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From: Food Quality & Safety magazine, August/September 2009

Pushing the Rapid Micro Envelope

Seeking true ‘real-time’ methods of pathogen detection

by Gina Shaw

Back in the 1970s, cop shows like “S.W.A.T.” would sometimes refer to the bulletproof vests that police officers wore. These days, you don’t hear much about bulletproof vests—now they’re called body armor or bullet-resistant vests because none of them are completely bulletproof. Tom Weschler feels the same way about the term “rapid microbiology” when it comes to pathogen detection. “Most of the current systems really aren’t that rapid,” said Weschler, president and founder of Strategic Consulting Inc., a leading diagnostic test consultant. “They may be more rapid than systems we’ve had in the past, but they’re certainly nowhere near real time.”

That is a problem for the food industry, which has gotten a series of black eyes from recent nationwide and multistate outbreaks involving Salmonella in peanut butter, spinach, and tomatoes, Escherichia coli O157:H7 in beef and spinach, and Listeria in deli meats. With new food safety legislation likely to pass Congress soon, inspections and other food safety activities will increase, and the urgent need for truly real-time methods for foodborne pathogen detection will also increase. “The longer it takes to get test results in, the more product you have to put on hold and store until you get the results,” said Michael Doyle, PhD, Regents Professor of food microbiology and director of the Center for Food Safety at the University of Georgia.

Two Current Pathogen Detection Tools

Currently, the more rapid microbiology tools for pathogen detection generally involve one of two options: antibody-based immunoassay or genetic amplification using real-time polymerase chain reaction (PCR). With both methods, time to result has shrunk exponentially compared with previous approaches, which often took from three days to a week to yield results.

For example, DuPont Qualicon’s BAX System Q7 now offers a reverse-transcriptase PCR assay that can detect Listeria in eight hours—the first commercial application of RNA technology to foodborne pathogen detection. This technique does not require the usual full enrichment step; instead, Listeria cells are resuscitated by heating in the collection buffer solution for four hours.

The company is now developing a BAX assay for E. coli O157:H7 that will drive time to result down to nine hours by shortening cycling time. “We’ve eliminated the post-cycling detection step by detecting signal at the end of each cycle and re-optimized the cycling conditions to shorten each cycle,” said George Tice, DuPont Qualicon’s director of research and development.

Another goal is greater integration, Tice said. “We’d like to be able to integrate at least sample prep and amplification detection, so that a customer could put on a sample, walk away, and come back and get the result. Of course, we have to be able to do that at a price that’s acceptable to the market.”

BioMérieux recently announced a new immunoassay in its VIDAS pathogen detection line, VIDAS UP (ultimate performance) for the detection of E. coli O157:H7. The company, which has a big presence in Europe, is extending its market in the U.S. Instead of using an antigen-antibody reaction to capture the bacteria, it uses a phage protein.

“We’re not actually working with bacteriophage per se, but the tip of the filaments that come off the phage that attach to the bacteria,” said J. Stan Bailey, PhD, director of scientific affairs for industrial diagnostics at bioMérieux. “From a German company that has been working on this technology for more than 10 years, we’ve licensed the protein materials on the tip of those fibers, which we use as a capture on the immunoassay. This allows us to have much better specificity, no cross-reactivity, and a very tight binding.”

For meat, produce, and irrigation water products, VIDAS UP has a seven-hour time to result with smaller sample sizes (around 25 grams), eight hours for larger sample sizes, and 10 hours for large pool samples.

“We’re developing the same general approach for next-generation VIDAS assays for Salmonella and Listeria, but they’re a year or so away,” Dr. Bailey said. “Our time to result goal for those is either same-day, eight- to 10-hour results as with E. coli, or at worst, an assay that you can start any time today, then walk in the door in the morning, run it, and in an hour have the results.”

In 2007, AES Chemunex acquired Warnex’s Genevision line of food-based diagnostic tools and now markets them under the name Adiafood. After an eight- to 18-hour enrichment phase, depending on the organism, the real-time PCR-based assay (simplex or duplex) takes 30 minutes for extraction and two hours for DNA amplification and analysis. Depending on the sample number, amplification can be done on a 96-well plate or strips.

“It’s a very simple system,” said AES Chemunex CEO Philippe Gadal, PhD. “You don’t really need a PCR specialist to run it. The thermal cycler does all the work for you, so it can be done by any technician in the lab.”

Enrichment Process Remains an Issue

But all of these tools, relatively rapid and advanced though they are, share one crucial rate-limiting step: the enrichment process. You can tweak your detection methods all you want, but you cannot change an organism’s doubling time. “From the fastest, E. coli at six to eight hours, to the slowest, Listeria at around 36 hours, getting the counts of the target organism up to what you need for reliable detection with these methods is what takes the time,” Weschler said.

“The major holdup right now is the need for enrichment procedures to get counts up to at least 103,” Dr. Doyle agreed. “None of them seem to do much better than that. Some can get down to 102 in an ideal system, but as we know, foods are not ideal. Depending on the organism and the type of food it is in, eight hours is probably the fastest time to response right now in terms of reliable tests using enrichments, and some have to go much longer than that. They can shorten the immunoassay or the PCR assay to minutes or a couple of hours, but that doesn’t reduce the enrichment time.”

One way to leapfrog the enrichment process might be to use concentration. Dr. Doyle points to research now being done by Peter Hesketh, PhD, a professor at Georgia Tech’s George W. Woodruff School of Mechanical Engineering, investigating magnetic beads as a means of concentrating the target in a microfluidic biosensor system.

“If you can concentrate the sample by 1-2 log, you’d have a direct reduction in the amount of time needed for enrichment,” Tice said. “Depending on the doubling time, a 2-log improvement in concentration could result in a two- to seven-hour decrease in enrichment time for zero-tolerance organisms like E. coli. If you apply it to non-zero tolerance organisms—like Campylobacter—a 2-log improvement in concentrating the sample would allow us to assay directly from, say, a poultry carcass, eliminating the enrichment and plating step entirely.”

Skip the Enrichment Step

Another option now in development may actually permit true real-time pathogen detection without an enrichment step at all—even for zero-tolerance organisms. It uses surface-enhanced Raman spectroscopy (SERS) to read the molecular fingerprints of pathogens.

Raman spectroscopy is a technique used to study vibrational, rotational, and other low-frequency modes in a system. It is performed by shining a near-infrared laser light onto an analyte and measuring the change in light frequency as it scatters off the analyte’s DNA or RNA. This frequency change is called the Raman shift (after the scientist who discovered it), and it is just as unique and distinct as a fingerprint. Unfortunately, Raman scatter has a very weak signal; researchers at the University of Georgia have spent the last five years developing a method of enhancing the signal using silver nanorod substrates.

“These nanorods allow us to increase the Raman signal that’s emitted by whatever pathogen we’re looking at by 100-million fold — basically, 1018 detection, at the sub-atomic level,” said Ralph Tripp, PhD, professor and Georgia Research Alliance Chair of Animal Health Vaccine Development at the College of Veterinary Medicine at the University of Georgia. “This means that we don’t have to modify the pathogen, do any form of amplification, or even go through extraction. We can just detect it in a specimen.”

To date, the majority of Dr. Tripp’s work has been with the Department of Defense and the Centers for Disease Control and Prevention, detecting bacteria and viruses such as respiratory viruses and HIV. But Dr. Tripp and the company he founded to advance the SERS pathogen detection technology, Argent Diagnostics Inc., are also leveraging it within the food industry, working with the U.S. Department of Agriculture on the detection of Salmonella, Listeria, and E. coli.

Molecular Fingerprinting in 60 Seconds

“We can do molecular fingerprinting on meat and poultry using SERS within 60 seconds,” Dr. Tripp said. “We usually use about two microliters of analyte for detection, but it’s possible to use one microliter or less.” Argent is now commercializing the technology in a biosensing unit for water baths in the poultry industry. Although it has mostly been studied in meat and poultry, Dr. Tripp does not foresee any major challenges to adapting the SERS technology for use in produce pathogen detection.

Compared to real-time PCR and immunoassay, the technology is not particularly expensive. “Essentially, we’re taking microscope slides and growing silver nanorods on them, and the cost for that can range between $1 and $3 a slide. On each slide you can do 36 individual analyses, so it’s very cost-effective,” Dr. Tripp said. “The instrument cost is essentially that of a confocal microscope—between $8,000 and $30,000, depending on whether you get the Cadillac version or not. We’re now building a lower-cost instrument specifically for the food industry to detect substrates.”

Tools like these may soon herald the end of PCR and immunoassay in food industry pathogen detection. “They’ve served us well and are the best we have right now, but they’ll be history in a couple of years,” Dr. Doyle said.

He points to recent outbreaks, such as the listeriosis outbreak in deli meats in Canada that killed 22 people. “Had they been testing end products for Listeria monocytogenes, they would have found it. When the testing of contaminated product was done, some not even by food testing companies, they were finding it in unopened samples. The same with the PCA peanut butter problem and other such outbreaks—if they had been testing finished products for pathogens, they likely would have identified the problem before the outbreak and could have taken corrective action.”

Truly real-time pathogen detection methods would make that much more feasible. “If companies aren’t volunteering to do more end-product testing, they will be forced into it either by regulators or the legal system, and in order to do this practically and economically, they’re going to have to have these shortened tests,” Dr. Doyle said. “Having assays that can give results in a matter of minutes will be real advantages both for industry and the consumer, and I don’t think it’s that far off. Right now, industry’s more interested than ever, and federal agencies certainly are as well.”

Shaw is a freelance writer based in Montclair, N.J. Reach her at ginashaw@vagabondmedia.com.

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