Efforts to Improve Rapid Microbiology Tools

“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.