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Rapid Micro Rolls On
The technology continues to revolutionize food safety
by Lori Valigra
Editor’s Note: This article on the history and impact of advances in rapid food microbiology is the second in a new series for Food Quality. In “FoodTech: Tools That Changed the Industry,” we look at various technologies and tools, such as rapid microbiological testing, that have played a key role in and had an indelible impact on the food industry.
In the science fiction series “Star Trek,” science officer Spock would merely point his tricorder at the surface of a new planet to instantly determine its composition and potential for human inhabitation. The medical version of the device could swiftly diagnose an illness in a Starfleet officer. While this holy grail of real-time testing has yet to be realized in today’s food and pharmaceutical industries, new and improved tools are moving toward that goal.
“We are on the leading edge of PCR (polymerase chain reaction) detection by now,” said Morgan Wallace, PhD, senior research microbiologist at DuPont Qualicon, Wilmington, Del. “It’s not yet a tricorder” in terms of immediate response, he added.
DuPont and other companies are racing to improve test responsiveness, both in turnaround speed and sensitivity. Part of the impetus is the heightened public awareness of food safety that followed notorious pathogen contaminations, some involving peanut butter and ground beef, which sickened and killed many people. Companies are not only sensitive about their corporate reputation but are also mindful of the huge amount of money required for recalls or for products stored in warehouses awaiting results from slow-growing cultures.
“The public health case is driving food microbiology now,” said Donald Zink, PhD, senior science advisor to the U.S. Food and Drug Administration’s Center for Food Safety and Applied Nutrition. Zink said rapid methods have progressed more slowly than expected, as sample-size preparations and amplification remain impediments to speedy results.
“It is critical to get enough of a pathogen to detect and also to determine whether the organisms are dead or alive,” he said. “We have yet to get away from some type of amplification.” He estimates cultured methods, either broth or agar, still account for more than 50% of testing, primarily because of the low cost.
Purnendu Vasavada, PhD, food safety and microbiology specialist at the University of Wisconsin in River Falls and a member of the Food Quality editorial advisory board, is more optimistic. “We’re pretty close to real-time testing.” With biosensors and other new technologies, it could happen in two to five years, he said.
Philippe Gadal, PhD, CEO of AES Chemunex, a company that offers Scan RDI, BactiFlow ALS, and D-Count products for food testing, said that even though Petri dishes remain the broad-based, basic tool for general testing, future biochips will test for multiple pathogens.
Mike Doyle, PhD, director of the University of Georgia’s Center for Food Safety, expects new technology to revolutionize the field. He pointed to ribosomal RNA, which can produce more copies than DNA. “There are more targets for molecular testing, and you don’t have to go through an extensive enrichment process,” Dr. Doyle said.
Microbiology testing volume has grown 17.8% since 2005, from 629.9 million to 738.3 million tests, said Tom Weschler, MBA, founder of Strategic Consulting Inc., in Woodstock, Vt. The total market for food microbiological testing surpassed $2 billion in 2008. “Food companies want to sell safe products, but they also want to protect their brand name,” he said.
Attention to pathogens is paying off. The overall incidence of E.coli O157:H7 has declined 42% since 1996, according to a report by the Centers for Disease Control and Prevention and others. The data also showed substantial declines in Campylobacter, Salmonella, and Yersinia that were attributed to enhanced surveillance and outbreak investigations.
Faster and Easier
Because the enrichment step is time-consuming, companies are taking steps to reduce that time. DuPont, for example, reduced amplification and detection of E. coli to less than one hour on its PCR-based BAX product, a significant reduction compared to the 3.5 hours needed with earlier machines. After enriching E. coli for about eight hours, the company saved time by taking a measurement at the end of each PCR cycle instead of at the end of every batch. The system is closed tube instead of gel-based to simplify the process, Dr. Wallace said, so that people with less training can perform the assay, and the results are better. The company has 13 PCR assays that target nine microbes. BAX can detect multiple species at the same time in one PCR reaction.
Increasing sample throughput is important during a contamination incident. One large poultry processor that routinely tested carcass rinses for Salmonella using rapid method assays noted an upward trend in positive results that it could not attribute to seasonality, according to DuPont. The plant examined its procedures to see if there was a contamination problem or whether its testing methods were faulty. The processor initially used cultures to monitor its interventions but could not wait five to seven days to get results.
With BAX’s DNA-based detection, the company could get results the next day and increased its weekly sample throughput by 25%. Within two months, the rate of positive Salmonella results fell from 18% to 5%, and soon thereafter to zero. DuPont said the fast turnaround of the results helped the company more quickly confirm the effectiveness of each change made in its process.
Simplifying testing procedures to make them less prone to human error and to ensure quicker results are strategies also adopted by Applied Biosystems Inc., part of Life Technologies Corp. “We are looking for ways to process the sample after enrichment to get faster time to results,” said Brooke Schwartz, MBA, senior director of Food and Environmental Testing at Life Technologies.
Although she did not divulge information about upcoming products, she said “some exciting technology is in the pipeline. We are working on reducing time to results and increasing ease of use in our entire workflow.”
The company also uses a PCR-based technique with its MicroSEQ detection kits that run on the Applied Biosystems 7500 Pathogen Detection System. The company believes PCR is more specific than immunoassays, which are also used to detect pathogens. “PCR is more specific and has a faster time to result (than immunoassay),” said Manohar Furtado, PhD, vice president of research and development at Life Technologies.
Key to the company’s focus is the use of lyophilized beads during PCR. The reagents used in its MicroSEQ Listeria monocytogenes detection kit, for example, are lyophilized into pre-formatted assay beads that hold the active enzyme, the target-specific primer and probe set, and other reagents for “fast” PCR. Listeria monocytogenes bacteria grow slowly and are difficult to distinguish from other species of Listeria on agar. Dr. Furtado said the beads eliminate some of the potential human error or contamination, and the automated sample prep leads to faster results.
The assay process is closed, meaning that the tubes are closed until detection is complete, reducing contamination potential. Listeria testing with traditional culture-based methods can take up to five days, Dr. Furtado said. Older PCR methods that took 2.5 hours have been reduced to 45 minutes by automating workflow and using the beads. The company is also looking at multiple types of growth media to reduce the time to results.
Tracing the Origin of Contamination
Recent food contamination outbreaks may suggest that the food supply is not safe, but that is not so, said J. Stan Bailey, PhD, director of scientific affairs at bioMérieux in Athens, Ga. “We are better now at identifying where the pathogen came from, and fewer people get sick,” he said. For example, the Peter Pan peanut butter outbreak was narrowed down to a particular brand at a particular plant. The Salmonella outbreak at Peanut Corporation of America in the summer of 2008 took longer, he said, because the outbreak wasn’t in a specific brand. Instead, it was a minor ingredient that went into hundreds of other products, including trail mix and other processed foods. The same was true of the melamine found in cat food. Now, government surveillance networks and tests more quickly track down the culprit. BioMérieux has an automated pathogen detection system called Vidas and an automated enumeration system for quality indicators called Tempo.
Life Technologies recently focused on strain typing, which it believes will be useful in tracing the origin of contaminants. Currently, pulse-field gel electrophoresis is used to type strains when there is an outbreak. Life Technologies is studying the roughly 20 existing sequenced strains of Salmonella, while using its own solid sequencing technology to sequence another 40 strains in house.
“The idea is to use all this information to come up with a single nucleotide polymorphism (SNP)-based or sequence-based genotyping format that would be faster and more accurate than current pulse-field gel electrophoresis,” Dr. Furtado said. “The general hypothesis is to collect enough data to come up with certain gene sets or SNP sets that will be able to specify the strain type,” he said, adding that it may be another six to eight months before the company comes up with defined SNP signature patterns. “Then we’ll need to test it with a large number of strains to make sure it works,” he said.
The Association of Official Analytical Chemists (AOAC) Research Institute validated the MicroSEQ Listeria monocytogenes detection kit, and Life Technologies plans to submit its other key products for validation, which the company said will help assure food manufacturers that they are using effective methods. “We’re putting the full portfolio of high-volume pathogen tests out this year and seeking validation through the AOAC,” said Schwartz.
Congress, which is also working to improve food safety, is now considering the Food Safety Enhancement Act. “We expect it will set standards for more testing and production and food safety in plants that didn’t have it before,” Schwartz said. “It will be good for food safety.” ■