AMHERST, Mass. − In work that has implications for the food safety industry, scientists, and environmental and public health agencies, University of Massachusetts Amherst researchers have developed a molecular-based method that distinguishes live bacterial cells from dead ones. The study was published online June 1 in the Journal of Microbiological Methods.
Developed by microbiologist Robert Levin, food science, and doctoral student Shishan Wang, the new method adds a level of specificity to DNA detection and could be applied to a suite of pathogens, perhaps preventing massive recalls of meat carrying E. coli, or enhancing tests that check for contaminants in drinking water.
“You aren’t only protecting the consumer” with such tests, says Levin, “you could save thousands of dollars.”
The research is supported by a Special Seafood Safety grant from the U. S. Department of Agriculture.
The new method takes advantage of a technique called polymerase chain reaction (PCR), which scientists use to make lots of copies of a small, specific stretch of DNA. PCR generates large quantities of DNA from tiny samples, and is used widely by scientists studying everything from birds to humans to bacteria.
Levin and Wang have used PCR to screen seafood for the DNA of Vibrio vulnificus, a disease-causing bacterium from the same family as those that cause cholera. But PCR just copies the designated DNA, it doesn’t indicate whether the DNA came from a cell that was dead or alive, critical information when testing food or water for organisms that make people sick.
The first step of PCR is heating the sample that contains the DNA of interest. At the right temperature, the two strands that make up a DNA molecule separate, and only then can they be copied. But Levin and Wang weren’t interested in copying all the V. vulnificus DNA in their sample, just the DNA from bacteria that were alive.
So the researchers treated their bacteria samples with ethidium bromide monoazide (EMA), a chemical that winds its way in between the strands and building blocks of a DNA molecule. EMA will insert itself into any DNA it finds, but it can’t get through the cell membranes of healthy, living bacteria. However, EMA can easily get to the DNA of a dead or dying bacterium with a damaged cell membrane.
After dosing the bacteria with EMA, the researchers zapped their samples with high-intensity visible light causing the EMA to form strong, cross-linking bonds with the DNA it’s tangled up in. These bonds prevent the DNA molecules from separating, so they can’t be copied during PCR. Only DNA from live cells will be copied, alerting the testers to the presence of living bacteria.
“Once you’ve determined the optimum concentrations of EMA you can completely inhibit amplification of DNA from dead cells,” says Levin.
The scientists have worked out the protocols for testing for V. vulnificus, and with minor adjustments the method could be applied to other disease-causing critters.
“This could take PCR one giant step forward,” says Levin.