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Challenges to Developing Real-TimeMethods to Detect Pathogens in FoodsDespite progress, real-time nucleic acid-based assays to detectpathogens in foods are not yet suitable for routine use

Lee-Ann Jaykus

Even with improved methods for de-tecting pathogens in foods and envi-ronmental samples, microbiologistsso mandated often face a “needle-in-a-haystack” challenge. How does one

detect small numbers of pathogens amid largenumbers of harmless background microflora ina large and complex sample matrix? Traditionalpathogen detection methods rely on culture en-richment, selective and differential plating, andadditional biochemical and serological meth-ods, making for analyses that may easily extendseveral days. Over the years, more rapid meth-ods have replaced plating steps with DNA hy-bridization or enzyme immunoassays. However,even these methods detect at best 103-104 CFU/gof target pathogens, meaning that culture en-richment steps are still necessary, as is confirma-tion for presumptively positive results. Theoverall time savings is minimal.

However, enzyme-based nucleic acid amplifi-cation methods, including the polymerase chainreaction (PCR) and nucleic acid sequence-basedamplification (NASBA), represent a significantadvance, one that has the potential to speed theoverall analysis by replacing culture enrichmentprocedures with those that amplify specific nu-cleic acid sequences. Moreover, these new meth-ods are highly specific and can be used to iden-tify microorganisms that cannot be readilycultured.

Important Technical Challenges in

Applying Rapid Methods to Food Samples

Despite these advantages, however, those hop-ing to routinely use nucleic acid amplificationmethods for detecting pathogens in food andenvironmental samples still face several techni-

cal challenges, including: (i) low levels of con-taminating pathogens; (ii) high volumes or highmass (�25 ml or grams) compared to amplifica-tion volumes (�10 �l); and (iii) residual matrixcomponents that inhibit enzymatic reactions.Additional challenges include the need to con-firm findings by time-consuming proceduresand satisfying industry and regulatory concernswhen nucleic acid sequences from nonviablepathogens are detected.

Thus, researchers in this field often report thatPCR or NASBA detection limits prove no betterthan 102-103 CFU/g of food product, which isonly slightly better than what they report usingELISA and DNA hybridization methods. By andlarge, culture enrichments are still necessary toamplify targets before they can be detected usingnucleic acid amplification procedures.

Recent developments, referred to as real-timenucleic acid amplification technologies, can fur-ther reduce overall test times by replacing time-consuming postamplification electrophoresis orhybridization methods with methods based onfluorescence resonance energy transfer (FRET)(Fig. 1). Of particular note are two commercialsystems—the TaqMan®, which is available fromApplied Biosystems in Foster City, Calif., (http://home.appliedbiosystems.com) and the Nucli-Sens®, which is available from bioMerieux ofDurham, N.C. (http://www.biomerieux.com).

The TaqMan® assay capitalizes on the endo-geonous 5�3 3� exonuclease activity of TaqDNA polymerase by including a dual fluoro-phore-labeled oligonucleotide probe during thePCR amplification cycle. The fluorescence pro-duced by the 5� reporter dye ordinarily isquenched by the 3� quencher dye. However, if theprobe hybridizes to its complementary amplicon

Lee-Ann Jaykus isan associate profes-sor in the Depart-ments of Food Sci-ence andMicrobiology, Col-lege of Agricultureand Life Sciences,North CarolinaState University,Raleigh.

Volume 69, Number 7, 2003 / ASM News Y 341

“mate” during the PCR reactions, the 5� nucleaseactivity of Taq polymerase lops off the fluorescentdye molecule, which fluoresces freely in the ab-sence of a neighboring quencher molecule.

Meanwhile, the NucliSens® Basic Kit is basedon NASBA, a transcription-driven isothermalRNA amplification method. bioMerieux recentlyintroduced its NucliSens® EasyQ system, whichcombines NASBA with molecular beacons, whichconsists of a dual fluorophore-labeled oligonucle-otide probe that is incorporated into the amplifi-cation cocktail. The molecular beacon probe se-quence is flanked by a hairpin stem that is formedby two complementary (yet unrelated) arm se-quences, forming a stem-and-loop structure. Oneof the arms is labeled with a fluorescent moiety,the other with a quencher that is kept in proximitybecause of the complementarity of the arm se-quences. The probe is added prior to NASBA, andif hybridization to specific amplicons occurs dur-ing amplification, this proximity is disrupted, leav-ing the fluorophore free to fluoresce.

These assays give results in real time becausethe amplicon can be detected and confirmed

while it is being amplified. In theory, these assaydesigns could enable nucleic acid amplificationto replace culture enrichment, while the newlygenerated amplicons that hybridize to fluores-cent probes and are immediately detected couldreplace otherwise time-consuming culture con-firmation steps. Because these two steps arecombined, total testing time could be droppedfrom days to hours (Fig. 2). However, despitethe promise of these real-time detection strate-gies, their routine use for detecting pathogens infood and environmental matrices will remainlimited until scientists are able to adequatelyaddress the needle-in-a-haystack dilemma.

Concentrating Pathogens Initially Can

Improve Overall Analysis

Separating, concentrating, and purifying food-borne pathogens from sample matrices beforeundertaking nucleic acid amplification steps im-proves the overall analysis (Fig. 3). Such proce-dures are necessary when detecting viral agentsfrom foods because, unlike those bacterial

F I G U R E 1

Representative detection and confirmation methods for nucleic acid amplification. (A) Traditional agarose gel electrophoresis and Southernhybridization; (B) Real-time PCR detection of the tdh gene using the TaqMan® assay in serial 10-fold dilutions of an overnight V.parahaemolyticus culture (courtesy of Angelo DePaola, FDA Gulf Coast Seafood Laboratory); (C) Fluoroscein (FAM)-labeled molecularbeacon used in NASBA reaction for the assessment of enterotoxin gene expression in Bacillus cereus grown in skim milk in late log/earlystationary phase (courtesy of John McKillip, Louisiana Tech University).

342 Y ASM News / Volume 69, Number 7, 2003

pathogens that can be cultured, viruses are inertin food matrices. Unfortunately, separating andconcentrating bacterial pathogens from foodscan prove difficult because, unlike many viruses,bacterial cells are highly sensitive to agents such

as organic solvents and detergents that are usedto remove matrix-associated interfering com-pounds.

Approaches for concentrating bacteria needto address three issues that plague environmen-

Hold the ShellfishLee-Ann Jaykus says emphaticallythat she will not eat two foods,raw shellfish and sprouts. Her se-lective abstinence is not surprisingbecause her research focuses onmicroorganisms that contaminatefoods–and both these foods arenotorious for being contaminatedby nasty microorganisms. “I tellmy students that when you areeating raw shellfish, you’re eatingpoop and all,” she says, laughing.

She learned this as a graduatestudent when she studied rawshellfish as part of her doctoralresearch. Her aversion to sproutscame later, about six years ago,when her own undergraduate stu-dents decided to study the micro-biological quality of sprouts astheir class project. “I was amazedwhen the results came in.” shesays. “When I saw the levels ofbacteria, I stopped eating them.It’s very difficult to control bacte-rial contamination on seeds. Theway they are grown—in high mois-ture at body temperature— it’s likeputting bacteria in an incubatorwith a bunch of food and lettingthem go crazy.”

Nevertheless, Jaykus has greatfaith in the safety of the U.S. foodsupply, although she wishes therewere faster methods for detectingproblems when they crop up. Shebecame aware of the need forfaster tests during the mid-to late1980s, a time of several high-pro-file food-poisoning outbreaks.She was working in a lab inModesto, Calif., where her dutiesincluded conducting tests for bac-

teria in food products. “It was justtaking too long,” she says, refer-ring to the wait before re-sults were ready. “The processorswanted their results, and wecouldn’t turn it over fast enough.

“We really have to develop al-ternative sample processing meth-ods,” she adds. “We need morescientists to be working in thatarea, and better technology todeal with these issues. We areworking with a couple of differenttechnologies to try to concentratebacteria out of representativefood sample sizes, and also tech-nologies to concentrate and purifynucleic acids that would provideevidence of contamination.”

Jaykus developed an interest infood science during her under-graduate days at Purdue Univer-sity in West Lafayette, Indiana,where she began studying medicaltechnology. “I was a little worriedI was going to be bored,” she says.“Somebody told me I should lookinto food science because it wasall of these different disciplinestogether, including biology, chem-istry, and engineering. So I tookmy first microbiology course andloved it. I really liked that youcould see the test tubes turn differ-ent colors during experiments. Itwas very definitive–if it turned acolor, you got what you wanted.Now that I’m a Ph.D., I know thatit’s not always that straightfor-ward.” What she likes today is theability to combine her formal train-ing in food science with her love ofbiology “and apply it in a very

practical matter. Everybody is in-terested because everybody eats.”

Jaykus grew up in Ridgefield,Conn., the eldest of three girls.Her father is a land surveyor, hermother teaches the fifth grade.She earned her B.S. and M.S. de-grees in food science at PurdueUniversity and her Ph.D. at theSchool of Public Health at theUniversity of North Carolina(UNC) in Chapel Hill. She cur-rently is an associate professor inthe food science department atNorth Carolina State University(NCSU) in Raleigh. She is marriedto a pediatric oncologist who is aprofessor at UNC Chapel Hill.Between them, they have fourteenagers. Before taking a posi-tion at NCSU, Jaykus worked as aquality control manager for Frito-Lay, Inc. and as the microbiologydivision manager for Dairy andFood Labs, Inc., in Modesto.

Jaykus good-naturedly fieldscomments about her cooking andeating habits. “My husband says Icook meat to death,” she says.“We eat a lot of baked Frito-Layproducts. It took a while to getused to them, but once you do, itbecomes part of your diet. At 44,with high cholesterol, I no longercan eat that high-fat stuff any-more.” However, she adds, “Sinceit was one of my guilty pleasures, Ihave to say this: there is nothingbetter than hot Fritos, or potatochips, right out of the fryer.”

Marlene Cimons

Marlene Cimons is a freelance writerin Bethesda, Md.


tal and food microbiologists, namely (i) how toseparate pathogens from sample particulates;(ii) how to remove inhibitory compounds asso-ciated with the matrix; and (iii) how to reducethe sample size and also recover nearly 100% ofthe target organism(s). Depending upon theneeds of the analyst, methods may be designedto concentrate either entire bacterial popula-tions or only specific segments. Preferably, thesemethods will not destroy viability, meaning sub-sequent culture methods will work if needed.

Options for Concentrating Bacteria

Methods for separating bacteria from a foodmatrix and then concentrating them depend onseveral chemical, physical, and biological prin-ciples (Table 1). In general, the goal is to take a25–50-g sample and reduce its volume to lessthan 1 ml, with high recovery of viable targetbacteria and full removal of matrix-associatedinhibitory compounds. During these proce-dures, attractive forces between bacterial cellsand matrix components are disrupted and

blocked from recurring, preferably without kill-ing the bacteria. Enzymes, detergents, andchanges in pH or ionic conditions provide waysto dissociate bacteria from such matrices, albeitwith mixed success.

Centrifugation is a commonly used physicalmethod to separate and concentrate microor-ganisms from complex sample matrices. Often,samples are centrifuged at low speeds to sedi-ment food particulates, leaving bacterial cells inthe supernatant fluid. Alternatively, samples arecentrifuged at higher forces to sediment bacte-rial cells, although other particles of equal orgreater density will sediment as well. Differen-tial and density gradient centrifugation methodsalso may be used to separate bacteria from com-plex food matrices such as meats. Centrifuga-tion efficiencies can be improved if particle di-ameters are increased. One way involvesremoving electrostatic charges (typically bychanging pH) to allow particles to adhere andthus coagulate. Alternatively, adding smallamounts of high-molecular-weight, charged ma-terials that bridge oppositely charged particles

F I G U R E 2

A comparison of traditional and real-time detection methods.


enables loose aggregates to form, orflocculate, and these may be readily re-moved by centrifugation.

Filtration is another important toolfor concentrating bacteria. Filteringthrough cheesecloth, filter paper, or asimilar material can remove solid foodparticles from samples, while the bacte-ria are retained in the filtrates. Alterna-tively, a food product homogenate canbe passed through a filter designed toretain the microorganisms based ontheir size and chemical properties withsubsequent disposal of the filtrate. Fil-ter type, pore shape and dimension, andthe physical and chemical properties ofthe microorganisms all contribute torecovery efficiencies.

Bacteria also may be immobilized us-ing various materials, including ion ex-change resins, lectins, and metal hy-droxides. Some of these agents, such asmetal hydroxides, can be used in flocform, while others are adsorbed tobeads or affinity columns. After thebacteria within a food sample becomeattached to a solid support and are sub-sequently separated from the food ma-trice, they are desorbed and then con-centrated. Enzymes or changes in pH or ionicstrength may be used to desorb bacteria fromthese immobilizing materials, with various de-grees of efficiency.

Immunomagnetic separation (IMS) is cur-rently a widely used biologically based bacterialconcentration technique. IMS combines the useof monoclonal antibodies with magnetic spheresto select target cells from a mixed population.After allowing the antibody to bind target bac-terial antigens within a food matrix, target cellsare separated from mixtures by exposing themto a magnetic field. For instance, monosizedsuperparamagnetic polymer particles, known asDynabeads™, are available from Dynal Biotechof Oslo, Norway (http://www.dynal.no). IMShas proved an effective tool for isolating severalfoodborne pathogens, including Listeria mono-cytogenes, Escherichia coli O157:H7, and Sal-monella species. However, even when IMS pre-cedes nucleic acid amplification steps, detectionlimits are rarely better than 103 CFU/ml of thetarget bacteria in a food homogenate, meaningculture enrichment is still often required.

When considered together, many of the bac-terial concentration methods are complex, ex-pensive, and can be applied only to relativelylow-volume samples. Another common theme isthe need for initially treating samples to desorbbacteria from food matrices. Although achiev-ing a 50- to 100-fold sample concentration withrecovery of 100% of the microorganisms andcomplete removal of all matrix-related inhibi-tory compounds is desirable, this goal is difficultto achieve with current technologies.

Additional Sample Concentration through

Nucleic Acid Extraction

Effective nucleic acid extraction methods canfurther reduce sample volumes and remove ma-trix-associated inhibitors. Although nucleic acidextraction kits have become commercially avail-able during the last five years, many are madefor use on relatively uniform clinical samples.

These nucleic acid extraction methods typi-cally begin with either enzyme lysis or cell solu-blization using guanidinium isothiocyanate, fol-

F I G U R E 3

Schematic of the concept and power of bacterial concentration prior to the application ofreal-time detection.


lowed by cleanup steps using organic solvents,suspended silica, affinity purification columns,or proprietary compounds. Each additional stepadds time and complexity to the assay, andusually reduces nucleic acid yield.

A further consideration is that many of thesekits, even those marketed for environmental ma-trices such as soil, are designed for samples ofonly 0.1–1.0 g. This means that pathogens willneed to be concentrated prior to nucleic acidextraction for those samples containing rela-tively small numbers of target pathogens. None-theless, a good nucleic acid extraction step canreduce inhibitory substances and further reducesample volumes 10- to 100-fold. If preceded bya pathogen concentration step, a combined con-centration factor of 100-fold or more meansthat a 25-g sample can be reduced to 250 �l orless, a volume that is more suitable for typicalmolecular detection approaches (Fig. 3).

Residual Matrix-Associated

Amplification Inhibitors

Even with the best concentration and purifica-tion schemes, residual matrix-associated inhibi-tors typically remain in final extracts. Theseinhibitors either prevent amplification, resultingin false-negative results, or else reduce its effi-ciency, resulting in poor detection limits. Theseinhibitory effects sometimes are more pro-nounced when target template levels are partic-ularly low, which is precisely when one needshigher amplification efficiencies.

The list of potential matrix-associated inhibi-tors is nearly endless; few are well characterized,and others remain unidentified. Usage of effi-cient and more robust enzymes helps to over-come some problems with inhibitors. Also,some investigators add enhancement agents toincrease amplification efficiencies in the pres-ence of matrix-associated inhibitors. For in-stance, bovine serum albumin (BSA) is particu-larly effective in enhancing the efficiency ofDNA amplification from extracts with iron-con-taining molecules such as hemoglobin or humicacids, which typically are found in meat-con-taining foods and environmental water samples,respectively. BSA presumably scavenges theseinhibitory compounds, preventing them frombinding to and inactivating Taq DNA polymer-ase. Dimethylsulfoxide (DMSO), dithiothreitol

(DTT), and betaine are among other commonlyused enhancement agents.

Choosing the Appropriate

Amplification Target

Nucleic acid amplification assays fail to differ-entiate live from dead cells. Culture enrichmentsprior to PCR do not fully overcome this problembecause nucleic acids from dead pathogens maybe detected even after such enrichments. Al-though some researchers suggest that 16S ribo-somal RNA would be a good alternative ampli-fication target, this molecule is also relativelystable and therefore not a completely reliableindicator of cell viability.

Messenger RNA is considered a more prom-ising target for amplification. However, to be areliable indicator of viability, the target mRNAshould be species or strain specific, have a briefhalf-life, and be constitutively expressed, prefer-ably at high copy number. Meeting all three ofthese criteria is a challenge and, although somestudies are promising, target choice remains animportant consideration. For instance, investi-gators report 1,000-fold differences in assaysensitivity when using different mRNA targets,and transcription levels for one specific targetcan change with cell physiological state. Alsoproblematic is the fact that very little work hasbeen done to adapt these RNA-based amplifica-tion methods to detecting pathogens in food andenvironmental matrices.

Feasibility of Real-Time and Endpoint

Detection Approaches

Proponents of real-time nucleic acid amplifica-tions cite two major advantages to this ap-proach: an ability to detect products as they arebeing amplified and the potential to designquantitative assays. Although both prospectsappeal to food and environmental microbiolo-gists, many food safety regulations stipulate de-tecting either the presence or absence of partic-ular pathogens and are based on zero-tolerancestandards. These rules appear unlikely to changein the near future.

Also, although detecting a signal while a par-ticular nucleic acid is being amplified may speedup an assay by an hour or so, real-time detectionmight be less important than endpoint detection


in which the specific products are detected im-mediately following the termination of their am-plification. Even so, for some pathogens, quan-titative real-time assays may be applicable in thefuture.

For instance, having semiquantitative assaysfor pathogens such as Vibrio vulnificus andVibrio parahaemolyticus could improve themanagement of shellfish-harvesting waters, thusprotecting public health while helping the sea-food industry. Food and Drug Administration(FDA) standards specify that V. parahaemolyti-cus levels remain below 10,000 CFU/g in ready-to-eat seafoods. This microorganism can be de-tected directly in oyster mantle fluids (shellliquor) at a limit of 400 CFU/ml using real-timePCR, according to Andy DePaola of the FDAGulf Coast Seafood Laboratory, Dauphin Is-land, Ala. Moreover, with further concentrationand DNA purification, this detection sensitivitymay be increased by 10- to 100-fold with rela-tive ease.

Nonetheless, there are difficult issues to ad-dress before the food industry adopts this tech-nology for routine uses. First, this industry tra-ditionally insisted on culturing microorganismsto identify them and, thus, is wary of the reli-ability of molecular techniques. Second, the costof reagents used in real-time assays is high(sometimes over $25 per test), while the instru-ments cost from $30,000 to $50,000. Thesecosts are prohibitive for all but the largest com-panies. Finally, the industry is not equipped tohire staff that is trained to do such testing. The

methods simply have to become less expensive,more user-friendly, and more robust.

Applications to Bioterrorism, Other

Considerations

In facing bioterrorism threats, food microbio-logists are seeking to improve their ability toidentify microbial agents in foods as quickly aspossible. Much like natural microbial contami-nants, such agents could be widely dispersedamong different foods in low concentrations,posing the same needle-in-a-haystack challengesthat are faced when dealing with other food andenvironmental samples. Efforts to harness real-time detection strategies and couple them withmicroarray or DNA chip technologies couldhelp to meet these challenges.

Meanwhile, further research is critical. Specif-ically, research is needed to identify, develop,and refine prototype methods for evaluating themicrobiological safety of foods, water, and theenvironment (Table 2). These methods shouldconcentrate pathogens and remove matrix-asso-ciated inhibitors, and should be universal, sim-ple, rapid, and inexpensive. They would thuseliminate or reduce the need for culture enrich-ments, yielding analyses in less than one dayand, preferably, faster. They should also mini-mize the chance for false-positive results and beavailable on a commercial and fully certifiedbasis. Meeting these goals will require extensivecomparative studies between these new technol-ogies and current standard culture methods.

ACKNOWLEDGMENTS

This article is based on a talk presented at the 102nd ASM annual meeting. I thank Andy DePaola of the FDA Gulf CoastSeafood Laboratory for the figure for real-time detection of V. parahaemolyticus using the TaqMan® assay (Fig. 1B) and JohnMcKillip of Louisiana Tech University, Ruston, for the figure for real-time detection of Bacillus cereus using NASBA/molecular beacons (Fig. 1C). I also acknowledge Christina Moore, who prepared Fig. 1 and 2, and the USDA-CSREESNational Research Initiative for financial support.

This work represents paper number FSR 03-13 of the Journal Series of the Department of Food Science, North CarolinaState University, Raleigh NC 27695–7624. The mention of trademarked products in this paper does not imply anyendorsement by the North Carolina Agricultural Research Service or criticism of similar products that were not mentioned.

SUGGESTED READING

Enserink, M. 2001. News focus: biodefense hampered by inadequate tests. Science 294:1266–1267.Lantz, P. G., W. Abu al-Soud, R. Knutsson, B. Hahn-Hagerdal, and P. Radstrom. 2000. Biotechnical use of polymerase chainreaction for microbiological analysis of biological samples. Biotechnol. Ann. Rev. 5:87–130.Payne, M. J., and R. G. Kroll. 1991. Methods for the separation and concentration of bacteria from foods. Trends Food Sci.Technol. 2:315–319.Sharpe, A. N. 1997. Separation and concentration of pathogens from foods, p. 27–44. In M. L. Tortorello and S. M. Gendel(ed.), Food microbiological analysis: new technologies. Marcel Dekker, Inc., New York.Wilson, I. G. 1997. Inhibition and facilitation of nucleic acid amplification. Appl. Environ. Microbiol. 63:3741–3751.


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