Advances in Microbial Food Safety - American Chemical Society

Chapter 2

Overview of Rapid Methods for the Detection of Foodborne Pathogens and Toxins Downloaded by PENNSYLVANIA STATE UNIV on July 7, 2012 | http://pubs.acs.org Publication Date: April 6, 2006 | doi: 10.1021/bk-2006-0931.ch002

Peter C. H. Feng Division of Microbiological Studies, Food and Drug Administration, U.S. Department of Health and Human Services, 5100 Paint Branch Parkway, College Park, MD 20740-3835 Analysis of foods for pathogens and toxins is a standard practice that has been done using mostly conventional microbiological assays. Advances in technology however, changed food testing procedures by introducing "Rapid Methods" that use antibodies, nucleic acids, special substrates, etc, that can detect these contaminants faster, simpler and with more sensitivity and specificity than conventional tests. As a result, they are ideal for screening foods for the presence or absence of pathogens or toxins. But the complexities of foods continue to be problematic and some culture enrichment or extraction is still required prior to and analysis. Positive Analysis of foods for pathogens toxins is a rapid standard method results are often regarded as presumptive and require practice that has been done using mostly conventional confirmation. Also, assay efficiencies may vary depending on microbiological assays.need Advances in technology however, foods, hence methods to be comparatively evaluated or validatedfood beforetesting routine procedures use. More sensitive and faster assays changed by introducing "Rapid are being that developed, but the complexity of foods continues to Methods" use antibodies, nucleic acids, special substrates, present challenging problems. etc, that can detect these contaminants faster, simpler and with © 2006 American Chemical Society Feng more sensitivity and specificity than conventional tests. As a Division of Microbiological Studies, Food and Drug Administration, result, they are ideal for screening foods for the presence or U.S. Department of Health and Human Services, 5100 Paint Branch absence ofParkway, pathogensCollege or toxins. ButMthe complexities of foods Park, D 20740-3835 continue to be problematic and some culture enrichment or extraction is still required prior to analysis. Positive rapid method results are often regarded as presumptive and require confirmation. Also, assay efficiencies may vary depending on foods, hence methods need to be comparatively evaluated or validated before routine use. More sensitive and faster assays are being developed, but the complexity of foods continues to present challenging problems.

14

© 2006 American Chemical Society

In Advances in Microbial Food Safety; Juneja, V., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2006.

Downloaded by PENNSYLVANIA STATE UNIV on July 7, 2012 | http://pubs.acs.org Publication Date: April 6, 2006 | doi: 10.1021/bk-2006-0931.ch002

15 Microbiological testing is a standard practice used by the industry and regulatory agencies to monitor contamination in foods. But testing foods for pathogens or toxins is a challenging task due to the variations in food composition and matrices. To overcome these problems, conventional methods use media to enrich, select, isolate and identify pathogens in foods. Similarly, in microbial toxin testing, extractions and concentration steps had to be used prior to detection by serological or animal assays. Advances in biotechnology introduced new technologies, which had a tremendous impact on food testing methods. These assays, collectively known as "Rapid Methods" uses antibodies, nucleic acids, specialized substrates and automation, to detect pathogens and toxins specifically, sensitively and rapidly. However, in testing foods, they are not free of limitations, as rapid methods remain susceptible to food matrix problems, hence, necessitating enrichment and sample preparation procedures, which compromises speed of analysis. Foods come in many physical forms (powder, liquid, gel, solid, semi-solid, etc) and their composition is even more varied as they are made up of various combinations of ingredients like carbohydrates, proteins, fats, oils, and chemicals, some of which can interfere with mixing, resulting in heterogenous samples. Compounded by the fact that bacteria are not uniformly distributed in foods, an aliquot tested may not necessarily be representative of the overall sample, so the result may be irreproducible. In addition to matrix problems, normal microflora that are found in many foods and especially at high levels (10 cells/g) in raw foods, can interfere with the detection of pathogens, which are found at much lower levels but can still cause illness. Interference is further enhanced i f food processing procedures have stress-injured the pathogens and they may be out competed by flora during enrichment. To overcome these problems, sample preparation steps had to be modified or adapted for specific foods and samples had to be enriched to resuscitate injured cells, suppress normal flora and to growth-amplify the pathogens prior to detection. Normal flora poses less problems in toxins testing but the complexity of matrices, low toxin levels and processing, which can denature toxins and affect their antigenicity, are of concern, hence, extraction and concentration steps are required prior to detection. Although conventional methods are most often used in food testing, and long regarded as the gold standard, they are labor intensive and time-consuming and therefore, inadequate for making quick assessments on the microbiological quality and safety of foods. 8


16

Rapid Methods


Origin and Definition The emergence of rapid methods is linked directly to biotechnology, which in turn originated from the wealth of knowledge derived from years of basic molecular research (/). Although the term "rapid methods" has only existed in the literature for the past 20 years or so, the speed with which rapid methods developments took place surprised many. In 1981, international experts were invited to the Delphi Forecast to speculate on the future technologies that will be used for the detection of bacteria in foods (2). Most of the technologies predicted by the panel were accurate and are used today, but the potential application of antibodies and nucleic acids as diagnostic tools were not predicted by the panel, and yet these two technologies came to dominate the area of rapid diagnostic methods. Development of rapid methods is currently a competitive industry that enjoys popularity and interest worldwide. There is however, no set definition of what is a rapid method and to come up with such a definition is probably not feasible, as the term "rapid" is subject to interpretation. A s a result, "rapid methods" includes a large group of assays that uses various technologies and ranges from tests that can give results in minutes to those that simply shortens conventional assay procedures, which in some cases take several days or even up to a week to complete (3).

Formats and Technologies Because of the subjective definition of "rapid", the assay formats and technologies used in rapid methods are extremely diverse. But regardless o f format, most rapid methods used for detecting bacterial pathogens in foods still require culture enrichment and the assays for toxins still need extraction or concentration. So, the assay may be rapid but the testing of foods is much slower due to the sample preparation requirements. For example, miniaturized biochemical tests, including automated and other identification tests, can rapidly identify bacteria, sometimes within 4 hrs. However, the isolate has to be a pure culture and the isolation procedure remains conventional, requiring media to grow, select and isolate the colonies, which can take several days. Other rapid methods are modification of conventional methods but are less labor intensive and shortens analysis time. For instance, some assays use disposable cardboards with hydratable selective media so that preparations and



17 disposals before and after testing are greatly simplified. Some assays use specialized substrates in media and measure changes in optical density or other metabolic products from the growth of specific bacteria. Yet, others use fluorogenic or chromogenic substrates, which cause color changes in colonies to provide presumptive identification of bacteria that express specific enzymes (4, 5). A l l these assays simplify and shorten test times but, continue to require growth incubation. Recently, the use of adenosine triphosphate (ATP) to measure total bacterial load is another concept that has been introduced to food testing ((5). A T P assays do not require culture enrichment, hence, provide a quick indication on the sanitary quality of a food processing environment or a product within minutes. But, since all living cells have A T P , procedures have to be included to separate bacteria from yeast or mammalian cells. Similarly, because A T P assays could not differentiate between bacterial species, they were used solely for estimating total bacterial counts. But, some A T P assays have been modified by using specific antibodies and immunomagnetic separation to selectively capture target bacteria from foods then, used bacteriophages to lyse the specific bacterial hosts to release A T P for measurement. The two technologies that had the most impact on testing methods include D N A and antibody analysis, and these assays dominate the field of rapid methods (7). The three prevalent D N A assay formats are probe, cloned bacteriophages and P C R (Table I). Probe assays usually target ribosomal R N A (rRNA) to take advantage of the fact that the higher copy number of bacterial r R N A provides a naturally amplified target and affords greater sensitivity. To detect the specific hybridization of D N A probe to their targets, some assays couple their probes with a chemiluminescent label for detection via fluorescence, but others use biotin for detection by strepavidin- antibody conjugates using enzyme linked immunosorbent assays. Some D N A probe assays are designated for use solely for the identification of pure cultures of bacteria, but others are used for testing for the presence of pathogens in food enrichment cultures. The specific interaction of phage with its bacterial host has also been used to develop assays for detecting pathogens. Two examples are ice nucleation (8) and bioluminescence (//0157:H7 PCR ΒΑΧ Probelia PCR Probelia Warnex rtPCR Genevision Perkin Elmer rtPC TaqMan Neogen probe GENETRAK Alaska Diag. IMS/ATP AK-Phage Neogen probe Listeria spp. GENETRAK Oualicon PCR ΒΑΧ MicroTech L L C probe OligoScan Don Whitlev Sci probe RABIT Warnex rtPCR Genevision Alaska Diag IMS/ATP AK-Phage Sanofi Pasteur PCR L. monocytogenes Probelia Oualicon PCR ΒΑΧ Gen-Probe probe AccuProbe Biotecon Diag Foodproof PCR Neogen probe GENETRAK Warnex rtPCR Genevision Alaska Diag. IMS/ATP AK-Phage rtPCR/probe Roche LightCvcler Neogen Probe Salmonella GENETRAK Oualicon ΒΑΧ PCR Idexx phage BIND Sanofi Pasteur PCR Probelia Warnex rtPCR Genevision Perkin Elmer rtPCR TaqMan rtPCR/probe Roche LightCvcler Biotecon Diag PCR Foodproof DonWhitlev Sci probe RABIT Alaska Diag. IMS/ATP AK-Phage Biotecon Diag PCR Shigella Foodproof Neogen probe Staphylococcus aureus GENETRAK Gen-Probe probe AccuProbe Wamex rtPCR Genevision Neoeen GENETRAK probe Yersinia enterocolitica Probe: DNA probe; PCR: polymerase chain reaction; rtPCR: real-time PCR; IMS/ATP: immunomagentic separation Adenosine triphosphate.


Bacteria Campylobacter

0

a



19 friendly enough for a food diagnostic setting. However, advances in instrumentations enabled automation of P C R assays and furthermore, the introduction of real-time P C R assays that not only enabled even faster amplification, but also provided real-time results, has greatly increased the potential of using P C R to detect for pathogens in foods (9, 70). Several P C R and real-time P C R assays using various detection systems, such as Sybrgreen, F R E T probes, TaqMan, and molecular beacon, are already commercially-available for testing for pathogens in foods. Furthermore, P C R is a critical component of next generation assays such as microarray that are being developed, which enable simultaneous detection of multiple genes on a single chip (11,12). P C R can theoretically amplify a copy of D N A a million fold in a few hours; hence this technology has the potential to eliminate the need for enrichment to growth-amplify bacteria (13). But, numerous attempts to use P C R in food testing have found that many foods contained substances that inhibited or interfered with P C R (73, 14). As a result, the sensitivity achievable by P C R with pure cultures, were often reduced when testing foods and that some cultural enrichment was still required prior to P C R analysis. The specific binding of antibody to antigen and the simplicity of this interaction has facilitated the design of many assays and formats and they comprise the largest group of rapid methods used in food testing (75). Latex agglutination (LA) is the simplest antibody test, where antibodycoated colored latex beads or colloidal gold are used to test cell suspensions of pure bacterial cultures. The presence of specific antigens is indicated by clumping and the reaction takes less than a minute, so it is a very rapid and useful serological typing tool. Reverse passive latex agglutination (RPLA) is a variation of L A ; the main difference being that in L A , the antigens (cells) are insoluble, whereas in R P L A , the antigens (proteins) are soluble, so it is used mostly in testing for toxins. Enzyme-linked immunosorbent assay (ELISA) is a popular antibody assay format and usually designed as a "sandwich" assay, where an antibody is used to capture the antigen and a second antibody conjugated with an enzyme is used for detection. The basic concept of E L I S A has been adapted to various formats and even automated and it can be done in microtiter plate wells, dipsticks, paddles, membranes, pipet tips, etc., and using a variety of detection systems, including chromogenic and fluorogenic substrates and fluorescent or chemiluminescent labels. Recently, immunoprecipitation or immunochromatography assays have become popular for detecting pathogens in foods. The assay is also a "sandwich" antibody test but, instead of conjugates, the detection antibody is labeled with colored latex beads or with colloidal gold to give a visible band of immunoprecipitation. Fashioned after home pregnancy tests, these assays use small, disposable plastic strips or dipsticks that require no washing or



20 manipulations, so are extremely simple, and can yield results within minutes, post enrichment. In food testing, another use for antibodies is for selective capture of bacteria (16, 17). In immunomagnetic separation (IMS), antibody bound to magnetic beads are used to selectively capture bacteria from enrichment media thereby, shortening culture enrichment time. IMS is analogous to selective enrichment, except that it can be done within an hour, so it is faster and does not use harsh chemicals or antibiotics that may cause cell stress or injury. Although IMS will not yield a pure culture, the target organism is greatly concentrated and can be further tested by plating, serological, genetic or other tests. Coupling IMS to other tests generally improves overall detection efficiency of assays. Antibodies are also used extensively as the specificity component in next generation tests like biosensors that detect physicochemical changes in a matrix caused by antigen-antibody binding (18, 19, 20). Biosensors for detecting food borne pathogens are already commercially-available and although most still require a short enrichment step in the analysis of foods, biosensors may potentially enable in-line monitoring for pathogens and toxins during food processing (27). Antibody-based assays for detecting bacteria and toxins are shown in Tables II and ΙΠ, respectively.

Applications, Validation, and Impact of Rapid Methods Most rapid methods are single target tests and continue to require some culture enrichment prior to testing. The benefits of enrichment however, outweigh the sacrifices in speed of analysis, as enrichment dilutes out effects of inhibitors, allows the repair of stress-injured cells, and also helps to differentiate viable from non-viable cells. But even with the enrichment steps, rapid methods are still faster, more sensitive, and more specific than conventional methods that are being used for the detection of pathogen and toxins in foods. A s a result, they are well suited for screening large numbers of food samples for the presence or absence of a particular target. In use as a screening tool, negative results by rapid method are accepted but positive results are regarded only as presumptive and needs to be confirmed. Since confirmation is often done by conventional methods, it is time consuming and will extend analysis time by a few days. This however, may not be an imposing requirement as negative results are most often encountered in food testing. Because rapid methods use various technologies, their detection sensitivities vary greatly (Table IV) and may be food dependant as some assays work better in some foods than others. It is therefore, critical that rapid methods are evaluated to ensure effective performance in specific foods. Comparatively, testing of rapid versus standard method, as done in validation studies, are also critical to determine false-positive or false-negative rates. Since negative results from rapid methods are accepted, false-negatives i f not recognized, are


21 Table II. Partial Listing of Antibody-Based Rapid Methods


Bacteria Campylobacter

Escherichia coli 0103 0111

0128 0145 026

091 H7 0157

Assay Campyslide Meritec Microscreen Dryspot Pathatrix Assurance Gold TransiaPlate Alert VIA EIAFoss VIDAS Singlepath PATHIGEN DIA/PRO DETEX SeroCheck Dyanbeads Olll-F SeroCheck Dyanbeads SeroCheck SeroCheck Dyanbeads 026-F SeroCheck Dyanbeads SeroCheck RIM Wellcolex RIM Dryspot Prolex Ecolex0157 Wellcolex 0157-AD Captivate Microscreen ANI£. co//0157 Pathatrix Dyanbeads VIP Reveal NOW QUIX ImmunoCardSTAT PATH-STIK TransiaCard RapidChek Singlepath

Formaf LA LA LA LA IMS ELISA ELISA ELISA ELISA ELISA ELFA Ab-ppt ECL biosensor UMEDIK ElectroIA LA IMS LA LA IMS LA LA IMS LA LA IMS LA LA LA LA LA LA LA LA LA LA LA LA IMS IMS Ab-ppt Ab-ppt Ab-ppt Ab-ppt Ab-ppt Ab-ppt Ab-ppt Ab-ppt Ab-ppt

Maker Becton Dickinson Meridian Microgen Oxoid Matrix Microscience BioControl Diffchamb AB Enojen TECRA FOSS bioMerieux Merck BioVeris

Molecular Circuitry Oxoid Dynal Denka Seiken Oxoid Dynal Oxoid Oxoid Dynal Denka Seiken Oxoid Dynal Oxoid REMEL Murex REMEL Oxoid Pro-Lab Orion Diagnostica Murex Denka Seiken IDG/LabM Ltd Microgen ANI Biotech Matrix Microscience Dynal BioControl Neogen Binax Univ. Health Watch Meridian Diag. Celsis Diffchamb AB Strategic Diag. Inc Merck

Continued on next page.


22 Table II. Continued.


Bacteria

0157/026 Listeria spp.

L. monocytogenes Salmonella

Assay Eclipse 0157.H7 0157 Antigen SMART-II 0157 Coli-Strip PetrifilmHEC EZcoli Assurance HEC0157 TECRA E. coli0157 Premier0157 Transia Plate 0157 Ridascreen Colortrix EIAFoss VIDAS VIDAS ICE PATHIGEN DETEX DIA/PRO RBD3000 EHEC-Tek Microscreen ListerTest Dyanbeads Pathatrix Singlepath VIP Clearview Reveal Listeria-TEK TECRA VIA Assurance Transia Plate VIDAS LIS EIAFoss UNIQUE PATHIGEN RBD3000 DIA/PRO DETEX VIDAS LMO TransiaPlate Bactigen Spectate Microscreen Wellcolex Serobact RapidTest ANI Salmonella Salmonella Verify Salmonella Seiken

Formaf Ab-ppt Ab-ppt Ab-ppt Ab-ppt blotEIA tubeEIA ELISA ELISA ELISA ELISA ELISA ELISA ELISA ELISA ELISA ELFA ELFA ECL electroIA biosensor biosensor ELISA LA IMS IMS IMS Ab-ppt Ab-ppt Ab-ppt Ab-ppt ELISA ELISA ELISA ELISA ELFA ELISA cap.EIA ECL biosensor biosensor ElectroIA ELFA ELISA LA LA LA LA LA LA LA LA LA

Maker Eichrom Technologies Morningstar Diag. New Horizon Coris BioConcept 3M Difco BioControl 3M Canada Tecra LMD Meridian Diffchamb AB rBiopharma Matrix Microscience FOSS bioMerieux bioMerieux BioVeris Molecular Circuitry UMEDIK AATI Organon-Teknika Microgen VICAM Dynal Matrix Microscience Merck BioControl Unipath Neogen Organon Teknika TECRA BioControl Diffchamb AB bioMerieux FOSS TECRA BioVeris AATI UMEDIK Molecular Circuitry bioMerieux Diffchamb AB Wampole Rhone-Poulenc Microgen Lab. Wellcome REMEL Unipath ANI Biotech VICAM Denka Seiken


23 Table II. Continued. Maker Formaf Assay Dynal IMS Dynabeads VICAM IMS Salmonella Screen Matrix Microscience IMS PATHATRIX Organon Tek. Salmonella-TEK ELISA TECRA ELISA TECRA VIA Binax ELISA EQUATE KPL ELISA BacTrace BioControl ELISA Assurance GEM ELISA Salmonella Rhone-Poulenc ELISA LOCATE Matrix Microscience Colortrix ELISA Bioline/Mast Diag. ELISA Salmonella Diffchamb AB ELISA Transia Plate Gold Labor Diag. Leipzig ELISA Salmotype FOSS ELISA EIAFoss bioMerieux ELFA VIDAS SLM bioMerieux ELFA VIDAS ICS BioVeris ECL PATHIGEN UMEDIK biosensor DIA/PRO AATI biosensor RBD3000 BioControl Ab-diff. Salmonella 1-2 KPL blot CHECKPOINT TECRA cap.EIA UNIQUE Celsis Ab-ppt PATH-STIK Neogen Ab-ppt Reveal Unipath Ab-ppt Clearview Diffchamb AB Ab-ppt TransiaCard Merck Ab-ppt Singlepath New Horizon Ab-ppt SMART-II VICAM LA S. enteritidis SE Verify BioControl Ab-diff. Salmonella 1-2 SE Bommeli Diag. ELISA CHEKIT IDEXX ELISA FlockChek Wampole LA Bactigen Shigella LabWellcome LA Wellcolex Becton Dick. LA Staphylococcus Aureus Staphyloslide LA Trisum AureusTest Oxoid LA StaphyTest plus LA Microgen Microscreen ANI Biotech LA ANI S. aureus TECRA ELISA TECRA Denka Seiken LA V. cholera 01-AD Vibrio cholera ANI Biotech LA Yersinia enterocolitica ANI Microgen LA Microscreen LA: latex agglutination; IMS: immunomagnetic separation; ELISA: enzyme linked immunosorbent assay;ELFA: enzyme linked fluorescence assay; Ab-ppt: immuno-precipitation; ECL: electrochemiluminescence; ElectroIA : electroimmunoassay; blot EIA : blot enzyme immunoassay; cap.EIA : capture EIA; Ab-diff. : antibody diffusion.


Bacteria

a


24

Table ΙΠ. Partial Listing of Rapid Methods for Bacterial Toxins Bacteria Bacillus cereus


Toxin diarrheal enterotoxin Clostridum botulinum A,B,E,F

C. perfringens Escherichia coli

enterotoxin Shiga toxin

Labiletoxin Stabletoxin Staphylococcus aureus

enterotoxin

SEB Vibrio cholera V. parahaemolyticus β

CT hemolysin

Assay BDEVIA BCET ELCA Bot toxin Smart-II BTA PET Verotest Premier VTEC Screen TaqMan Ridascreen Duopath ProSpecT Transiaplate VET COLIST E. coli ST SET

Format ELISA RPLA ELISA ELISA Ab-ppt Ab-ppt RPLA ELISA ELISA RPLA LA rtPCR ELISA Ab-ppt ELISA ELISA RPLA ELISA ELISA RPLA

Company TECRA Denka Seiken Elcatech METAbiologics NewHorizon Alexeter Tech Denka Seiken Microcarb Meridian DenkaSeiken DenkaSeiken Perkin Elmer rBiopharma Merck K g a A REMEL Diffchamb A B DenkaSeiken DenkaSeiken Oxoid Denka Seiken

SETVIA SETID Transiatube TransiaPlate Ridascreen VidasSET SMART BTA VET KAP

ELISA ELISA ELISA ELISA ELISA ELFA Ab-ppt Ab-ppt RPLA RPLA

TECRA TECRA Diffchamb A B Diffchamb A B rBiopharma bioMerieux New Horizon Alexeter Tech Denka Seiken DenkaSeiken

See previous tables.


25 Table IV. Detection Sensitivities of Various Assays


Assay Culture Adenosine triphosphate (ATP) Latex agglutination (LA) Reverse Passive L A (RPLA) Enzyme linked immunosorbent assay Immunomagnetic seperation (IMS) Immunodiffusion (1-2 Test) Immunoprecipitation (Ab-ppt) D N A Probe phage (lux or ina) PCR Biosensor a

Bacteria (cells/g) 1 0 - 10 10 10 NA 1 0 - 10

Advances in Microbial Food Safety - American Chemical Society

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