Getting Really Rapid Test Results: Advances in Pathogen and Toxin Detection for the Food Industry
By Purnendu C. Vasavada, Ph.D.
In the past few years, the food processing industry has witnessed the introduction of a dizzying array of new or improved rapid methods for the detection of foodborne pathogens and toxins. As the use of the Hazard Analysis & Critical Control Points (HACCP) model and other food safety systems have become firmly entrenched in quality assurance/quality control (QA/QC) programs, the industry’s need for “faster, better, cheaper” real-time test results has also increased.
The penultimate goal for rapid methods (particularly, microbiological methods which constitute the major emphasis in pathogen detection advances) is to provide an operation with answers in “real time.” The HACCP model’s primary goal—on- or at-line monitoring and control—has indeed driven the focus on the role of rapid methods for use in such programs, Although rapid methods can be used in developing a HACCP system and for verifying that the system is working, the technology at this time has not reached the point where real-time monitoring and control is possible.
Several recent rapid and automated method developments are reviewed here—testaments to the food industry’s commitment to close the gap between rapid detection of foodborne contaminants and instantaneous, on-line monitoring to ensure the safety and wholesomeness of foods.
The AOAC Test Kit Database
Keeping updated on the methods available that will enhance efforts to identify which rapid methods really are rapid can be a daunting task. One excellent source of information about rapid test kits is the AOAC Research Institute (AOAC RI), which has certified more than 40 test kits since 1991 when the Performance Tested Methods program was created to provide an independent third-party review of test kit performance claims. Performance Tested Methods status assures the test kit user than independent assessment has been conducted and the kit performs as claimed.
The Performance Tested Methods Program provides a rapid entry point into the AOAC validation process and provides a rapid approval process. The AOAC RI streamlined its Performance Tested Methods program in January 2000 to shorten review times to four to six months while still maintaining high standards, In December 1999, the AOAC RI Board of Directors approved a new consulting service allowing the AOAC RI to work directly with kit manufacturers to develop complete validation programs for test kits, In the past, the AOAC RI provided generic validation guidelines that could be difficult to interpret and apply. Now, with the new consulting service, the AOAC RI can design entire validation programs that meet AOAC validation requirements and are tailored specifically for individual test kits. This service significantly reduces the amount of resources required to collect validation data, increases the quality of data submitted for review and reduces review times.
The AOAC RI also developed and introduced a parallel independent testing procedure. In the past, validation data collected by kit manufacturers were first submitted to and reviewed by the AOAC RI, and then independent data would be collected by the AOAC RI. The new parallel independent testing program lets the AOAC RI and test kit manufacturers collect validation data at the same time. This innovation has significantly reduced the overall time needed to validate a test kit method.
Since last year, eight test kits have been approved under this program and a few others have undergone modifications. These kits, approved by the AOAC RI, have all been granted Performance Tested Methods status and are all licensed to use the AOAC RI Performance Tested Methods certification mark. It should be noted that AOAC RI Performance Tested Methods must meet the same requirements for validation as AOAC International Official Methods, with the exception of reproducibility data.
A selected list of Performance Tested test kits organized by pathogen and toxin is provided in Table 1. A detailed list of AOAC Official Methods and Performance Tested Kits is available on the AOAC website (www.aoac.org).
Advancing Pathogen Detection Techniques
Some of the most significant microorganisms of concern to today’s food industry are E. coli O157:H7, Salmonella, Listeria monocytogenes and Campylobacter jejuni. The rapid methods developed to address these microorganisms have, in many cases, supplanted traditional methods during the course of the last five years. These methodologies utilize novel culture media and miniaturized kits and technologies such as bioluminescence, impedance/conductance, immunoassay and gene probes to detect and identify bacterial populations and specific organisms.
Novel Media and Kits. Conventional methods for the isolation and characterization of microorganisms, especially pathogens, entail the use of special enrichment and cultivation, selective and differential media and a wide range of biochemical tests. Miniaturized methods, diagnostic kits and sophisticated instruments have been developed and successfully used by the food industry to rapidly identify foodborne pathogens. Many diagnostic kits developed for clinical applications have been successfully adopted for the identification of food isolates. The API Bacterial Identification system and the mini-API (bioMérieux), Micro-ID system (Organon Teknika) and Minitek and Crystal systems (BBL Microbiology Systems) are examples of miniaturized kits currently available for use in the food industry. These systems are convenient, efficient, economical and easy to use. They are also 90-95% accurate when compared to conventional methods, and many of these systems and kits have undergone collaborative validation.
New developments in selective culture media used for pathogen detection in foods also are helping to improve efficiencies in food testing. New chromogens and media ingredients have been used to develop novel media such as BBL-ChromAgar (ED Diagnostics), Rainbow Agar (Biolog) and FloroCult agars (Merck) that allow differential detection of pathogens on the same agar plate. Enzyme activity indicators such as 4-methylumbilliferryl-b-d-glucuronidase (MUG) also are valuable in developing rapid tests for differential detection and enumeration of E. coli/coliforms, Staphylococcus aureus, and other foodborne pathogens and toxins.
Bioluminescence. All living organisms contain adensoine triphosphate (ATP). Bioluminescent systems measure the presence of ATP in a sample using an enzyme system from fireflies, luciferin-luciferinase. This enzyme system, plus a little oxygen and magnesium, will emit light if ATP is present. The amount of light may be related to the concentration of ATP in the sample. Bioluminescent methods in the form of luminometers and ATP swabs/sponges have found several niches in the food processing industry. The most prominent use is as a tool to monitor the efficacy of sanitation. The application also allows efficacy of sanitation to be used as a critical control point (CCP) in HACCP because results can be obtained quickly and easily. If a positive reading is revealed, the QC staff can get the clean-up crew to re-clean the suspect area.
There are a number of companies with ATP test systems for hygiene monitoring on the market that offer very rapid indicator readings in a hand-held format, including the Uni-Lite (BioTrace), a luminometer/reagent system that determines sample contamination in 30 seconds and the Lightning Cleaning Validation System (BioControl), a luminometer/swab technique that contamination in less than 20 seconds, and the Zygiene 100 Rapid Hygiene System (BioTest), which combines a sensitive luminometer with a proprietary reagent kit that detects the ATP bioluminescence signature of somatic and bacterial cells in less than one minute. The 3M Quick Swab test, which consists of a five-inch-long, rayon-tipped swab that, used wet or dry, delivers approximately 1.0 mL of sample directly onto a Petrifilm Plate or 3M Redigel Test. The swab contains letheen neutralizing buffer to facilitate the recovery of bacteria and to neutralize residual sanitizers that can remain on surfaces after cleaning.
Perhaps the most notable development in ATP hygiene monitoring systems is the ability to perform electronic data handling, tracking and trending of measured hygiene levels at selected sites. For example, the systemSURE luminometer (Celsis) can store up to 1,200 reproducible results that can be downloaded onto a computer, analyzed and stored.
There are also swab alternatives available such as the Hycheck hygiene contact paddles (BD Diagnostics), which are two-sided media paddles that are touched onto surfaces or dipped onto liquids, incubated and colony counted to estimate bioburden, and contact plate/dip slide formats such as HYCON Contact Slides and Dip Slides (BioTest) and Oxoid Dip Slides and Contact Plates, which rapidly measure contamination on equipment, surfaces and in liquids on-site. The SpotCheck (Celsis) is used to assess surface cleanliness, but is based on a color-change reaction when ATP is present, rather than by a reaction to light.
A word of caution, though, regarding the use of ATP systems: Attempts to correlate ATP levels with the total microbial counts may or may not be successful because the amount of light generated does not always reflect microbial numbers. Most hygiene monitoring devices measure total ATP, which includes both microbes and the ATP present in the sample or food residue. ATP levels may vary depending on the metabolic activities of the organisms and different groups of mixed microbial populations. Also, sanitizer residues on food contact surfaces or certain food components may interfere with or quench the ATP reaction.
There may be a better, faster or cheaper ATP system just around the corner. But you may not want to wait until a model that is “just right” comes along. Chosen wisely, current ATP bioluminescence methods can help your approach to plant sanitation and improve quality and safety of the products. (Some criteria for selecting an ATP system are listed in the sidebar on p. 34).
Impedance/Conductance. Sophisticated systems such as the Bactometer (bioMérieux), the Malthus (Malthus Diagnostics) and the RABIT (Microbiology International / DW Scientific Ltd.) have been developed for the rapid detection of microorganisms based on electrical impedance, conductance and capacitance of the medium. As microorganisms grow, they break down relatively uncharged nutrients, such as proteins or fats, into smaller, highly charged molecules like amino acids and fatty acids. Such instruments measure changes in the growth medium at regular intervals (for example, six minutes for the Bactometer) and record the time required for sharp and significant changes in the impedance as the detection time (DT) or impedance detection time (IDT). DTs and IDTs are inversely proportional to the initial microbial load in the sample. The IDT and slope of the impedance curves provide valuable information about the initial levels, generation times and growth densities in the sample.
Impedance and conductance systems are computerized, multitasking, multi- user systems that allow electronic data recording and handling, displaying the status of a sample in modes of “Accept,” “Caution,” “Reject,” or “Pass/Fail,” as well as trending or tracking of microbiological data. These instruments can be used to detect poor quality or substandard samples in less than eight hours, offer low cost per test, and offer high capacity in terms of simultaneous tests (up to 512 in the case of the RABIT). Both the Bactometer and the Malthus systems provide screen displays of green, yellow and red colors, indicating “accept”, “reject”, or “pass”, “fail” levels for routine monitoring of microbiological quality of samples being tested. The computerized data handling, storage and retrieval is an attractive feature offered by these systems.
Numerous applications of the impedance and conductance methods in dairy microbiology have been recently developed. The Bactometer has been used for detection of abnormal milk, estimation of bacteria in raw and pasteurized milk and dairy products, detection of antibiotics, measurement of starter culture acitivity and determination of bacteriophage, and monitoring milk quality and predicting the shelf life of pasteurized milk. The Malthus system has been used for detection of post-pasteurization contamination of pasteurized milk, estimation of lactic acid bacteria in fermented milks, detection of psychrotrophic bacteria in raw milk, and detection of microorganisms in powdered milks. Recently, conductance methods for detecting Salmonella and for the enumeration of Enterobacteriaceae in milk have been reported.
Immunoassay. The immunoassay is based on the qualitative or quantitative determination of antigen or antibody present in a sample by specific antigen- antibody reaction, which can be visualized in terms of clumping or agglutination, color or fluorescent development from an enzyme reaction or formation of an immunoband. The development of monoclonal antibodies directed against specific antigenic fractions of targeted microorganisms or toxins has led to the application of a number of immunological techniques to the industry.
The enzyme immunoassay (EIA) and the enzyme-linked immunosorbent assay (ELISA) are the most commonly used techniques for rapid detection of pathogen and toxins. ELISAs have been designed for the detection of specific pathogens, toxins and enterotoxins, antibiotics, drug and pesticide residues, and some are designed to detect specific organisms such as Salmonella Enteritidis or Listeria monocytogenes from food or environmental samples.
There are many types of immunoassay from which to choose, as well. The convenience of the portable, self-contained “dipstick” sampling systems have proved beneficial to plant operations; these devices have gone a long way toward the HACCP ideal of on-line or at-line monitoring. The highly automated and sensitive laboratory bench-top instruments based on immunoassay have increased time to result substantially over the last few years, as well. The VIDAS and mini VIDAs (bioMérieux) is a good example of these automated immunoanalyzers. These instruments are based on enzyme-linked fluorescent assay (ELFA) technology in a solid phase receptable (SPR) and pipetting device. VIDAS, which can be used to detect bacteria and toxins including Salmonella, Listeria, E. coli O157:H7 and Staphylococcal enterotoxin, can handle up to four analytical modules at once for a total testing capacity of 240 tests per hour. The use of immunomagnetic beads coated with antibodies specific for target organisms, once considered novel, are also now widely used.
Some of the most recently approved AOAC RI Performance Tested Methods are good examples of advances made in the rapid immunoassay to address safety and QA/QC challenges in food processing:
Listeria. A recent spate of food product recalls due to Listeria contamination, particularly L. monocytogenes, and the U.S.’s stated objective to reduce the number of Listeria-related illnesses by 50% in the next four years have increased the focus on this statistically rare but lethal pathogen. The EiaFoss Listeria ELISA assay (Foss Electric A/S) was granted Performance Tested Method status by the AOAC RI last year. The kit was validated to detect Listeria spp. (except for Listeria grayii) in a variety of foods, including ground beef, ground pork, ground chicken, prepared salad, peas, luncheon meat, hot dogs, smoked fish, crab sticks, cottage cheese, ice cream, pasteurized milk, paprika, dried eggs, orange juice, tomato juice, fresh pasta, pet food, yogurt, peanut butter and chocolate.
The assay is a qualitative ELISA, which involves three enrichment steps totaling 48 hours and a two-hour ELISA analysis. Antibodies are coated onto magnetic beads that are used in the assay to capture the antigen. This system separates the cells from the food debris. The antigen reacts with the alkaline phosphatase conjugated antibody. The substrate is added resulting in a fluorescent signal. Samples are read on an EiaFoss Type 23000 instrument in which a program card is inserted to control specific ELISA kit operating parameters such as incubation times and cut-off points.
Q-Laboratories Ltd. in the U.K. served as the independent laboratory and performed an extensive study comparing the EiaFoss Listeria ELISA assay to the USDA/FSIS method. The independent laboratory compared the results of 640 samples analyzed using both the EiaFoss Listeria ELISA assay and the USDA/FSIS method for Listeria. The two methods agreed on 627 out of 640 analyses. Method agreement was 98%.
Also of note are two AOAC International Official Methods previously validated for food testing that were also recently granted method applicability extensions to include Listeria environmental surface testing. The VIP for Listeria and the Assurance Listeria EIA (BioControl Systems) underwent a 30-laboratory collaborative study following which results demonstrated both methods as equivalent to the reference culture procedure. The assays, which provide visual indicators within minutes, are the first two rapid tests to have been collaboratively validated and approved for this purpose.
E. coli O157:H7. This a pathogen for which many immunoassay-based systems and kits have been developed and
successfully used by food manufacturers. In the past six months three such immunoassays have been granted AOAC RI Performance Tested Methods status, the TECRA E. coli O157 Visual Immunoassay (TECRA International), the EiaFoss E. coli O157 (Foss Electric A/S), and the Detex System MC-18 for E. coli O157, including H7 (Molecular Circuitry, Inc.). The TECRA screening test enables the user to screen out negative samples within 20 hours and can be used to comply with the USDA/FSIS test directive for the detection of E. coli O157:H7. The test requires a flail 18-hour enrichment step that ensures that very low levels of sub-lethally injured cells are recovered and detected. The method has been validated for composite 375-gram ground beef samples to reduce the amount of testing to be performed.
The EiaFoss E. coli O157 requires two steps: an enrichment phase where the selective growth of the specific analyte is performed using a recommended enrichment broth E. coli, followed by an automated ELISA using analyte-specific antibodies. The results are displayed as a simple positive/negative answer. The system can run up to 27 samples per run and the test time on the analyzer after enrichment is less than two hours, with a total test time, including enrichment, of 24 hours.
The Detex System MC-18 for E. coli O157, including H7 test kit, combines a unique signaling procedure with a modern immunoassay capture technique to distinguish the presence or absence of food pathogens in enriched samples, received verified performance claims of 98°/o sensitivity, 90% specificity, and 96% accuracy. The overall method agreement between the Detex assay and the USDA/FSIS method was 96%. These data indicate that the Detex method is comparable to the USDA/FSIS method for detection of E. coli O157 in raw ground beef and raw ground poultry. The Detex system The MC-18 system is designed to simultaneously assay up to 27 samples at a time, and allows the analyst to test for multiple pathogens at the same time with results in approximately two hours.
The rapidity and sensitivity of immunoassay-based test kits and systems have come a long way in the past few years due to developments in immuno-precipitation devices, lateral flow devices and immunomagnetic separation (IMS) techniques. For example, one can combine the IMS and differential plating media to isolate and detect pathogens such as Listeria monocytogenes, E. coli O157:H7 and Cryptosporidium in 36 to 72 hours. The following are some examples of different formats and devices developed for these “user friendly” immuno-assays: REVEAL for Salmonella (Neogen Corp.), which utilizes a lateral flow test device to detect the pathogen in 21 hours; Alert for Salmonella (Neogen), a microwell ELISA format designed for high-volume rapid screening of samples (up to 94 samples per test kit); VIP Salmonella and VIP Listeria (BioControl); Micro-ID Listeria and Rapid Test for Salmonella and E. coli O157:H7 (REMEL); EHEC-Tek and Listeria-Tek (Organon Teknika); Clearview Listeria Test (Oxoid); and the TECRA Staphylococcal Enterotoxin VIA, which offers direct detection of enterotoxins in four hours.
Polymerase Chain Reaction (PCR). This technique has been established for quite some time, but recently has enjoyed renewed interest by food industry scientists. PCR involves detection of specific gene fragments by in-vitro enzymatic amplification of the target DNA, followed by detection of the amplified DNA molecule by electrophoresis, ELISA or other techniques. The PCR method is a highly specific and sensitive method allowing the detection of low numbers of microorganisms. In the past, the general limitations of the technique have included difficulty in obtaining specific DNA primers and production of nonspecific PCR products. Also, organisms that have been killed during processing were not recognized as dead if their DNA was still present, thus giving false-positive reactions. Automation and improvements in PCR systems have addressed some of these problems and made the technique an extremely attractive option.
One example of such improvements is the AOAC RI Performance Tested Method status granted to the BAX for Screening/Listeria monocytogenes (DuPont Qualicon) last year. This test showed greater than 98% accuracy and sensitivity and greater than 97% specificity as compared to the BAM, USDA/FSIS and AOAC Official Method for this organism. Validated to detect L. monocytogenes in a variety of foods such as chicken, crabmeat and milk, the BAX system targets and makes copies of a DNA fragment unique to L. monocytogenes, and not present in other bacteria. Detection of these DNA fragments provides a highly reliable indicator that L. monocytogenes is present in food samples. The BAX system incorporates all necessary reagents, primers, and polymerase into a single tablet that is prepackaged in a PCR tube. The test is easy to run, requiring only basic microbiology skills, and takes about three hours of processing time after pre-enrichment broth samples are incubated.
Qualicon conducted an internal evaluation on 16 food types analyzed with the BAX assay, and the ISO method for the detection of L. monocytogenes (ISO). No statistically significant difference in performance between the BAX and ISO methods was observed. Inclusivity/ exclusivity testing showed that the BAX system was able to detect 97 of 97 (100%) of L. monocytogenes strains tested. None of the 56 other Listeria or non-Listeria spp. tested gave a reproducible positive BAX result.
The AOAC RI compared the performance of the BAX system to standard culture methods for the detection of L. monocytogenes in chicken (USDA/FSIS method), crabmeat (BAM method), and milk (AOAC Official Method 993.12) at Silliker Laboratories, Inc., Homewood, IL. These independent studies validated BAX system performance claims, and the BAX system was demonstrated to be equivalent to, or better than, each of the reference methods.
Advances in Rapid Toxin Detection
The Codex Alimentarius Committee on Food Additives and Contaminants (CCFAC), which establishes standards, maximum levels allowed for contaminants and food additive levels, identified last year priorities for evaluation by the Joint Expert Committee on Food Additives (JECFA), the scientific advisory committee to CCFAC, for toxicological evaluations. JECFA is the body responsible for conducting risk assessments and setting food additive composition specifications. Of top emerging international contaminants identified, mycotoxins such as ochratoxin A, zearalenone, aflatoxin and fumonisins led the priority list for risk assessment. Also, trichothecenes, such as deoxynivalenol (DON), which can adversely affect wheat and barley, were identified as an emerging area of interest.
Last year, a rapid assay for DON was granted AOAC RI Performance Tested Methods status. The RIDASCREEN FAST DON Assay (R-Biopharm, Inc.) is a competitive ETA based on a deoxynivalenol specific antibody for the rapid quantitation of deoxynivalenol (DON) in cereals. A pre-ground sample can be analyzed in less than 30 minutes. The standard calibration curve of the ELISA covers a range from 0.2 to 6.0 ppm DON. The test was proven to comply with the requirements of the USDA design criteria and test performance specification for quantitative deoxynivalenol test kits. Trilogy Analytical Laboratory, Inc., Washington, MO, performed independent testing under the direction of the AOAC RI. The AOAC RI found that the RIDASCREEN FAST DON Assay and the reference HPLC method for deoxynivalenol gave nearly identical results.
The assay consists of a microtitration plate coated with sheep anti-rabbit IgG antibodies to which a deoxynivalenol standard, or a test sample, arc added, along with deoxynivalenol-enzyme conjugate, and rabbit anti-deoxynivalenol antibody. The mixture is incubated for five minutes. DON and DON-enzyme conjugate compete for the antibody-deoxynivalenol binding site. Unbound enzyme conjugate is removed by a washing step. Chromogen/substrate is added to the wells and incubated for three minutes. Bound enzyme converts the chromogen into a blue product. The addition of the stop reagent inhibits the enzymatic process and causes the colored product to turn yellow. Measurement is performed photometrically at 450 nm. The resulting absorbance values are inversely proportional to the concentration of deoxynivalenol of the sample.
Other established rapid methods for natural toxins include the Veratox product line (Neogen Corp.) for T-2 Toxin, Aflatoxin, Ochratoxin, Fumonisins B1, B2 and B3, and Zearalenone, which take about 20 minutes and utilize both competitive and non-competitive ELISA formats; and the RIDASCREEN FAST T-2 Toxin Assay (R-Biopharm, GmbH) for the rapid detection of T-2 toxins in corn, wheat and mixed feeds, which uses a standard competitive immunoassay technique using monoclonal antibodies specific to the T-2 toxin and takes 15 to 20 minutes.
The Veratox for DON (Neogen) was was also approved by the USDA’s Grain Inspection, Packers and Stockyards Administration (GIPSA) for official testing in the national grain inspection system. The test requires two five-minute incubations and offers high sensitivity and reproducibility at lower detection levels. Also, recently introduced by Rhone Diagnostics is the Fuminoplate, a plate for the quantitative determination of fumonisins B1, B2 and B3 in maize and maize-based products that can detect the toxins at contamination levels between 0.3 ppm and 10 ppm in approximately 40 minutes.
Many of the testing methodology and technological advances described here fall under the descriptive term “rapid.” Do they have a place in your operations? That is a decision that individual companies will have to make carefully as the array of choices available in the form of improved systems and techniques shows no sign of abating. Clearly, the ever-increasing availability of “better, faster, cheaper” pathogen and toxin technology is one way for a small food company to develop food safety and QA/QC programs without spending an arm and a leg, and for larger operations to reduce bottom-line costs while assuring the safety and wholesomeness of their product lines.
The molecular methods for detection and characterization of pathogens and toxins offer advantages in terms of specificity and selectivity not possible by conventional methods, and as such, these are very useful in epidemiological investigations of foodborne illness. However, they are not yet common in routine microbiological analysis of foods. They do, however, represent the latest trend in analytical microbiology and offer great promise for food industry applications.
Purnendu C. Vasavada, Ph.D., is professor of food science at University of Wisconsin-River Falls. As an extension specialist in the area of food safety and microbiology, he has worked with rapid methods and been an instructor in programs the world over. Vasavada directs the three-day course, “Current Concepts in Foodborne Pathogen and Rapid Methods and Automation in Food Microbiology,” designed to provide food processors with up-to-date information on rapid methods, testing protocols and technologies most suitable for their operations.
This paper is a contribution from the College of Agriculture, Food and Environmental Sciences and the Cooperative Extension Service of the University of Wisconsin. The consultations, personal communications, cooperation and support of colleagues and companies in the U.S. and abroad are gratefully acknowledged.
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Editor’s Note: The mention of specific methods or instruments in this article does not constitute an endorsement of these products and technologies by the author or Food Safety Magazine.