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From: Food Quality & Safety magazine, April/May 2011

Here, Now, and Soon to Be: A Suite of Food Lab Innovations

Industry leaders working feverishly to hone new methods of identifying pathogens, contaminants, species, and more

by Neil Canavan

Here, Now, and Soon to Be: A Suite of Food Lab Innovations

It’s often said that the future is now, which may explain why so many people are unprepared for it.

The phrase is meant to reflect the immediacy of modern life, but it can be taken to explain why the attributes of the present seem so often unmanageable. Take the globalization of the food supply: It’s here. It’s off and running. And now we have to catch up with it.

For starters, as pointed out by Paul Zavitsanos, product marketing manager for Agilent Technologies in Santa Clara, Calif., food-related microbiology has a lot of catching up to do.

“When I entered this field 35 years ago,” Zavitsanos said, “a large portion of work in chemical labs involved glassware and boiling solutions and condensing vapor … making colors. But now, you dissolve the stuff and shoot it into an instrument.” This is not the case in microbiology. “Today we still test largely the way Pasteur used to”: isolate, enrich, and, in some low-tech way, identify—often using a confirmatory metabolic test. Which is not to say the right tools aren’t out there; in the immediate future, Zavitsanos wants to bring the already available and near-term novel instrumentations to microbiology.

To that end, Agilent recently entered into a collaboration with the University of California at Davis to develop a new process of pathogen identification that, from field to finding, takes a matter of hours rather than days. The technology, called MassCode PCR, will use mass-tagged PCR primers to amplify target genes-of-interest, liquid chromatography (LC) to resolve the target, and mass spectrometry (MS) to identify the bug in question, with the current project to focus on Salmonella spp. The technology will combine the sensitivity of the LC/MS with the reliability of the dual barcode approach to polymerase chain reaction (PCR) that eliminates false positives.

Zavitsanos is certain this technology will shorten the duration of, if not prevent, outbreaks of certain foodborne diseases. “With traditional technologies, it takes two weeks to get the first round identification of a bug, and then verifications must be performed.” With MassCode quickly nabbing the suspect, exposure time for the general population might be cut in half.

“It’s tough to make predictions, especially about the future.”

—Y. Berra

That’s Rich

As with all things new, this bug-finding strategy may have its own bugs. Zavitsanos is uncertain, for example, of the effect MassCode’s potential sensitivity will have on the enrichment phase—the step prior to loading a PCR. “It might be sensitive enough and selective enough where certain enrichment technologies are no longer required. We just don’t know.”

Should enrichment remain an issue, as is likely, PhD candidate Brian Poe, of the University of Virginia at Charlottesville, may have a suggestion. Poe just presented his data regarding an acoustic cell trapping method that rapidly concentrates a pathogenic sampling at Pittcon, the laboratory science conference.

“We used ultrasound to create low-pressure zones within a microfluidic device,” explained Poe, “and in those zones any particles, or cells, are localized into that site.” The idea is to improve the time and circumstances involved in current sample preparation in the monitoring of wash water used in food production. Currently, the time is long, and the place is a bulky centrifuge.

In his investigation, Poe tethered the acoustic trap to another innovation, an ultra-rapid, infrared-mediated microchip PCR now being commercialized by Poe’s adviser, James Landers, PhD, chief scientific officer of the biotech company ZyGEM. “We integrated the two steps: Flow the sample through the cell-concentrating trap, then perform heat lysis and sequence amplification—all within the same device.” The process is rapid and the device portable.

Pittcon results suggest the need for more specificity, however. Poe will address this limitation by using higher frequencies that will theoretically trap with a greater discriminating force.

Bug Out

The future capabilities Poe describes are primer-dependent, the bug having already been thoroughly characterized. Some strains of E. coli would frustrate current detection methods, however. A cooperative research and development agreement between the U.S. Department of Agriculture (USDA) Research Service and DuPont Qualicon has recently been inked to address new toxic E. coli players in the food safety game—the so-called non-O157 Shiga toxin-producing E. coli. “There are six of these that are significant in terms of foodborne illness, ” said Megan DeStefano, global marketing manager for DuPont. “One of the issues is that they don’t have differentiating phenotypic characteristics.”

Although the six bugs in question are not so much new as, say, novel mutations, their increased detection in the food supply has recently intensified concern. DuPont’s mission is to create and/or enhance methods for both phenotypic and genetic detection. “This holistic look is really important because just doing the detection piece is not accurate enough to keep our food safe.”

“The biggest hurdle is you’re looking for six different (bugs), all of which act differently. To be able to find an enrichment and detection method that can equally identify all of them even in the presence of each other and other background flora is challenging.”

— Megan DeStefano, global marketing manager for DuPont

The three areas of research interest are enrichment, detection, and culture confirmation. “Culture confirmation has been a challenge for government and industry,” said DeStefano. “It really isn’t well-developed.” And the deficit is critical, because, currently, a PCR result is not definitive for the government agencies.

“The biggest hurdle is you’re looking for six different (bugs), all of which act differently,” DeStefano noted. “To be able to find an enrichment and detection method that can equally identify all of them, even in the presence of each other and other background flora, is challenging.” DuPont does have a leg up on the mission; the company’s BAX PCR system is the standard instrument used by the USDA Food Safety and Inspections Service for E. coli 157:H7 detection. So, for the moment, it’s not the machine, it’s the method.

Something Fishy

Species identification is also the business of Applied Food Technologies (AFT) of Alachua, Fla., a company that wants to make sure that the fish you ordered is what made it to your plate. “I’ve worked in seafood industry for 20-plus years” said AFT CEO LeeAnn Applewhite, “and I was very interested in converting traditional microbial tests used in seafood industry to molecular diagnostics. We developed the first molecular diagnostics to identify the species of fish.” Also presenting at Pittcon 2011, Applewhite highlighted the utility of the company’s AUTHENTI-KIT DNA technology platform.

“The reason I was asked to speak at Pittcon is because over the last eight years there has been a lot of media regarding the mislabeling of seafood products in the marketplace.” Some occurs by accident—a lot of fish do look alike—but increasingly, it’s a matter of economic fraud.

The Agilent 6460 Triple Quadrupole LC/MS. Agilent recently entered into a collaboration with the University of California at Davis to develop a new process of pathogen identification that, from field to finding, takes hours rather than days.
The Agilent 6460 Triple Quadrupole LC/MS. Agilent recently entered into a collaboration with the University of California at Davis to develop a new process of pathogen identification that, from field to finding, takes hours rather than days.

Applewhite long ago saw the writing on the global seafood wall and started to collect whole fish, imported from all over the world, and have them taxonomically validated. “We worked with the Smithsonian and others to create reference specimens from which we derived a DNA library of validated gene targets. No one else has this.”

This is both good and bad. Other labs rely on public databases, like GenBank; however, the Food and Drug Administration (FDA) does not recognize these sources as valid for compliance testing. Yet, for the moment, species testing with AFT profiles must be done in house. Though Applewhite has been approached by multiple global commercial entities, she has not made this unique data set, or the derived primers, available for fear of reverse engineering. “We were selling it mainly to state agencies for use in their labs, but there’s no way to protect our gene target,” explained Applewhite. Other avenues are being explored, but in the eyes of AFT attorneys, the terms of future licensing opportunities are not yet focused.

Meanwhile, at Eurofins, an international laboratory services company, the seafood unknowns of concern are of a chemical nature, as evidenced by their Pittcon presentation, High Sensitivity Multi-drug Residue Analysis in Seafood by LC-MS/MS: Developing Robust Methods that Meet Industry Regulatory Needs. “We first started developing this line of testing in 2002 when chloramphenicol was discovered in some seafood from Asia,” explained the president and director of Eurofins’ central analytical lab, John Reuther. “As time went on, other agents were found and we continued to develop capability to test for those drugs.”

The challenge came in the form of an ever-lengthening list of drugs to look for.

Right now, the industry typically uses a single-class drug-residue method for identification. “The problem with that approach is that, oftentimes, there are so many different possibilities of drug residues present in a particular species or fish that by the time you get finished running every class of drug, the cost of the job (in time and money) is astronomical,” Reuther said.

Once again, the solution is in the method; available hardware can already do multi-drug analysis. “There’s enough power in currently available instruments,” Reuther said. “The reason why this is an issue is that the methods that have been handed down to date have been centered on class-specific drugs, and no one has taken the approach of trying to make it more efficient.” The novel procedures for multi-class LC-MS/MS developed by Eurofins and reported at Pittcon will, once validated, be freely distributed. “These methods will not be exclusive to a single system,” Reuther assured. “I’m sure they can be adapted.”

The Enemy You Don’t Know

Methods are also on the mind of Vincent Paez, director of food safety business development at Thermo Fisher. “In the past, we were considered instrument vendors, never a lab, and we’ve been trying hard in last three to four years to change that image and to become technical and scientific partners in the industry.”

Thermo Fisher is making strides toward detecting traces of contaminants down to parts per billion with its Orbitrap LC-MS technology.
Thermo Fisher is making strides toward detecting traces of contaminants down to parts per billion with its Orbitrap LC-MS technology.

Witness the recent activity at Thermo Fisher’s Food Safety Response Center in Dreieich, Germany, where two new analytical screening methods to detect petroleum contamination in oysters and fish were developed in response to the 2010 oil spill in the Gulf of Mexico.

Other methods on the drawing board will address the contaminants you may not have thought to consider. “Today, when it comes to analytical instruments, you really need to tell it what to look for—that’s called targeted analysis,” said Paez. “What we will see in the future is non-targeted screening.” As currently imagined, already efficient detection instruments (MS variants) will see all there is to see, identify known species, then call out the unknown but prominent peaks. Beyond this, a technician will track the culprit using data mining.

The big obstacle right now is sensitivity. Is the system sensitive enough to detect traces, even down to parts per billion, that might be toxic? “Producers will say the technology’s not there, but most likely someone will soon tackle it,” Paez assured. “And that will blow a hole through the ceiling of analysis.”

For now, Thermo Fisher has made significant contributions with its Exactive Orbitrap LC-MS technology, the main drawback of which is portability. “The technology of the future is in portables,” Paez noted. “The industry is clamoring for the ability to take a device and go right out to the farm or dock or truck and do a quick analysis.” Though such an innovation would have to pass regulatory approval, Paez sees the machines and protocols in place within the next five years.

Neil Canavan, a freelance science/medical writer based in Brooklyn, N.Y., holds a master’s degree in molecular biology. In addition to press coverage of medical meetings, Canavan has been writing about pharmaceutical science for more than 10 years.

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