Getting Really Rapid Test Results: Advances in Pathogen and Toxin Detection for the Food Industry
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.[1] 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).