Collaborators Required to Commercialize DNA Based Identification Tests

ARS scientists in Peoria, Illinois, have devised a new DNA-based approach for genetically identifying pathogens that's faster, easier to use, and more precise than some currently used methods.

For example, pulsed-field gel electrophoresis (PFGE) is considered the gold standard for genetically identifying L. monocytogenes. But PFGE is difficult to run, takes about 3 days, and has several disadvantages that complicate efforts to determine the relationships between different isolates.

In contrast, "Our method can be performed in a single day," says microbiologist Todd Ward, at ARS's National Center for Agricultural Utilization Research, in Peoria, "and can target single nucleotide variations within specific genes."

Such variations help distinguish one strain of L. monocytogenes from another. By targeting genes for virulence, for example, the test could enable a user to understand what makes some strains more harmful or better adapted to a particular environment than others.

This feature could prove especially useful in hazards analysis and critical-control-point programs at food-processing plants. "The ability to identify Listeria that have colonized your duction plant can help determine whether food products are contaminated before coming into the plant or within the plant by resident strains," says Ward.

Brent Page, a molecular biologist and colleague in the ARS center's Microbial Genomics and Bioprocessing Research Unit, and research leader Cletus Kurtzman say the test could be used to check for yeasts that cause food and beverage spoilage. It could also speed the search for yeasts adept at fermenting cornstarch into ethanol or those used for biocontrol of fruit-storage rots.

Clinical applications may include, identifying the 30 to 40 Candida species that can cause human infections, particularly in immunocompromised people. Page says the various culture-plate testing methods now used to diagnose Candida infection are limited to a few species--notably C. albicansand the turnaround time is 24 hours to a few weeks, delaying treatment. Genetic fingerprinting tests used in some labs may be faster, but they too detect only a few Candida species.
By comparison, the test from Kurtzman's unit identifies 32 total species--simultaneously and in less than 5 hours.

The method uses a flow cytometer, which can handle up to 100 samples at a time, probes find and bind to corresponding pieces of species-specific DNA. The researchers created the probes using DNA-sequence information from their unit's microbial-genomics database. The probes have a fluorescent marker that tells the cytometer which DNA sequence was detected. The machine clearly displays the species' identity as color-coded bar graphs.

Such accuracy stems from Kurtzman's and University of Miami professor Jack Fell's co-development of gene-sequence databases for all known yeast species, leading to the current effort, which was initiated with funding from the National Institutes of Health.

Commercial collaborators are being sought to develop the resulting tests in kit form.

Todd J. Ward, Brent T. Page, and Cletus P. Kurtzman are with
the USDA-ARS National Center for Agricultural Utilization
Research, 1815 North University St., Peoria, IL 61604; phone
(309) 681-6560, fax (309) 681-6672, e-mail wardtj@ncaur.
usda.gov, pageb@ncaur.usda.gov, kurtzman@ncaur.usda.
gov.