From immunoassays to more advanced DNA detection, today’s microbial tests offer faster results and increased accuracy.
Years ago, meat and poultry processors testing for pathogens such as E. coli, salmonella, and listeria were faced with lengthy wait times for results and limited testing options. These obstacles, some argued, would compromise the efficiency of pathogen testing and, thus, food safety overall. Today, the industry has come full circle, with more processors taking advantage of quicker microbial testing methods and more advanced technology.
During the last several years, there has been a movement by processors to use rapid pathogen testing methods to decrease result time. Yet, real-time results remain out of reach, due to the necessary incubation periods required by these tests.
At present, there are two common types of rapid pathogen tests. Immunoassays are used to identify salmonella, listeria and E. coli. These tests utilize an antibody specific for a target pathogen to "capture" the pathogen. A second solution containing an indicator will change color to indicate the presence of the pathogen. The antibody specific for a target antigen is sprayed and immobilized in a line on the surface of a membrane comprising a "test line" (in lateral flow devices) or is coated onto the inside of a well on a micro titer plate (for ELISA assays). A second antibody reagent, also recognizing the target pathogen and labeled with a color indicator, causes the color change so that the result can be read.
In the past, immunoassays had the propensity to give false positive results, so pathogen confirmation needed to be confirmed with standard culture methods. However, new lateral-flow technology significantly reduces false positive rates and shortens testing time.
The newest type of rapid testing, called DNA-based assays, include a variety of recent DNA technologies, including the popular Polymerase Chain Reaction (PCR). The advantage of this test is that only a few organisms are needed for detection, and this can occur in as little as three hours. There also are fewer false positives with these tests, sources say.
Despite utilizing the newest technologies and because product samples have to incubate in broth, the enrichment period hampers the speed of these tests.
Defining rapid tests
This incubation or enrichment period is required by all pathogen tests and has been the main roadblock in establishing a real-time method of detection, experts say. “To my knowledge, there currently are no real-time methods for pathogen detection or quantitative analysis of microbial populations in food products,” says Geoff Bright, microbiology product manager at BioControl Systems, a Bellevue, WA-based provider of food-safety tests.
He says that it’s not just matter of getting fast results with rapid tests. “They have to be easy to use and interpret and be accurate,” he adds.
Because the meaning of ‘rapid’ in this case is somewhat ubiquitous, many describe tests in this category as those that decrease result time compared to traditional methods.
“As of now, confirmatory rapid tests normally require 24 hours for reliable results,” says Julie Buddemeyer, marketing and sales associate for Frederick, MD-based Microbiology International, a distributor of automated tools for microbiologists,. “When comparing tests, it is important to consider the steps required before a rapid test can be employed. For example, some rapid tests require a purity plate for a MacFarland calibration, while others do not. This extra step adds an additional twenty-four hours before the rapid test can be used. There are some real-time tests currently available, such as latex agglutination tests, like the ones provided by Microgen BioProducts for Campylobacter, listeria, salmonella, and E. coli. Although they will confirm, for example, a listeria species in several minutes, these tests cannot identify which type of listeria species is present.”
Bill Hoerner, senior director of marketing at Neogen, a Lansing, MI-based provider of food-safety test kits, says the term rapid test has expanded in the last five years. “Rapid testing refers to a broad range of tests that can be done in a matter of seconds or in forty-eight hours, depending on the pathogen you’re looking for,” he says. “But real-time is not specific for a bug. These tests will tell you if surfaces are clean or dirty, but they won’t identify pathogens.”
He adds that the issue is the time it takes to grow pathogens to detectable levels for testing. “If you look at our E. coli test, it needs to enrich for eight hours before the fifteen-minute reaction. There are different enrichment times for each pathogen, and no one has been able to crack them.”
Many agree, however, that the industry is moving faster toward more rapid detection. “We’re still talking hours for running these tests from start to finish,” says Ravi Ramadhar, global marketing manager at Wilmington, DE-based Dupont Qualicon, a provider of genetic-based diagnostic products. “However, we’ve done a lot in decreasing the time it takes to run these tests.”
The total testing time for a rapid test will be determined by the pathogen needing to be identified. Most E. coli O157 methods typically have an eight-hour enrichment period followed by the detection step, whereas salmonella enrichments can take as long as 48 hours utilizing a two-three step enrichment process, says Orla Cloak, product manager at Strategic Diagnostics (SDI) in Newark, DE, a developer and manufacturer of immunoassay-based test kits.
“For E. coli O157, about ninety-five percent of all the tests utilized are rapid, and this is largely due to the “hold and release” policy associated with this organism’s presence in meat. For salmonella, it’s more a fifty-fifty split between rapid and cultural methods,” she says.
Unlike with meat and poultry, rapid testing for surface sanitation on processing equipment surfaces is measured in real-time. “In less than thirty seconds, we obtain results if a surface is clean or dirty. This could be either microbial contamination or product residue,” says Sandy Bull, senior marketing manager for food and beverage at Ecolab, a St. Paul, MN-based developer of cleaning, sanitizing, pest elimination and water management products and services.
“A positive test may not trigger more microbial testing, but instead a recleaning that would improve the overall sanitation in a plant,” says Robert Salter, vice president of regulatory and industrial affairs with Lawrence, MA-based Charm Sciences, a manufacturer of rapid food safety tests. “In thirty seconds, you have a positive to react to.”
In terms of detecting contaminates aside from pathogens, such as pesticides, herbicides, and hormones, Waters Corp. in Milford, MA, offers rapid tests that provide chemical methods of analysis. “We use liquid chromatography coupled with mass spectrometry. The actual test can take less than twenty minutes, but measurements can take several hours to perform,” says Jim Willis, director of market development, chemical analysis.
With the rapid testing market getting smaller due to consolidation, companies in this industry are looking at innovative ways to detect pathogens that will also fit in with a routine testing program. “There are many new tests coming out with two main goals; companies are constantly looking at ways to reduce both the enrichment and detection time of the test,” says SDI’s Cloak.
Until recently, the major obstacle in offering improved testing methods was gaining approval from regulating/validating agencies, such as the Food and Drug Administration, the Agriculture Department, and the AOAC (Association of Official Analytical Chemists, a 120-year-old not-for-profit scientific association committed to worldwide confidence in analytical results.)
However, Buddemeyer says that many of these rapid tests are now being considered standard methods and/or receiving validation approval. “For example, BBL CHROMagar Listeria is listed in the FDA/ BAM manual as a recommended method and has received AOAC-RI approval. BBL CHROMagar Salmonella and the Microgen Listeria ID kit have also been AOAC-RI approved,” she says.
One of the most talked about new developments in rapid testing is BioControl’s Assurance GDS genetic detection system. “It is the fastest test for pathogen detection, allowing users to get results for E. coli O157:H7 in eight hours,” says Bright. “It also is the first PCR-based test for E. coli O157:H7 to receive AOAC Official Methods approval, which validates the performance of rapid testing methods.
In addition to faster results, Assurance GDS also offers greater accuracy in the form of multiple layers of specificity. These include Immunomagnetic separation (IMS), highly specific DNA primers, and a patented DNA probe system.
Ramadhar at Dupont Qualicon says the industry is discovering more about E. coli’s different pathogenic strains. “There are more strains that are pathogenic and found within the meat industry, but some that are not [in this industry],” he explains.
The company recently signed an agreement with Applied Biosystems Group by which the two businesses will jointly develop and market new BAX® system applications for the food industry. This pathogen detection system uses PCR to test for multiple bacteria simultaneously. Results are achieved within 24 hours, with automated detection minimizing human error and false positives.
Another new rapid test to emerge for the meat and poultry industry is a 24-well, test strip specifically for the identification and confirmation of Bacillus, says Buddemeyer at Microbiology International. “Bacillus ID from Microgen can identify all twenty-three Bacilli and closely related genera that are commonly implicated as causes of food poisoning and food spoilage. This is a great improvement upon a previous method, which combined two separate tests to create a seventy-well test strip that is very cost prohibitive,” she says.
For helping to eliminate pathogens from plant surfaces and equipment, the synergy of ATP and microbe testing is a proven technology that provides added assurance that surfaces are clean. ATP is a chemical found in all biologic systems. When it is detected on a swab, ATP activates a firefly enzyme that puts out light. “In the past, with pure microbial testing, surfaces were swabbed and the wait for results was forty-eight hours. Product would be held for results or, if it made its way to the distribution center, meat would need to be recalled. With ATP technology, processors can get real-time results and ship right away,” says Bull.
Charm and Ecolab have developed electronic equipment that simplifies documentation, providing time-date stamps on food safety tests. “We integrated these into computer systems so documentation can be stored electronically and used for HACCP program compliance,” says Bull.
On-site vs. off-site testing
Cost is a factor when plants are considering whether to conduct rapid testing in-house or use an off-site lab’s services. Hoerner at Neogen says that five years ago, it appeared that more meat processors were conducting tests in-house. “Five years ago, we broke off our commercial lab division, putting limited resources in it because we saw an increase in in-house labs. But now this division has tripled in sales, [so we’re seeing more outside testing],” he says, adding that it depends on the individual company, its size, and how much liability they want to take on. “The price per test is less expensive when done in-house, but the overhead for housing a lab makes it more cost prohibitive to test in-house.”
Gina Bellinger, president of Food Safety Net Services Ltd., a San Antonio, TX-based provider of laboratory, consulting, auditing, and educational services to the food industry, says she is seeing more outsourcing of rapid testing for pathogens. “Companies are finding that it is not economical to keep testing in-house. Also, more processors want to start using labs that have ISO 17025 accreditation (general requirements for the competence of calibration and testing laboratories).
Yet, some say that the speed of rapid testing can be compromised when it’s done off-site. “I’ve seen more rapid tests being conducted in-house for added control and also the time issue involved in shipping samples to a lab,” says Cloak at SDI. “In the same vein, where testing is conducted depends on the pathogen in question, and the individual’s testing capabilities and resources. A larger amount of E. coli testing is done on site, but O157 samples, confirmation of presumptive positives and testing of other pathogens such as salmonella and listeria can be completed at commercial laboratories that are very experienced in the multitude of tests available in the marketplace.”
Real-time results will eventually be achieved with rapid tests, predicts Neogen’s Hoerner. “The way science is developing, someone will figure it out,” he says. “It’s just a matter of time.” NP
Lisa White is a freelance writer based in the Chicago area.