodbome diseases and those used to wage biological warfare are very important. The need for advanced detection systems is further reinforced by the constant appearance of new strains.
“Microbial contamination of fresh fruits or vegetables is a growing concern in the food industry. This is due to increased consumption of these products and recognition of new foodborne pathogens like Escherychia coli o157:H7 and Campylobacter jejuni (Tauxe 1992). While federal agencies have recommended safe food handling practices to minimize risk, they are still unable to provide rapid and accurate methods of detecting pathogens in production, processing and distribution systems. Particular concern when monitoring food safety is the identification of bacteria that causes the majority food-related deaths in America, such as Salmonella, Escherychiacoli O157.H7, Escherychia monocytogenes, Escherychia pneumoniaes, and Campylobacter.
Bioluminescent methods are used in the food industry to detect bacterial contamination. One technology that relies on the detection of ATP is based on the biochemical requirements of bacteria to use ATP for energy production. This is essential for growth and survival. The ATP detection method does not distinguish between bacterial species and does not distinguish pathogens from non-pathogenic ones (Vanne et al. 1996).
“Several reports have reported bioluminescent detections of target bacteria using bacteriophage infection. These procedures have identified a select number of pathogens, as shown in Table 1.
“TABLE 1\nBioluminescence detection of bacterial pathogens by\nbacteriophage containing a luxAB insert.\nPathogen Bacteriophage Detection Limit Test Source Reference\nEnterobacteriaceae Unspecified 10 cells/g/cm2 Surface and meat Kodikara\ncarcass swabs et al.,\n1991\nEscherichia coli ? Charon 100 cells/ml Milk Ulitzer and\nspecies Kuhn,\n1987\nEscherichia coli ? V10 Not determined Pure culture Waddell\nO157:H7 and\nPoppe,\n1999\nListeria A511 10 cells/g Cheese, pudding, Loessner\nmonocytogenes cabbage et al.,\n1996\nSalmonella P22 10 cfu/ml Eggs Chen and\nspecies Griffiths,\n1996\nSalmonella P22 100 cells/ml Pure culture Stewart et\ntyphimurium al., 1989\nStaphylococcus Unspecified 1000 cfu/ml Pure culture Pagotto et\naureus al., 1996”
“In all these cases, the bacteriaiophage contained only an incomplete lux gene (i.e. luxAB. Although useful for the detection of certain pathogenic species, there are several drawbacks to this technique. For detection of the bioluminescent response, it is necessary to have an exogenous source for the aldehyde substrate required for the luciferase reaction if only the luxAB genes were used. This can cause problems when attempting to detect the bioluminescent response. Additional problems arise when conditions like the amount of inducer used may need to be changed. This can be especially problematic if you use the methods in environments such as farms, where the environment is not conducive to running the tests and the end-user may not have the necessary training.
“An additional problem with bacterial detection is the fact that pathogens can often be found in low levels. Existing bioluminescent techniques may not be able to detect such low levels of pathogens. There are many non-bioluminescent methods that can be used to detect low levels of bacteria. These methods include amplification steps such as sample pre-enrichment to increase pathogen concentrations to detectable levels or DNA-based polymerase chains reaction (PCR). These amplification steps have a disadvantage: they require extensive user training, and costly instrumentation.
“There is a need to develop methods and devices that can detect specific bacteria, especially pathogens, and do so selectively, accurately, quickly, and with high sensitivity. To monitor a wide range of common pathogens such as those that are implicated in food safety, food processing, hospital environments, and biological warfare, new devices are required.
“The present invention addresses some of the deficiencies in the methods and devices presently employed in detecting individual species of bacteria, by providing a novel internally amplified bioluminescent bacteriophage/bioreporter system. The disclosed devices allow for rapid and sensitive detections of specific pathogens using a fully integrated, easy-to-use system that requires no more than sample addition. A signal amplifier mechanism is integrated into the design to increase the device’s sensitivity. Two cooperating elements are included in the invention, i.e. The invention includes two cooperating elements, i.e. biosensor and bioreporter element that work together in a unique two-step process. The invention’s biosensor elements are demonstrated by genetically modified bacteria, while the bioreporter element may be any one of many genetically modified cell line. The biosensor bacteriophage infects a selected pathogen (e.g., bacteria) and causes an inducer to cause the bioreporter cells line to express the Lux gene cassette. This results in amplified bioluminescence which is easily detectable.
“In certain embodiments, the invention uses a bacteriophage that has been genetically modified to have a luxI-gene. The luxI gene encodes the protein product, acyl heteroserine latone synthetase. This enzyme performs a condensation reaction between cell metabolites that results in the production acyl en homoserine (AHL) lactone. The genetically modified bacteria is introduced to a selected target bacterium. The phage luxI gene of the bacterium is transcribed into the bacterium. This results in the expression of the LuxI protein in infected cells. The target cell produces AHL molecules that diffuse into the surrounding medium.
“Infected bacteria is caused by the presence of bioreporter cell, which have been genetically engineered to produce light when stimulated with AHL. The bioreporter cells don’t produce any light if the AHL inducer is absent. The bioreporter cell’s ability to absorb AHL molecules from the environment after phage infection causes the production of bioluminescent protein. This is because the bioreporter cells are genetically engineered with a lux gene cassette (luxR+luxI+luxCDABE), which is responsive to AHL. AHL is an autoinducer which positively regulates lux operon. The AHL complex stimulates the lux genes of the bioreporter cells, which results in the production light.
“The invention’s unique feature is the amplifying of bioluminescence by the presence of lux-modified bioreporter cell. The induction of the light-producing genes in one bioreporter cells results in the production not only of light-producing protein but also AHL molecules. These AHL molecules diffuse from the light-producing bioreporter cell and induce the expression of the lux gene in nearby bioreporter cell. Intense bioluminescence is caused by this cascade effect that involves multiple bioreporter cell neighbors. Infection of a target bacteria results in a chain reaction that produces bioluminescence in many bioreporter cell. This allows for detection of extremely low levels of target bacteria. This innovative integrative method allows rapid detection of pathogens without the need for sample enrichment.
“The bacteriophage/bioreporter system employs a luxI-integrated bacteriophage that infects only a particular bacterium. The invention involves the first selection of a target bacterium. Next, the identification of a bacteriophage that is specific for the target bacterium is made and then genetic engineering the bacteriophage with the luxI gene. You can use the specificity of phage infections to detect, monitor and identify certain species of bacteria.
“In most situations in real life, the target bacteria is in a natural environment. This can often be in the presence of microbes or other contaminants. The bacteriophage/bioreporter system addresses this problem by directing the luxI bacteriophage against specific strains of bacteria. You can choose from many commonly recognized pathogens to target bacteria. Several types of bacteria are often linked to food contamination. These include Salmonella, Escherychia coli, O157:H7, Listeria moncytogenes and enterobacteriaceae.
Many other bacteria can be detected with the methods and devices described, provided that an appropriate infectious phage is identified or engineered. In practice, the first step in identifying a pathogen is to identify a pathogen-specific bacteria. There are many. For example, bacteriophage m13 which infects E. coli is one of them. Additional examples of bacteriophage which specifically infect pathogenic bacteria species are found in Table 4.
“An important consideration is the identification of a species-specific bacteriophage that harbors the luxI cassette, and the ability to penetrate the bacterial cells so that the luxI gene can be expressed in the target bacterium. E.coli can be infected by bacteriophage, M13. coli.”
“Some embodiments of this invention allow simultaneous contact with a sample using multiple bacteriophage-biosensors. Each of these biosensors specifically recognizes a specific bacterium and is kept in a separate compartment. Multiple target cells may be detected simultaneously in this manner. The bacteriophage/bioreporter elements can be integrated onto a chip surface to provide a convenient, easily handled device.”
“Some embodiments include multi-component packaged kit kits that contain both sensor and detector elements to detect select strains. These kits can be used to detect bacterial presence in samples. They contain one or more types genetically engineered bacteriaophages, each of which is designed to infect a specific bacterium and, upon infecting, cause the bacterium to express an inducer molecule. The bacteriophage may contain a luxI genetic product that results in the formation of AHL, which is an inducer of lux genes in bioluminescent cells. The kits contain a number of genetically engineered bacteria bioreporter cells that are capable of producing bioluminescence when they are stimulated with the inducer. Particular embodiments of the bioreporter bacteria include a luxR?luxpro/luxI/luxCDABE gene that is stimulated with AHL to produce light. Instructions for use and optionally a device to measure the generated light may be included in kits. This could include an integrated circuit that detects a bioluminescent signal. An integrated circuit could include a photodetector and low-noise electronics (e.g. On-chip wireless communication system, biocompatible housing and semi-permeable membrane that covers the bioreporter area.”
The disclosed methods and devices are based on biologically-based sensor technology, which can be easily adapted to quality control programs and pathogen detection. This system is extremely easy to use because the necessary elements, biosensors and autoamplifying biologicalluminescent bioreporters, are integrated into the detector device’s compartments. The system is simple to use. Simply contact a sample with the detector device’s sample chamber and allow the device to process it and send the results. This technology is a new way to detect, monitor, and prevent biological contamination.
“4.1 Vibrio Fischeri Bioluminescence”
“Genetically engineered bioluminescent bioreporter cell cells are used in the invention. These cells are capable of producing light due to the incorporation of lux genes. They are responsible for bioluminescence produced by the marine bacterium Vibrio Fischeri. FIG. FIG. 1. This is a schematic representation illustrating the lux genes. It shows positive regulation by the luxI- and luxR gene product. The luciferase gene (luxAB), encodes proteins that generate bioluminescence, while the synthetase and reductase(luxC), transferase/luxD genes code for proteins required for the production of an aldehyde substrate. FIG. FIG. The luxR, luxI gene regulatory elements are represented by small circles. Acyl-homoserine (AHL), synthetase is the expressed product of luxI gene. Its product, acyl-homoserine lactone [AHL], acts as an inducer for the bioluminescent response. AHL (represented by black circles) forms a complex that is bound to the expression product of the LuxR gene, i.e. The LuxR transcriptional regulator is represented by white circles. This complex (black and wavy circles) binds with the promoter of the luxI (black box). Inducing transcription is done in the direction indicated by the lower arrow of luxI or luxCDABE. These codes code for proteins involved in biochemical reactions that produce 490 nm of light. This mechanism of positive regulation by the LuxR?AHL complex of the lux operon occurs in both native Vibrio fischeri as well as in the recombinantly added lux gene cassette in bioreporter cells.
Autoinduction is a mechanism that amplifies light generation in Vibrio cells and bioreporter cells. AHL is produced by the luxI gene. This causes AHL to diffuse into the extracellular environment and induce luxI and luxCDABE transcription in nearby bioluminescent cells. This cascade effect eventually generates intense bioluminescence. Autoinduction is a mechanism that increases the production of light by engaging many cells. This invention uses autoinduction to increase the production of light using genetically engineered lux -based bioluminescent bioreporter cell.
“4.2 Bioluminescent Bioreporter System”
“As shown in FIG. “As seen in FIG. 2, the invention uses two elements, i.e. A biosensor and a Bioreporter are the two elements of the invention. The biosensor serves two purposes. A luxI-integrated bacteriophage is an example of a biosensor that infects a specific strain of bacteria. The biosensor DNA infects the target bacterial cell and causes it to produce the gene products encoded by biosensor DNA. The luxI-integrated bacteriophage case shows that the infected bacterium generates the luxI gene product acylhomoserine lactone synthetase. This leads to the ultimate production of AHL.
“A second element of the bacteriophage/bioreporter system is the bioreporter cell. The bioreporter serves two purposes. It responds to the biosensor signal and amplifies that signal to allow multiple bioreporters to be responsive to the signal from infected targets cells. A bioreporter is a bacterial line that has been genetically engineered so it can produce light when stimulated by the target cell signal. AHL is the signal used in systems that use luxI bacteriophage.
“FIG. 2 shows a bacteriophage/bioreporter system in which the bacteriophage biosensor incorporates a luxI construct and the bacterial bioreporter cell incorporates the lux R+I+CDABE constructs. By including the entire luxCDABE gene cassette, the bioreporter element can produce light without the need to add exogenous chemicals. This is because the luxI-luxR genes positively regulate the lux operon in Vibrio as well as in genetically engineered bioreporter cell cells. (FIG. 1).”
“Using the autoinduction mechanism Vibrio fischeri to amplify the bioluminescent signal, invention is achieved through diffusion and uptake by multiple bioreporter cell bioreporters. Nearby cells absorb the AHL molecules released by one cell from the medium. This AHL binds with the luxR binding points in neighboring cells. This causes lux gene transcription to be initiated from the promotor (Plux), and more AHL production by these cells. The number of LuxR binding episodes increases with increasing AHL concentrations. Bioluminescence is intensified when multiple bioluminescing cell are involved.
“4.3 Materials and Methods to Build Bacteriophage Bacterial Cell Lines
Table 2 lists “Plasmids” and “Bacteria strains that are suitable for the practice of the invention.”
“TABLE 2\nPlasmids and bacterial strains utilized in bioluminescent bioreporter and luxI\nbacteriophage construction strategies.\nRelevant genotype/characteristics Reference\nPlasmids\nPCR? ?II 3.9kb cloning vector to PCR products with three? A Invitrogen\noverhangs, ApR, KmR Carlsbad, CA.\npUTK214 pUT/mini-Tn5KmNX ,ApR, KmR Applegate et\nal., 1998\npUTK222 pUT/mini-Tn5KNX-lux containing the promoterless lux Hay et al.,\ngene cassette with unique NotI-XbaI cloning sites for 2000\npromoter insertion, ApR, KmR\nBacterial Strains\nE. coli SV17- ?pir, recA, thi, pro, hsdR?M+, RP4:2-Tc:Mu:Km DeLorenzo et\n1(?pir) Tn7TpRSmR; mobilizing strain for pUT mini-Tn5 al., 1990,\nderivatives 1993\nE. coli INVIF? Strain used in combination with TA Cloning Vector, pCR? ?II Invitrogen\nF? ?80lacZ? ?M15 ? (lacZYA argF)U169 recA1 Carlsbad CAnendA1 HsdR17(rK?, mK+) phoA?E44? ?thi-1 gvrA96\nrelA1\nP. Fluorescens 5R Naphthalene metabolizing Strain, harboring the archetypal Sanseverino etnNAH plasmid pKA1 (93)
“4.3.1 Bioluminescent Bioreporter Line Responsive To AHL Inducer Molecules.”
“Only a single bioluminescent bioreporter cell line needs to be constructed since its function, to respond to AHL molecules, remains the same regardless of the bacteriophage/pathogen system with which it is coupled. These methods can be used to construct several bioluminescent cell lines for chemical sensing (Table 3).
“TABLE 3\nWhole cell bioluminescent reporters constructed utilizing\nthe MiniTn5NXlux transposon.\nBioluminescent\nReporter lux fusion Reference\nPseudomonas putida chromosomal-based tod-lux fusion for the Applegate et\nTVA8 detection of toluene al., 1998\nRalstonia eutropha tfd-lux to detect the herbicide 2,4- Hay et al., 2000\nJMP134-32 dichlorophenoxyacetic acid (2,4-D)\nPseudomonas putida Ferric uptake regulatory (fur) responsive Bright et al.,\nFeLux-1 promoter fused to lux to determine the 2000\nbioavailability of Fe in aqueous systems”
“The bioluminescent bioreporter is constructed by a promoterless luxCDABE genetic cassette in a MiniTn5 transposon named MiniTn5NXlux. (Applegate et al. 1998). This construct has a unique NotI/XbaI Cloning Site that allows for direct insert of promoter fragments. MiniTN5 transposon that contains luxR and an associated promoter element (hereafter luxR?luxproluxI) can be constructed by amplifying luxR and divergent promoter with appropriate primers containing base modification. These restriction sites allow for directional cloning. 3). Touchdown PCR is used to amplify the fragment to accept the primer modifications and decrease spurious products (Don et. al., 1991).
“The MiniTn5/luxR/luxpro transposon was transformed into E. coli SV17-1(pir) and biparentally mated to Pseudomonas Fluorescens 5R. P. fluorescens5R is the best strain for this purpose, as it produces the highest levels light (King and al. 1990). Transconjugants can be selected using minimal media with 50 mg/L kanamycin and salicylate to provide a single carbon and energy source. The E.coli donor strain harbors the archetypal NAH DNA plasmid, pKA1, which permits the strain to use naphthalene or salicylate as carbon and/or energy sources. Salicylate metabolism allows for the isolation of the recombinant Pseudomonas. Transconjugants, which are not inserted into the chromosome, are tested for their ability to increase the number of genetic reporter genes. The plasmid is extremely stable and can be mobilized into other strains, if needed.
“4.3.2 LuxI Bacteriophage”
“A variety of bacteriophage can be genetically incorporated into the luxI gene to detect unique pathogenic species. Table 4 lists examples of pathogens and associated phage. These examples were chosen because they have been extensively used for epidemiological typing of the specific pathogen. Based on phage adsorption coefficients, latency times, and lysis time in pure culture studies, temperate and virulent phages are more likely to produce higher AHL concentrations upon host infection. Carriere and colleagues have conducted studies to support this conclusion. (1997) using luxAB reporter phage to Mycobacterium tuberculosis. It was shown that rapid cell lysis with virulent phage caused a rapid decrease in light output, while the temperate reporter phage produced longer light responses due to an accumulation of luciferase proteins in the host. L. monocytogenes temperate (A511) and virulent (A118) phages have been sequenced. They can be used in the homologous recombination process described below to create luxI integrated Phage.
“TABLE 4nBacteriophages and their corresponding host pathogensnHost Paragon ReferencenVirulent PhagenKH1 Escherichiacoli O157:H7 Kudva, 1999nE79Pseudomonas Aeruginosa Hayashi 1981nFelixO-1 Salmonella spp. Stewart et al., 1998\nTwort Staphylococcus aureus Loessner et al., 1998\n?4 Campylobacter spp. Frost et al., 1999\nA511 Listeria monocytogenes Loessner et al., 1996\nTemperate Phage\n?V10 Escherichia coli O157:H7 Khakhria et al., 1990\nG101 Pseudomonas aeruginosa Miller et al., 1974\nP22 Salmonella spp. Chen and Griffiths 1996n??11 Staphylococcus Aureus Stewart et.al., 1985n??C Campylobacter species. Bokkenheuser et al., 1979\nA118 Listeria monocytogenes Loessner et al., 2000; van der Mee-\nMarquet et al., 1997”
Primers may be used to amplify the luxI gene of V. fischeri using standard PCR techniques. The 5? The primer contains stop codons in the three reading frames for the luxI start codon, ribosomal binding sites and a primer to prevent frame shifting that could lead to fusion proteins. To ensure optimal expression in the target organism, ribosomal binding site are modified. The resulting fragments are cloned in the TA cloning vector PCR. 2. According to the manufacturer’s instructions. To verify the orientation and size of fragments in transformants with inserts, they are subject to restriction analysis.”
“Strains with inserts in the correct orientation (lacproluxI), are screened to produce the diffusible AHL Signal by testing the supernatant’s induction activities using an AHL-responsive, bioluminescent reporter strain. After growing E.coli cultures with the correct inserts, the assay is performed by centrifugation followed by growth to an optical density (1.0) at 546nm. By adding aliquots of the reporter strain to the supernatant, the supernatant can be tested. For verification, clones that produce functional AHL will be sequenced.
“4.4 Bioluminescent Response.”
“The bioluminescent light response, regardless of whether it is bacterial or bacteriophage, is usually measured using optical transducers like photomultiplier tubes or photodiodes. Additional equipment is required to transfer the bioluminescent signal from the transducer. This includes fiber optic cables, lenses or liquid light guides. The result is often a bulky, heavy instrument that can’t be used in the field. use. Azur Corporation (Carlsbad Calif.) has among other things developed hand-held, battery-operated photomultiplier units that can directly be interfaced with a laptop.
“?Field-friendly? “Field-friendly? The bioluminescent integrated circuits (BBICs), which are bioluminescent and bioreporter, have a 5 mm2 surface. They consist of two main components: photodetectors to capture bioluminescent signals on-chip and signal processors for managing the storage and management of bioluminescence information. Remote frequency (RF) transmitters may be added to the overall circuit design for wireless data relay. All elements required are contained within the BBIC. This allows for the BBIC’s operational capabilities to be realized simply by exposing it to the test sample.
“4.5 Use of Bioluminescent Pathogen Detection Systems”
“4.5.1 Bioreporter Line Cell Line”
“Detection limits and response times, saturation kinetics, and basal expression levels lux (Winson, 1998) were observed in bioreporter cells lines using standardized bioavailability tests (Heitzer, et. al. 1992). The bioreporter cells are grown in yeast extract-peptone-glucose (YEPG) medium to exponential phase (OD546=0.35) whereupon 100 ?l aliquots are transferred to 96-well microtiter plates. Microtiter wells are incubated with Acyl homoserine Lactone (AHL), at concentrations of 0.01 to 1000ppm. Light readings are continuously taken using a scintillator counter for 24 hours. Vials containing no AHL are used to determine the background levels of bioluminescence caused by basal expression.
“Plotting background-corrected Bioluminescence against time produces standard curves that indicate detection limits and response times. For the analysis of AHL concentrations, standard HPLC techniques can be used (Winson and al. 1998). After baseline measurements have been made, microtiter plates are used to perform tests using the bioreporter and varying levels of luxI bacteriophage or associated pathogen. These tests are done in similar formats in order to determine detection limits, response time, saturation kinetics and background induction.
“Measurements can also be taken with an Azur DeltaTox (Carlsbad. Calif.) photomultiplier device. The Azur photomultiplier, a battery-operated handheld unit, interfaces directly with a laptop computer. This makes it ideal for field measurements. monitoring. To identify potential effects on bioreporter responses, parameters such as pH and temperature are closely monitored. To account for AHL molecules intrinsic to the organism, a negative control is made up of samples that are free of bacteriophage.
An integrated circuit photodetector can also be used to analyze samples. A test bed with integrated circuits to measure the replicate amount of induced bioreporter-bioluminescence. This analysis uses integrated circuits that are connected to a flowcell system, through which the desired substance passes. The integrated circuit records the bioluminescent reactions and then downloads them to a computer interface.
“4.5. “4.5.
“The physiological status of bacteria can influence the degree bioreporter reaction, since luminescence requires active reproduction of the bacteriophage (i.e. acyl-homoserine latone synthetase) in the pathogen. Analyses using log-phase cells could underestimate the field conditions sensitivity of bioreporter systems. Studies are done with strains of interest in a variety of physiological conditions, including starvation and disinfectant treatment with chlorine. Starvation can be induced by extracting log-phase cells and then rinsing the cells three times. Finally, these cells are stored in minimal salts media. To provide a wide range in metabolic states, stored cells are tested after 0 hr to 1 hr and 1 hr to 3 d, 3 days, and 7 days (Morita 1982). Similar log-phase suspensions that have been washed are also treated with a variety of levels of chlorine (0,0.5, 1, 2 and 3 mg/L) for two minutes to create a gradient in active cells (Boulos, et al. 1999).
“Samples of the different starvation times are evaluated for bioreporter responses. Several types of cell counts are performed, including 1) total direct count using acridine orange staining (Hobbie and al. 1977); 2) viable cells using LIVE/DEAD (Molecular Probes Eugene, Oreg. ), and 3) respiring cells using 5-cyano-2.3-diotyl Tetrazoloium (5CTC). Bioreporter tests may utilize an Azur Deltatox photomultiplier unit following standard procedures defined during baseline studies. AO counts are one of the many cell counts. They provide an estimate of total cells, and should not change with starvation or chlorine treatment. CTC counts are a measure of active respiration in cells that have been exposed to the fluorescent formazan dye (CTC dye) and cells that have active electron transport activity. CTC response (both as a function of the number and fluorescence per cells) responds rapidly to carbon source availability (Cook & Garland, 1997) and stress such a chlorine treatment (Boulos, 1999).
The LIVE/DEAD Baclight Kit contains two nucleic acid-binding stain stains that can be used to distinguish between viable and dead cells. SYTO 9, which is a fluorescent green dye, passes through all cells. Propidium iodide can penetrate cells only with damaged membranes and stain them red. Dual staining gives separate estimates of dead (red) and live cells, using membrane integrity to distinguish them. LIVE/DEAD counts have shown less response to stress than CTC count estimates (Boulos and al. 1999; Braux and al. 1997). Analyzing samples simultaneously with the different methods allows for a direct assessment of the impact that respiration and viability play on the bioreporter’s response. It is crucial to determine the extent to which viable cells, but not actively breathing, respond to the biosensor assay. If viable cells respond weakly to the biosensor assay, it is possible that potentially virulent cells are not detected.
“4.5.3 The Effect of the Sample Matrix on Bioluminescence
The bioreporter’s response may be affected by the sample matrix, which is particulate matter that includes microorganisms. Particulate matter can bind to cells and prevent infection of the bacteria, which could lead to non-specific phage binding and/or general quenching. This could decrease the detection limit of a bioreporter. Non-specific infections, though unlikely, can lead to false positives.
“In certain embodiments of the invention it may be desirable for the bioreporter cell to be immobilized in a stabilizing matrix. Without adverse effects on viability, alginate has been used successfully to encapsulate cells. As long as alginate-encased cells are moist, it is possible to sustain long-term viability (weeks or months). Lyngberg et. al., 1999 reported that latex copolymers are also useful in immobilizing E. Coli as well as maintaining viability. Other matrices include carrageenan, acrylic vinyl acetate copolymer, polyvinyl chloride polymer, sol-gel, agar, agarose, micromachined nanoporous membranes, polydimethylsiloxane (PDMS), polyacrylamide, polyurethane/polycarbomyl sulfonate, or polyvinyl alcohol. You may also use electrophoretic deposition.
“4.5.4 Shelf-Life Assessment and Bioreporter Lyophilization”
“Lyophilization (freeze drying) is a significant advantage when using microorganisms to report on the environment. This allows for long-term storage, ranging from months to years. There is little risk of them losing viability. It is desirable that the bacteriophage/bioreporter system be placed in a physiological state amenable to long-term storage such that the end-user can simply revive a pellet of cells whenever measurements are required. These components are lyophilized and resuscitated at different intervals to determine the shelf life of the bioluminescent bioreporters. Bioluminescence is then measured in accordance with the above.
“5.0 EXAMPLES”
“5.1 Example 1”
“Amplification of Bioluminescent Sign in Bioreporter cells”
The detection scheme used to quantify pathogenic targets is based on the ability of AHL molecules induce bioluminescence so that it can be correlated and correlated with the original number AHL-producing targets in the sample. This technique is based on the same principles as quantitative PCR, except that the initial AHL concentrations (instead of nucleic acids) allow differential detection of the exponential rise in signal, i.e. bioluminescence in reporter cells (Heid and al., 1996).
“Amplification of bioluminescent responses of bioreporter cell occurs through the autoinduction mechanism V. fischeri AHL is released to the extracellular environment after target cell infection with the biosensor (i.e. a luxI bacteriophage). Light production is stimulated by the neighboring bioluminescent bioreporter cell’s uptake of AHL. This AHL stimulates the production of more light. This cascade effect results in intense bioluminescent light because of the involvement of multiple binding episodes within multiple bioluminescent cell types.
“FIG. “FIG. V. fischeri overnight culture was diluted to 0.01 OD546. Standard dilutions for N-(3-oxohexanoyl] homoserine lactone (Quorum Sciences were prepared by resuspending 213 mg in 1mL acidified ethyl alcohol. This gave rise to a 100mM stock, followed by dilutions with acidified ethyl alcohol. After placing 100 mL of N- (3-oxohexanoyl] homoserine lactone/ethyl-acetate solution in shell vials, the assays were carried out. The test vials were then filled with one mL of V. fischeri (prepared in the same manner). The vials were shaken at 140 RPM. Light measurements were taken at both time zero and 30-minute intervals with a Zylux portable lumenometer. The data were plotted in photons per second against time. This was the average of three replicates.
“A threshold line is used in data analysis to distinguish between samples (FIG. 4). This line indicates the photons per second at a given time when the bioluminescent bioreporter has been autoinduced. The curve characteristics on the graph determine this value. Autoinduction occurs when the curve’s slope increases rapidly. To determine the threshold line for the assay, the autoinduction of the control specimen is used. After determining the threshold value, the sample data can be analyzed to determine the time when autoinduction occurred. Autoinduction occurs sooner if the inducer concentration is higher. You can use this in either a quantitative or qualitative format, depending on your application.
The results showed that homoserine lactone concentrations are more important than light production. 4) All samples achieved the same light levels. However, it is only the beginning of the geometric increase of light that allows quantification of inducers. These results show that homoserine-lactone molecules increase the lux-based, bioluminescent signal in quantifiable ways.
“In the bacteriophage/bioreporter system, the target bacterium is infected by a specific bacteriophage carrying the luxI gene. The bacterium becomes capable of producing the inducer, acylhomoserine lactone, (AHL) after infection. The higher the number of phage infections, the more AHL is produced and the shorter time it takes for the light to be produced in bioluminescent bioreporter cell cells. To measure the amount or number of bacteria infected with the luxIbacteriophage, the time difference between the samples and the control is used.
“5.2 Example 2”
“Exemplary strains of LuxI Bacteriophage
“Because the genomes A511 and A118 of L. monocytogenes phages A511 have been characterized, luxI incorporation in these bacteriophage can be achieved through homologous recombination. The A511:luxAB phage was described by Loessner et. al. (1996). This is the basic strategy. The luxI construct is described in section 4.3.2. It contains appropriate ribosome binding site for L. monocytogenes. A set of primers containing flanking sequences at the 3? End of the cps gene at phages A511 and 118. The product is amplified, and then inserted into the Phage by Recombination. Bacteria are screened and enhanced essentially according to Loessner and colleagues (1996). Supernatants from primary lysates are tested for their ability inducing the lux genes of the reporter strain, since AHL production is the preferred phenotype. RFLP analysis is used to verify that the phage containing luxI has been selected.
For uncharacterized phage genes, the transposon mutagenesis technique of Waddel & Poppe, 1999 can be used to generate luxI phage constructs. The MiniTn5 transposon contains a promoterless gene for luxI that is used to mutagenize the phage. The MiniTn5luxI transposon can be constructed by inserting the appropriate luxI previously constructed into the unique cloning location of pUTK214. (Applegate and al., 1998). The construct is converted into E. coli SV17-1 (pir). The restriction fragment analysis is used to screen transformants for inserts. Once MiniTn5luxI transposons have been identified, they can be used to mutagenize (via biparental marriage) the appropriate phages for that specific application. The screening of phages is the same as described by Waddell and Poppe (1999), with the exception that kanamycin is used to select. The bioluminescent reporter strain is used to screen Phage that show the KmR genotype for production of acylhomoserine lactone. The detection assays are performed on phage that produce significant amounts of AHL.
“The M13 bacteriaiophage is an example of a system that can detect various pathogens. luxI genes can also be modified to be expressed by the following organisms: E. coli o157:H7 L. monocytogenes Salmonella spp. Campylobacter spp. B. anthracis B. thuringiensis B. thuringiensis B. subtilus.”
“5.3 Example 3”
“Detection and Treatment of Bacterial Pathogens In Food”
Salmonella, Listeria monocytogenes and Escherichiacoli O157-H7 are four pathogens that are particularly concerning to the food industry. Recent estimates show that these four types of bacteria are responsible for more than 95% of all food-related death in the U.S. (Mead and al. 1999). Cantaloupe (Riess and al. 1990), alfalfa seeds (Mahon and al. 1997), tomatoes (Hedberg und al. 1994) as well as watermelon (del Rosario and al. 1995). Salmonella-related illnesses have been linked to cantaloupe. A wide range of fresh vegetables have been found to contain salmonella, including artichoke and cabbage, celery and eggplant as well as lettuce mustard cress and parsley (Sumner & Peters, 1997).
E. coli O157.H7 is an emerging human pathogen that was first linked to outbreaks of food illness (e.g., fast food hamburgers). It can cause death in children and adults, especially if it produces enterohemorrhagic toxic toxins. Although cattle appear to be the primary reservoir, E.coli O157H7 has been linked with outbreaks in cantaloupe and broccoli (Sumner & Peters 1997). Cubed watermelon and cubed melon can be used to grow the organism (del Rosario & Beuchat 1995). It has also been isolated from celery, cilantro, coriander, and cabbage.
“Campylobacter, an emerging pathogen that causes acute stomachitis and Guillan-Barre syndrome (an acute neurological disorder), has been recognized as a common antecedent. Most often, illness is associated with contaminated poultry or raw milk. However, Campylobacter can also be found in raw fruits and vegetables (Bean et al. 1990; Harris et. al. 1986).
“The invention can be used to detect food-borne pathogens, taking into account the nature of the sample being tested. Sample matrix, which is particulate material that results from the rinsing and blending of vegetable matter, can affect the bioreporter’s response. Particulate material can bind to target cells, block infection by the bacteria, and/or cause general quenching in the light signal from the bioreporter cell. To test the effect of the sample matrix on the bioreporter, samples from tomato and lettuce may be tested. Lettuce, tomato, and other plant types are used as examples. They are both commonly eaten fresh produce that has been contaminated with some or all of the pathogens important to the food industry. The two crops are also distinct types of plant material (i.e. leafy vegetables versus fruit), with potentially unique matrix characteristics.
“Surface washings are made of lettuce by shaking aseptically-cut strips in sterile tris buffer for 2 hr. Then, homogenized samples can be obtained using a stomacher mixer (Donegan et. al., 1991; Jacques and Morris 1995). Tomatoes can be surface rinsed using gentle hand rub and placed in sterile bags with sterile buffer. The stomacher blender homogenizes them (Zhuang, et al. 1995). Others use mixed samples of tomato and lettuce. Log-phase cells can be introduced at levels that are well within the detection limits established by baseline studies. Results with additional vegetable matter are also compared to controls.
“Other tests provide baseline information about the effectiveness of the biosensor for detecting contamination in plant production, processing and distribution systems. Plant material is given specific amounts of pathogens either during or shortly after the plant’s growth cycle. The survival of the pathogen can be compared using the biosensor to monitor its survival up until harvest and after storage. This is then compared with the results obtained by reverse transcriptase – quantitative PCR (Heid, 1996) as described below.
“Tomatoes and lettuce are examples of how the invention can be used to detect foodborne pathogens. The typical production cycle of lettuce (cv Waldmann?s Green) takes 28 days in controlled environmental chambers (Wheeler and al., 1994). The production cycle of tomato (cv. Reimann Phillip) is grown in controlled environment chambers for 90 days. However, ripe fruits can be harvested as early as day 60 (Mackowiak and al., 1999). The plants are inoculated for pathogens on the day 60-aged fruit, and they are then removed from the plant after 1, 7, 14 and 21 days or at other appropriate intervals.
“Reverse transcriptase quantitative PCR (RTQPCR)” is a method that can be used to determine the copy number of DNA in a sample of nucleic acid extracts (Heid et. al. 1996). This method uses either PCR or RNA to target amplification and kinetic detection of PCR product at each cycle of the ABI 7700 Sequence Detection System in Foster City, Calif. The specific target molecule can be detected in a single PCR reaction. This involves primers that flank the area of interest, a dual-labeled diagnostic probe with a fluorescent reporter dye as well as a quencher. The 5?-nuclease activity (Taq) cleaves perfectly annealed probes. The result is an increase in reporter signal. Laser-induced fluorometry (LCD) and charge coupled devices are used to detect the fluorescent signal produced by PCR products labeled. This instrumentation and method are compatible with multiple reporter chemicals.
“The TaqMan? The assay (Applied Biosystems Foster City, Calif.), can detect 5 copies of the target against a background of 500 ng non-target DNA/RNA. This is 5 copies per sample and has a dynamic range of five orders of magnitude (i.e. 5 to 106 copies). RTQPCR is currently the most sensitive method to quantify and detect molecular targets in pathogen detection. It is therefore the best method to evaluate biosensor sensitivity.
“Primer-directed PCR amplification combined with probe hybridization has a higher specificity than membrane-bound nucleic acids hybridization. It allows for the quantitative detection of DNA and RNA. RT-PCR can be used to quantify mRNA or rRNA. This allows us to identify potential pathogens in agricultural products and determine their relative proportions. Both mRNA and rRNA are related to the physiological state of the cell/community; rRNA generally increases with growth rate.
Two primer-probe detection systems are used to detect Salmonella in QPCR. They target different molecular markers that are specific for Salmonella. The TaqMan? The TaqMan? in raw meat products (Chen et al., 1997; Kimura et al., 1999). The second primer set amplifies either a 173 bp (or 107 bp) product from a Salmonella typhimurium-specific region of the phase 1-flagellin filament gene. Its application has been demonstrated in the quantitative detection in soil systems of viable, but non-culturable S. Typhimurium populations.
QPCR detects Listeria monocytogenes by targeting the hemolysin-A (hlyA), transcript, which is unique to Listeria. Norton and Batt (1999) developed the hlyA detection method. It allows for the quantitative detection and quantification of viable Listeria populations using a 210-bp segment from the transcribed hlyA genes as a target.
“Escherichiacoli O157H7 detection for QPCR uses the 5? Oberst et al. developed a nuclease test. (Oberst and al., 1998) which targets the intimin-protein encoded by eaeA. The eaeA test generates a 631 bp section of the gene that contains target sequence, which may be detected using one of four probe sequences.
“Campylobacter Jejuni detection uses the nestedPCR approach of Winters and al. (1998, 2000) In a modified 5 Nuclease assay. Initial PCR amplification produces a 159-bp product, which can be used as a template for a second one using the original forward primer and a complementary reverse primer to a region within the 3. End of the first amplification product. The nested product produces a C. jejuni product with a 122-bp C. product, but no C. coli.”
“5.4 Example 4”
Click here to view the patent on Google Patents.