APPLEGATE Project Rationale SAVIKHIN - Purdue …€¦ · · 2005-04-18Project Rationale This research is ... of DNA probes for the detection of specific target DNA sequences associated

Project Rationale This research is based on the specificity of DNA probes for the detection of specifictarget DNA sequences associated with thevirulence of the identified foodbornepathogens to be detected. Antibody methodsrely on the detection of surface antigens buthave numerous drawbacks including cross-reactivity and antigen expression may also be affected by environmental conditions. The use of multiplexed PCR offers an alter-native approach to current antibody methodsallowing detection of a suite genes present in a pathogen providing redundancy andelimination of false negatives and positives.Multiplexed PCR relies on the amplificationof multiple target sequences which arepathogen specific. Once amplified the fragments are analyzed by electrophoresis for the indicative banding pattern, which is definitive. The technology being developedis a fluorescence-based wavelength shift assayutilizing Fluorescence Resonance EnergyTransfer (FRET) to detect hybridization of a set of target DNA sequences associatedwith identified pathogens. The use of thisspatial detection format (arrayed probes) will allow for the simultaneous detection of products from multiplexed PCR reactionsmore rapidly than could be achieved withelectrophoresis. The spatial isolation ofprobes allows for simultaneous detection of a larger number of targets than would feasible with current real time PCR.

M U LTI P L EX E D D E TE C TI O N O F PATH O G E N SU S I N G F LU O R E S C E N C E R E S O N A N C E E N E RGYTR A N S F E R I N A S PATI A L D E TE C TI O N F O R M AT.

Project Objectives

Design of molecular beacon incorporating appropriate FRET pairing to provide maximum emission separations with excitation overlap.

Design of molecular beacon target sequence and corresponding ampliconprimer pairs for identified target sequences to use in a multiplex PCR.

Construct a molecular beacon array and optimization of hybridization conditions with PCR amplicons.

Construction of the field-deployable detection systems and validation of the integrated system.

Project HighlightsWe determined the sequences for the ampli-cons and molecular beacon probes for thesimultaneous PCR of 15 different toxin genesassociated with the pathogens in the study.The primer and probe sets were synthesizedfor evaluation in the multiplex PCR reaction.The amplicon primer sets were tested forcross reactivity with individual strain DNAand mixtures in a multiplex PCR format andno cross reactivity was determined. This wasa critical accomplishment for the project as it provides the DNA to be analyzed in theproposed spatial format detection platform.We also determined the minimal primer concentrations to amplify the target ampli-cons to equivalent detectable levels (in themultiplex format) using fluorescence analyzedby capillary electrophoresis. Subsequentdetection limits were also determined (102copies of target). Primers were synthesizedwith fluorescence tags to allow identificationof peaks and used in the multiplex PCR reaction using capillary electrophoresis asopposed to electrophoresis since the frag-ments are similar sizes and can not resolvedby electrophoresis. The optimization allowsthe amplification of target amplicons in varying initial concentrations without loss ofdetection of lower concentration ampliconswhich is observed when primers are utilizedin excess concentrations. This is an importantdevelopment in the overall goal of the projectbut could also be utilized in current amplifi-cation detection methods currently in use.

Bruce Applegate (Principal) and Sergei Savikhin (School of Agriculture, School of Science) � INVESTIGATORS

A P P L E GATE

SAV I K H I N

“Multiplexed PCR

relies on the

amplification

of multiple

target sequences

which are pathogen

specific.”

Annual_Approved_Web 10/25/04 5:43 PM Page 2

Project Rationale Numerous outbreaks of foodborne outbreakshave been associated with fresh and minimal-ly processed produce, such as green onions(Hepatitis A), lettuce (E. coli O157:H7,Listeria monocytogenes), sprouts (E. coliO157:H7), cantaloupes (Salmonella spp.),and tomatoes (L. monocytogenes). Improvingproduce safety has been challenging for theproduce industry due to low effectiveness (<2 log reduction) of current decontamina-tion treatments, such as washing with chlori-nated water and other aqueous sanitizers.Chlorine dioxide (ClO2) gas treatments have emerged as a very promising non-ther-mal sanitation technology with high efficacyfor pathogen reduction on produce surfaces.Considerable work has been done within a laboratory setting, however, more research is needed to understand how this technologycan be utilized for food industry applications.

D E CO NTA M I N ATI O N O F P RO D U C E U S I N GC H LO R I N E D I OX I D E GAS TR EATM E NT

Project Objectives

Develop a pilot scale ClO2 gas treatment system for produce.

Determine the microbial and quality effects of continuous ClO2

gas treatments for selected produce models.

Determine inactivation kinetics of aqueous and gaseous ClO2

treatments for selected bacteria and bacterial spores.

Initiate study to better understand the mechanisms of inactivation by ClO2.

Project HighlightsA pilot scale ClO2 gas treatment system has been developed. That features automatedClO2 gas generation, concentration, exposuretime, and relative humidity control and continuous monitoring, and a 30,000 squarefeet capacity. More than a 5-log reduction of selected pathogens (E. coli O157:H7, L. monocytogenes, and Salmonella spp.) havebeen achieved on green peppers, strawberries,mushroom, and oranges using this system.Treated products have an increased shelf-life,showed no negative changes in surface colorand have minimal residues (<1mg/kg) ofoxidative species. The kinetics during aque-ous and gaseous ClO2 treatments for inacti-vating E. coli O157:H7, L. monocytogenes,Bacillus spores, and recombinant biolumines-cent E. coli O157:H7 has been studied. The results suggested that moisture contentin the environment surrounding cells playsan important role in microbial inactivationby ClO2 gas. Early mechanism studies indicate that visible damage was primarilyobserved in the interior of the cell rather than on the cell membrane. More studies on mechanisms will be carried out next year.

INVESTIGATORS � Richard Linton (Principal), Philip Nelson, Bruce Applegate, David Gerrard, Yingchang Han and Travis Selby (School of Agriculture)

L I NTO N

N E LS O N

A P P L E GATE

H A N

S E L BY

“More than a 5-log

reduction of

selected pathogens

(E. coli O157:H7, L.

monocytogenes,

and Salmonella ssp.)

have been achieved

on green peppers,

strawberries, mushroom

and oranges using

this system.”


Project Rationale Currently, the cost for health departments to develop fish consumption advisories isprohibitive. There is a need to develop newtechnologies that will reduce or eliminate the equipment for direct sampling of fish to determine residue levels. Our researchgroup is developing new methods for measur-ing contaminants (PCBs and mercury) at a lower cost and with a faster throughput. By reducing the cost for analysis, federal and state agencies will have the ability to collect and analyze a greater number of samples and improve the accuracy of theiradvisories or surveys. The overall objective of this effort is to protect the fetus or nursinginfant from exposure to harmful chemicalcontaminants in fish. Specifically, we areinterested in developing rapid methods to measure total PCB and mercury in fish tissue so that health departments can conductmore complete risk assessments related tochemical contaminants in sportfish and commercial fish.

R A P I D D E TE CTI O N O F TOTA L P C B S A N D TOX I C IT Y E Q U I VA L E N C E Q U OTI E NT ( TE Q ) I N F I S H TI S S U E F ROM I N D I A N A WATE R S A N D U S E O F A N OV E L D EV I C E TO P R E D I CTCO NTA M I N A NT LOA D I N F I S H .

Project Objectives

Finalize the validation of a lower cost, rapid assay for the measurement of PCBs in fish tissue that could be used by States when developing fish consumption advisories. This assay would allow States to analyze more fish samples and develop more comprehensive advisories.

Measure the levels of PCBs and omega-3 fatty acids in fish oil dietary supplements using the ELISA that was validated previously. Since consumers are more interestedin dietary supplements, we wanted to assess levels of PCBs in fish oil supplements.

Compare mercury residues in fish tissue as measured by a standard method in comparison to a new analytical method. Improving the throughput and lowering the cost for mercury testing can help during the development of fish consumption advisories and improve the oversight of commercial fish that may contain high levels of mercury.

Measure mercury and omega-3 fatty acids in canned fish and in retail fish sandwiches using this newer mercury analysis method. Determining the risks and benefits of these fish products would help during the development of comprehensive fish consumption advisory.

Project HighlightsThis project has shown that it is possible to simplify the analytical method, reduce the cost and increase the throughput whentesting for PCBs and mercury in fish tissue.These methods are important when develop-ing sportfish consumption advisories or whenmonitoring fish that enters into interstatetrade. The project could dramaticallyimprove the quality of fish consumptionadvisories which had previously been limited to a small number of samples. Thus, advisories would be more accurate and helpful to anglers and their families. In addition, it would help to prevent fishthat has elevated levels of contaminants from entering the marketplace.

INVESTIGATORS � Charles Santerre (Department of Foods and Nutrition)

SA NTE R R E

“Currently,

the cost for health

departments to develop

fish consumption

advisories is prohibitive.”


Project RationaleL. monocytogenes has emerged as one of the most important food pathogens with a “zero tolerance” for it in ready-to eatprocessed meats and dairy foods. This bacterium not only causes serious illness but also is also lethal in infants, people over60, and immune-compromised individuals.

Current methods of detecting this bacteriumrely upon enrichment in the numbers of bac-teria present in a sample. The food or foodextract is incubated in specific growth mediafor 12 to 24 h, and, the resulting culture istested for L. monocytogenes using proceduresthat require an additional 3 to 24 h.

Rapid and affordable technologies to detect low numbers of L. monocytogenescells directly from food, and which distin-guish living from dead cells, are needed.

Project Objectives

This multi-disciplinary, multi-departmentalresearch project is addressing the funda-mental engineering and science requiredfor development of microchip, bio-basedassays that are transportable to the field,useable in a manufacturing plant environ-ment and capable of rapidly detecting L. monocytogenes at the point of use. This research has the goal of microscaledetection on a real-time or near real-timebasis with a time-to-result of 4 hours.Our research is addressing the develop-

E N G I N E E R I N G O F B I O SYSTE M S F O R TH ED E TE C TI O N O F L I STE R I A M O N O CY TO G E N E SI N FO O D S

ment, engineering and validation of such a microchip system that combinesbioseparations technology for rapid concentration and recovery of microbialcells and bionanotechnology to constructsystems capable of interrogating fluidsfor pathogens. Although the focus is on rapid detection of L. monocytogenesby a combination of technologies that will ultimately give a time to result in a few hours, our approach is resulting in a technology platform capable ofdetecting other types of foodborne and medically relevant pathogens.

Project HighlightsA second generation silicon-based microflu-idic biochip was fabricated that enables collection and concentration of cells accompanied by impedance-based detectionof their presence on the chip. The chip was designed, fabricated, and tested in amanner that integrates off-chip processingwith on-chip microfluidic handling andinterrogation of a small sample volume. This is an important step forward towardsachieving the goal of systems integration for a laboratory prototype for L. monocyto-genes detection in samples derived fromready-to-eat meat products. Research alsodemonstrates how environmental stressreduces binding of L. monocytogenes to its bioreceptor and consequently reduces sensitivity for detection of the pathogen. This work showed how enrichment mediamight be formulated to optimize the physiological status of the microorganismand to enhance its binding to a pathogenspecific antibody while minimizing “cloak-ing,” thereby reducing probability of a falsenegative result. The outcome of this workwill ultimately enhance reliability and sensitivity of biochip-based detection of L. monocytogenes by formulating specialmedia or buffers for this purpose.

Michael Ladisch (Principal), Rashid Bashir, Arun Bhunia and J. Paul Robinson (School of Engineering, School of Agriculture) � INVESTIGATORS

L A D I S C H

BAS H I R

B H U N I A

RO B I N S O N

“This research has

the goal of microscale

detection on a real-time

or near real-time basis

with a time-to-result

of 4 hours.”


Project Rationale To keep the food supply safer, food production, processing, andretail establishments must be able to better identify and then control or eliminate microbial foodbornecontaminants such as pathogenicSalmonella, Campylobacter jejuni, and Escherichia coli O157:H7. The Centers for Disease Control and Prevention (CDC) estimates

that annual foodborne-related outbreaksresult in 76 million cases of illness,325,000 hospitalizations, and 5,000 deaths(Mead et al, 2000). Conventional detec-tion methods take at least 24 to 48 hoursto differentiate and identify microorgan-isms; therefore, measures taken to counter-act food contamination must wait at leastthat long. In order to facilitate timely inter-vention measures, the food industry needsmore rapid detection methods and a sensorable to accurately and rapidly identify lowlevels of microbial foodborne contaminantswithin food systems or cultural media. Our research group is investigating the efficacy of infrared (IR) technology as a means of rapid detection of select bacterial pathogens.

I N F R A R E D S E N S O R S F O R R A P I D D E TE C TI O N O F S E L E C T M I C RO B I A L F O O D B O R N E CO NTA M I N A NTS.

Project Objectives

Create a library of FT-IR spectra of bacterial cell wall components and whole cells (from Salmonella, Campylobacter jejuni, and Escherichia coli O157:H7) needed for cell identification and differentiation.

Develop FT-IR methods for cell identification and quantification in water, cultural media, and foods.

Develop a limited wavelength approach for cell identification.

Build and validate an IR sensor based on the most promising few-wavelengthalgorithm developed using FT-IR techniques developed in the first two milestones.

Project HighlightsWe successfully developed an approach forsample preparation, FTIR spectral collection,and data analysis that is able to both quantifyand identify Salmonella and E. coli O157:H7from microbial culture media. This approachwill be used as the format for development of a portable-use IR sensor. To develop thisapproach, we evaluated sample preparationmethods (including preenrichment, selectiveenrichment, filtration and immunomagneticseparation techniques), FTIR data collectionmethods (ATR, transmission, reflection,Continuum IR microscope), and analysis of raw spectra. Further development of thisapproach will enable the design of a sensorthat can be used in a production or retailfacility to characterize a food sample as contaminated or free of select pathogenicbacteria in less time than current methods for detection.

Lisa Mauer (Principal), Maribeth Cousin, Jay Gore, Jean Guard-Petter, Brad Reuhsand Sivakumar Santhanakrishnan (School of Agriculture, School of Engineering) � INVESTIGATORS

M AU E R

CO U S I N

G O R E

G UA R D - P E T TE R

R E U H S

SA NTH A N A K R I S H N A N

“Our research group

is investigating the

efficacy of infrared (IR)

technology…”


Project Rationale Most Fusarium species of molds producemycotoxins in cereal grains and foods withthree mycotoxins, fumonisins, trichothecenesand zearalenone, being linked to potentialhuman health problems. These mycotoxinsare produced worldwide in grains duringgrowth in the fields, storage in grain elevatorsand processing of some foods. These myco-toxins are difficult to destroy in grains andfoods because they resist most food process-ing operations. There is a need to detectFusarium species in grains and foods beforethey can grow and produce mycotoxins.Currently, there are no rapid methods todetect Fusarium species. Our earlier work led to development of an enzyme-linkedimmunosorbent assay (ELISA) and poly-merase chain reaction (PCR) assay that could detect Fusarium species as a generalgroup and also a multiplex PCR was devel-oped to detect the major species of Fusariumthat produce three major mycotoxins:Fusarium verticillioides and Fusarium proliferatum that produce fumonisins andFusarium culmorum, Fusarium graminearumand Fusarium sporotrichioides that producetrichothecenes and zearalenone. We havecontinued the research by using the antibod-ies for immunocapture of the Fusariumspecies that then can be detected by multi-plex real-time PCR. The overall goals of our current work are to develop a biosensorprotocol based on the antibodies that wereproduced to capture antigens of mycotoxin-producing Fusarium species and the combi-nation of these antibodies with the real-timePCR assay to rapidly identify Fusariumspecies.

D EV E LO PM E NT O F I M M U N O CA PTU R E R EA L-TI M E P C R TO D E TE C T FU SA R I UMS P E C I E S I N G R A I N S A N D F O O D S

Project Objectives

To develop real-time PCR to detect Fusarium species.

To develop real-time PCR to specifically detect Fusariumspecies that produce fumonisins and trichothecenes.

To capture Fusarium species with antibodies.

To combine immunoassay with PCR to specifically detect Fusarium verticillioides that produce fumonisins and Fusarium graminearum that produce trichothecenes.

Project HighlightsResearch was done to develop an immuno-capture method for Fusarium species by using different solid support systems: microcentrifuge tubes, magnetic and non-magnetic beads, and microtiter plates. It is important to develop a simple method to take the mold away from the food particlesbefore going to PCR. A modified ELISAusing microtiter plates gave the best captureand was used before going to PCR. If amethod could be developed to easily andquickly detect Fusarium species, then thesemolds could be prevented from producingmycotoxins in agricultural commodities and foods.

INVESTIGATORS � Maribeth Cousin (Principal) and Charles Woloshuk (School of Agriculture)

CO U S I N

WO LO S H U K

“There is a need to

detect Fusarium species

in grains and foods

before they can grow

and produce

mycotoxins.”


Project RationaleListeria monocytogenes and enterohemorrhagicEscherichia coli are important foodbornepathogens because of their low infective dose.For the detection and evaluation of foodscontaminated with these pathogens, USDA/FSIS recommends initial enrichment andsubsequent plating on a selective agar media,which follows further identification by biochemical, serological or genetic means.However, these latter steps are time consuming and may take more than 2-3days. Our main objective was to develop a simple noncontact light scattering sensorymethod that would reduce the time of identification of these pathogens after platingon microbiological agar plates. The method has now been improved and is able to differ-entiate Listeria species colonies directly from the agar plates based on the light scatteringpatterns. There have been increased food-borne illnesses, outbreaks, product recalls,and loss of lives in recent years. Pathogencontamination in products not only puts the public at risk, it is also costly to the due to the product recalls and liability. If successful, development of a simple detection device such as a light scatterometercan make pathogen detection an easy, lesscumbersome and inexpensive process.

L I G HT S CAT TE R I N G S E N S O RY M E TH O DF O R R A P I D A S S E S S M E NT O F F O O D B O R N EBAC TE R I A L CO NTA M I N A NTS.

Project Objectives

To determine how angle resolved elastic light scattering can be used to detect bacteria and distinguish particular foodborne pathogenic bacteria from nonpathogenic bacteria. Target pathogens include Listeria and enterohemorrhagic E. coli.

To determine if angle-dependent scattering can rapidly detect and identify bacterial microcolonies on selective agar plates.

To generate scattering characteristics based on growth time, density, strains and pathogencity.

Project HighlightsA scatterometer was developed to measurethe angel-resolved forward scattering charac-teristics of surfaces of bacterial colonies andwas successfully adapted to measure and differentiate specific patterns of differentListeria cultures. Several approaches to scatterometry were explored using prototypeinstruments that were developed. After completion of proof-of-principle experimentson a limited number of cultures, the mostpromising instrument (based on forward scattering) was assembled where the scatter-ing properties of wide range of pathogenicbacteria are conducted. We measured the forward scattering characteristics of 50 strains of L. monocytogenes, 20 L. innocua,14 L. ivanovii, 6 L. seeligeri, 3 L. grayi, and 3 L. welshimeri. After visual examination,most of the non-L. monocytogenes producedreproducible unique patterns; however, patterns for L. monocytogenes strains werediverse with some showing patterns that overlap with L. innocua or L. ivanovii.We anticipate a sensitive pattern recognitionsystem would be able to improve data analy-sis thus improving culture identification. The technology seems very promising andattractive for use with pathogen detectionand identification, which require furtherinvestigation.

Dan Hirleman (Principal) and Arun Bhunia (School of Engineering, School of Agriculture) � INVESTIGATORS

H I R L E M A N

B H U N I A

“Light scatterometer

can make pathogen

detection an easy,

less cumbersome and

inexpensive process.”


Project Rationale Conventional methods for the detection of pathogen from food are greatly restrictedby prolonged assay times (up to 7 days),requiring initial enrichment to bolster targetpathogen numbers. Antibody-based detectionmay greatly reduce the assay time but it stilllacks the ability to detect biomolecules in“real-time.” Biosensors use a combination ofbiological receptors and physical or chemicaltransducers, which represent a new andunique technology with great potential tomeet the need for the rapid detection of lowlevels of biomolecules. Fiber-optic biosensorsexploit the measurement of fluorescent lightexcited by an evanescent wave generated by a laser to quantitatively detect biomoleculesimmobilized on the fiber surface. The assayprinciple is based on a sandwich immunoas-say, using a capture antibody, immobilizedonto the optical fibers, and a Cyanine 5(Cy5)-labeled antibody for detection usingthe Analyte 2000. Sensitivity and specificityof antibodies are critical for immunodetec-tion of biomolecules in antibody-basedbiosensors. This project is focusing on: laseroptical detection of Listeria monocytogenes,Salmonella Enteritidis and E. coli O157:H7with fiber optic sensor, and (ii) antibodydirected silver staining technique for detection of E. coli O157:H7.

O PTI CA L B I O S E N S O R F O R F O O D PATH O G E N D E TE C TI O N

Project Objectives

The overall goal of this project was to develop and evaluate antibody-coupled optical biosensors for sensitive detection of pathogens from enriched food samples by:

Development of polyclonal antibodies to Salmonella and Listeria monocyto-genes.

Optimization of capture and detection of target pathogens on the fiber.

Testing of assembled fiber in pure culture of pathogens.

Evaluation of other potential reporters.

Testing spiked foods.

Project HighlightsA fiber optic sensor was developed for L. monocytogenes that can detect about 4.3 ¥103 to 4 x 104 CFU/ml after 2.5 h of sampling even in the presence of commonfood contaminants or stress conditions. Thissensor was able to detect L. monocytogenesfrom hotdogs or bologna that was naturallycontaminated or artificially inoculated with10 to 1000 CFU/g after enrichment inbuffered Listeria enrichment broth in lessthan 24 h. This is an important advancementfor detection of L. monocytogenes in realworld ready-to-eat foods since this sensor is capable of detecting cells in less than 24 h.

INVESTIGATORS � Arun Bhunia (Principal), Mark Morgan and Shu-I Tu (School of Agriculture, USDA-ARS)

B H U N I A

MORGAN

TU

“Biosensors use

a combination of

biological receptors

and physical or chemical

transducers, which

represent a new and

unique technology…”


Project RationaleFoodborne disease is one of the most com-mon causes of morbidity and mortality in theworld, and more than 200 known diseasesare transmitted through food. In the UnitedStates there are about 76 million cases and of which 325,000 require hospitalization and5,000 die each year. Foodborne pathogens of high concern include: E. coli O157:H7,Salmonella, Listeria monocytogenes, Toxoplasmaand Campylobacter. Therefore, detection of these pathogenic bacteria during food processing and storage is crucial for themicrobiological safety and prevention of possible outbreaks. Antibody-based detectionmethods are regarded as rapid and efficientand are widely used in conventional enzymelinked immuno-sorban assasys (ELISA) anddipstick methods. In recent years antibodieshave been successfully used in biosensor tools for lessening detection time. Therefore,specificity and avidity of a given antibody for the target bacteria is extremely important,specifically those originating from stressfulenvironments of food. It has been observedbefore stress can affect antigen expression onbacterial cells thus affecting antibody-baseddetection. The completion of this project will allow for development of a microbialdetection kit that can be used for multiplepathogens. This will save time and money for product testing and also help regulatoryagencies and the food industry evaluate products for key food pathogens.

M U LTI - PATH O G E N S C R E E N I N G A N D /O R CO N F I R M ATI O N V I A M I C ROA R R AY D E TE C TI O N

Project Objectives

Develop antibody specific for L. monocytogenes, Salmonella enterica and E. coli O157:H7.

Identify specific unique target antigensfor antibody development.

Synthesize peptides, generate rabbit polyclonal antibodies, and characterize.

Determine the effect of environmental and physiological stresses on antigen expression.

Develop sandwich ELISA for each pathogen.

Project HighlightsAfter genomic/proteomic analysis, nine rabbitpolyclonal antibodies (PAb) were developedagainst different target peptide antigens of L. monocytogenes. All but one showed a specificreaction with the target peptide. Of which,only PAb Lm404 (internalin B) and LmC639(ActA) (actin polymerization protein) showedspecific reactions for the proteins fromL. monocytogenes and not from any otherListeria species tested. However, the remainingPAbs reacted with multiple protein bandsand showed cross-reactions with otherListeria species. The lack of specific bindingcould be due to the denaturation and alteredfolding of the antigen on the cell surface.InlB and ActA peptides appeared to be themost promising targets for L. monocytogenesspecific antibody production. The majordrawback for these antibodies involved high background reactions with E. coliand Salmonella. We believe this may be due to high titer levels (for antibodies against several common microorganisms in the serum) in rabbits. Specific pathogen-free rabbits are currently being considered to improve antibody development for theseantigens.

Arun Bhunia (Principal) (School of Agriculture) � INVESTIGATORS

B H U N I A

“The completion of

this project will allow

for development of

a microbial detection

kit that can be used for

multiple pathogens.”


Project Rationale Less than 1,000 L. monocytogenes cells in processed ready-to-eat products could be a serious threat to susceptible consumerswith compromised immune system.Therefore, sensitive and specific detection of this pathogen is essential. Conventionalmicrobiological methods take too long(2-7 days) to detect and identify pathogens in food. A biosensor based approach presentsa promising and sensitive alternative tool todetect few bacterial cells in hours instead ofdays. Foods containing such low numbers of cells require enrichment, which is a time-consuming step. Immunoseparation methodsare inefficient in recovering large numbers of L. monocytogenes cells from food, thus a sensitive method of detecting low cell numbers is needed. A cell-based sensor wasdesigned to assess its ability to sensitivelydetect L. monocytogenes-induced cell membrane damage. Initially, we used a B- lymphocyte cell line, Ped-2E9 for thiseffort. Since these cells grow in suspensionand do not require contact dependent growthand producing inconsistent impedance signal.Therefore, we used a macrophage cell line,RAW, which propagates on solid substrateand elicits a cytotoxic response from L.monocytogenes as early as 2 h. Mechanism ofRAW cell death was also evaluated and wasfound to be due mainly to the action of thetoxin and only partially by cellular invasion. Using a binding solution of Arg-Gly-Asp and fibronection adhesion-promoting peptide, the RAW cells were grown on an interdigitated microsensor electrode(IME)-chip and exhibited a positive cytotoxic response from L. monocytogeneson-chip using the LDH release assayalthough significant differences as measured by impedance spectroscopy were not observed.

B I O S E N S O R BAS E D A P P ROAC H E S F O R R A P I D A N D S E N S ITI V E D E TE C TI O N O F L I STE R I A M O N O CY TO G E N E S

Project Objectives

Develop or evaluate cell line that wouldgrow on the interdigitated microsensorelectrode chip.

Measurement of pathogenic potential of Listeria off- and on-chip.

Adhesion and invasion properties of Listeria monocytogenes serotypes on adherant Caco-2 cells.

Use of frozen Ped-2E9 cells or cell analog (liposome) for fluorescence based or colorimetric based detection.

Test stressed Listeria cells (heat, cold, salt acid induced stress) with the two step detection method.

Develop a rapid cytotoxicity assay for Bacillus cereus toxin.

Project HighlightsA cell-based sensor was developed to detectviable pathogenic Listeria monocytogenesfrom food. An adherent macrophage cell line, RAW was used for cell based sensor.Interdigitated electrode chips were fabricatedand cell growth chambers were created byusing PDMS. Fibronectin adhesion promot-ing peptides and a tripeptide (Arg-Gly-Asp)were successfully used to promote the attach-ment of this cell line to interdigitated elec-trode chip. Data showed that this cell line is sensitive to L. monocytogenes and the cytopathic effect could be assayed in 2- 4 hdepending upon the initial L. monocytogenesconcentrations off chip or on chip; however,on chip measurement of cytotoxicity byimpedance spectroscopy was unsuccessful.The cytotoxic mode of action of L. monocyto-genes was found to be due mainly to theaction of the toxin and only partially to cellu-lar invasion. Although we were unsuccessfulon demonstrating a significant difference inthe impedance measurement for pathogenicand nonpathogenic species, several key funda-mental questions were resolved in this projectand this would pave the way for alternativemeasurement systems in the future.

INVESTIGATORS � Arun Bhunia (Principal), Mark Morgan and Rashid Bashir (School of Agriculture, School of Engineering)

B H U N I A

MORGAN

BAS H I R

“A biosensor

based approach

presents a

promising

and sensitive

alternative tool

to detect few

bacterial cells

in hours

instead of

days.”


Project Rationale Our long-term research goals are to developbioluminescent sensors for the detection ofpathogens associated with food and integra-tion of these bioreporters with emerging lightdetection technologies. The advancements in optical detection technologies will permitthe development of small, portable, andhighly sensitive optical transducers capable of measuring bioluminescent responses from bioreporter organisms. The integrationof these transducers with bioluminescentreporters will provide detection technology toimprove food safety. We are most interestedin the development of a suite of bacterio-phage-based bioluminescent bioreporters for the detection and monitoring of patho-genic bacterial species in raw or processedmeats, fruits, and vegetables. Bacteriophagebioluminescent bioreporters are capable ofinfecting specific host cells resulting in theproduction of visible light. This work is in response to microbial contamination of fresh fruits and vegetables and recent concerns related to foodborne pathogenssuch as Campylobacter jejuni, E. coliO157:H7, and L. monocytogenes.

D E TE C TI O N O F S P E C I F I C FO O D B O R N EPATH O G E N S U S I N G A T WO COM P O N E NTBAC TE R I O P H AG E / B I O LUM I N E S C E NTR E P O RTE R SYSTE M I N CO N J U N C TI O N W ITH A H A N D H E L D LUM I N OM E TE R .

Project Objectives

Genetically construct pathogen-specificbacteriophage capable of inducing bioluminescence from bioluminescent bioreporter cell populations for quanti-tative sensing of microbial pathogenic species in raw or minimally processed meats, fruits, and vegetables.

Analyze bacteriophage/bioluminescent bioreporter system with portable field-based photomultiplier unit for the development of a suite of simple, rapid, real-time, on-site testing mechanisms for bacterial pathogens in meats, fruit, and vegetables.

Project HighlightsWe completed construction of a P22/luxISalmonella Bacteriophage/BioluminescentReporter System for the detection ofSalmonella and integrated the MicroTox assaystrain into the assay. This construct allowsthe testing and validation of the overall project objectives of the luxI phage basedapproach for pathogen detection. Detectionlimits in the developed assay were around103 cfu (pure culture) in approximately 6 hours. The developed assay has the potential of immediate impact because it can be incorporated with equipment alreadydeployed and used routinely for ATP hygienetests. To facilitate the construction of aphage-based detection system the completegenome E. coli O157:H7 bacteriophagephiV10 was sequenced. Initial analysis hasidentified regions for modification allowinginsertion of reporter genes which do notaffect the phage life cycle. A recombinationsystem was developed for the modification of the E. coli O157:H7 bacteriophage phiV10by isolating a phiV10 lysogen of E. coliO157:H7. The strain was subsequently transformed with pKD46 which containingan arabinose inducible recombinase, allowingelectroporation of linear DNA for insertionof reporter genes into specific regions of the phage genome. This allowed assembly of a phiV10 ainS phage for the abovedescribed assay.

Bruce Applegate (Principal) (School of Agriculture) � INVESTIGATORS

A P P L E GATE

“The developed

assay has the potential of

immediate impact…”


Project Rationale The goal of this research is to harness thepower of bacterial phage display with affinitychromatography to develop a biologicalamplifier for the detection of small numbersof pathogenic organisms in foods. Theresearch will generate and purify phages thatare designed to selectively detect Salmonellaspp. Projects on biochips, antibody-basedassays coupled with impedance based spectroscopy, fluorescence microscopy,and enzyme-linked immuno-assays (ELISAs)are addressing the diverse needs of detectionmethods for food pathogens. Research under-way in our laboratories are addressing theprocessing of food samples, amplification of organisms using culture or rapid separa-tion techniques, and selective capture ofpathogenic organisms from a complex background of other organisms, proteinmacromolecules, and other substances. Theproject examines the biological fundamentalsof applying a phage display method with the use of affinity (liquid) chromatography to obtain purified phages for infectingSalmonella spp. and also separating phagesthat would be generated in the presence of Salmonella spp., thereby indicating thepresence of this food pathogen, even if other(non-pathogenic) bacteria are present. If successful, this technology platform could be directly coupled to the existing antibody-based assays.

B I OA M P L I F I CATI O N U S I N G P H AG E D I S P L AYF O R TH E D E TE C TI O N O F SA L M O N E L L A S P P.A N D ITS EVA LUATI O N A S A TE C H N O LO GYP L ATF O R M F O R TH E S I M U LTA N E O U S D E TE C TI O N O F M U LTI P L E PATH O G E N S I N TH E SA M E SA M P L E .

Project Objectives

Construct and propagate modified bacteriophages (MB) in host cells consisting of E. coli and Salmonella spp.

Purify modified bacteriophages using affinity chromatography.

Develop an assay using the modified bacteriophages to detect food samplesinfected with Salmonella and/or non-pathogenic organisms, capture, concentrate, and detect using affinity chromatography and other forms of liquid chromatography.

Project HighlightsInitial construction of the recombinant M13to be utilized in the assay was unsuccessful.Therefore, an alternate strategy was pursued.We obtained an M13 GIII phage displaylibrary and isolated phage containing a streptavidin binding epitope. These findingsallowed for the development of purificationprotocols and proof in principle assays for the ultimate success of the project. The P22 recombination vector was obtained and modified to allow insertion of tail spikeprotein. We constructed a P22 sensitiveSalmonella strain which overexpresses the Lac repressor to facilitate the production of the 6xhis tag modification P22 phage. The modified P22 successfully propagatedthe 6xhis tag modified phage withoutexpressing the modified tailspike proteinwhich will then only be expressed by infect-ing wild type strains. This was accomplishedby inserting a constitutively expressed lacIinto the chromosome of the Salmonellastrain. This was crucial to the specific application but also for validation of the overall concept of suppressing the displayed epitopes prior to infection of a wild type host.

INVESTIGATORS � Bruce Applegate (Principal) and Michael Ladisch (School of Agriculture, School of Engineering)

A P P L E GATE

L A D I S C H

“The research will

generate and purify

phages that are

designed to

selectively detect

Salmonella ssp.”


Documents

APPLEGATE Project Rationale SAVIKHIN - Purdue …€¦ · · 2005-04-18Project Rationale This research is ... of DNA probes for the detection of specific target DNA sequences associated