Penn State research is making it easier for food processors to detect a dangerous foodborne pathogen and keep contaminated products from entering the market.

Listeria monocytogenes can be found in such products as milk, meat, undercooked poultry, vegetables, raw or smoked fish, soft cheeses (such as feta, Camembert, or Brie), and deli meats such as bologna and hot dogs. Pasteurizing or cooking these foods will kill the pathogen, but even after pasteurization, contamination could occur before a product is packaged at the processing plant or after the package is opened at home.

Graduate student Sangeetha Paramaswaran worked with food scientist Steve Knabel to find an easier, faster way to detect Listeria in foods.

Current methods of testing food products for Listeria require technically trained lab workers. First, a food sample is placed into a special broth that enhances the pathogen’s growth. If the broth indicates the presence of Listeria, technicians perform a more definitive test to confirm the results. The DNA from the bacteria in the broth is put through a process called polymerase chain reaction (PCR), and if DNA from Listeria monocytogenes is present, millions of copies of unique DNA segments are created. Next, a process called gel electrophoresis separates these unique DNA segments by their size. Because Listeria monocytogenes has a unique DNA segment of a specific size, a lab worker can positively identify its presence in the food sample.

Some smaller companies may not be fortunate enough to employ workers with the expertise to perform these tests. The process is also time-consuming, which creates another challenge: the food being tested needs to be refrigerated for up to five days until the results come back, and most mid-sized meat processors cannot afford the extra refrigerated storage space to hold food pending test results.

Simpler, quicker detection methods would eliminate the need for storage areas as well as technical training for employees, making it easier for smaller companies to test for Listeria.

“The gel electrophoresis method is cumbersome for most lab workers, especially those not trained in biochemistry or molecular biology,” says food scientist Steve Knabel. “We wanted to figure out a quicker and simpler method that would still be sensitive and accurate enough to work in a food processing plant.”

L. monocytogenes sickens an estimated 2,500 people in the U.S. each year.

Under Knabel’s supervision, food science graduate student Sangeetha Parameswaran began studying the problem in 1998. Carey Brickner, a senior in microbiology, worked with Para-meswaran during the summer of 2000. First, Parameswaran optimized the PCR method using a commercial PCR test kit, and then she combined it with optimized PSU broth, which had been previously developed in Knabel’s laboratory. This resulted in rapid and consistent amplification of Listeria monocytogenes DNA present in the samples.

In looking for alternatives to gel electrophoresis, she found that the amplified DNA could be detected faster and more simply with a chemical called PicoGreen, a fluorescent green dye that binds to DNA segments. DNA bound to PicoGreen emits a higher wavelength of light than unbound DNA, and that light can be measured by an instrument called a spectrofluorometer. A high level of emitted light confirms the presence of Listeria monocytogenes. “The main advantage to the PicoGreen is simplicity,” Parameswaran says. “The lab workers add the chemical to the DNA, let it stand for three minutes, then put the sample in the fluorometer and measure the fluorescence. It’s much easier than using gel electrophoresis.”

Extensive tests of the PicoGreen detection method have proved that it works for detection of low levels of Listeria monocytogenes in both pasteurized milk and hot dogs. Knabel and Parameswaran are submitting their research results to the Journal of Applied and Environmental Microbiology, and they expect the new detection method to be adopted by the dairy industry this year.

—Rebecca Zeiber