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Use of the DCode System to Detect the Food-Borne Bacterial Pathogen Listeria monocytogenes

on. The denatured molecules with the same G+C content accumulate at the same location in the gel to form a discrete band.

Bands tend to sharpen as their rate of migration is reduced because molecules at the trailing edge of a band move faster than molecules at the leading edge of the band. As a result, DGGE appears to be less sensitive to loading conditions (volume of sample loaded, ionic strength of sample, etc.) than other polyacrylamide gel electrophoresis procedures.7 To further sharpen the bands, an acrylamide gradient can be incorporated into the gel. After electrophoresis, the bands are visualized by reacting the gel with fluorogenic, chromogenic, or radioactive molecules that bind to DNA. Fragments containing sequences that differ by as little as one base-pair have been resolved in this manner.6 This sensitivity offers the opportunity to detect and distinguish closely-related bacteria present in a sample.

There are two different ways to perform DGGE: perpendicular and parallel gels. Perpendicular gels have an increasing gradient of denaturants from left to right, perpendicular to the direction of electrophoresis. Parallel gels have an increasing gradient of denaturants from top to bottom, parallel to the direction of electrophoresis. Perpendicular gels are normally used to determine the optimal denaturant gradient to use in the gel, while parallel gels are normally used for final separation of different fragments. Determination of the denaturant gradient can also be achieved using parallel gels.

BACTERIAL DNA EXTRACTION AND QUANTIFICATION
Pure cultures of L. monocytogenes (ATCC 15313), Listeria innocua (ATCC 33090), and Listeria seeligeri (ATCC 35967) s
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