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

erved as the source of 16S rDNA for this study. Broth cultures of the three species of bacteria were serially diluted and lysed by heating at 94 C for 10 min and stored at -40 C until used. DNA concentration in the various dilutions was determined by reacting 25 ml of sample with 5 ml of a 50 ul/ml volume of 4', 6-diamidino-2-phenylindole (DAPI), a fluorogenic molecule that selectively binds DNA. The fluorescence intensity of the reaction product was determined in a fluorescence spectrometer at 435 nm using an excitation wavelength of 360 nm. Calf thymus DNA was used as a standard.

POLYMERASE CHAIN REACTION PCR
was performed with a Bio-Rad Gene Cycler using PfuTM or PfuTurbo polymerase. Two different primer pairs, obtained from Macromolecular Resources, Colorado State University, Fort Colins, CO, were used: 341F (with GC-clamp) and 534R, which target the V3 region and 1055 F and 1406 R (GC-clamp), which target the V9 region of the 16S rRNA gene. The DNA fragments produced using these primers contained sequences unique to L. monocytogenes. Amplification was carried out in a mixture containing 5 ml of 10x cloned Pfu polymerase buffer (Strategene, San Diego, CA), 50 mM of each dNTP (Sigma Chemical Co., St. Louis, MO) 0.6 mM of each primer pair, 1 ml containing 2.5 units of cloned Pfu or PfuTurbo (DNA polymerase Strategene, San Diego, CA), 1 ml of sample DNA, and deionized water (Sigma Chemical Co., St. Louis, MO) added to achieve a final reaction mixture volume of 50 ml.

To minimize non-specific annealing of the primers, Pfu or PfuTurbo polymerase was added after the first denaturing step, at a temperature of 80 C, a technique referred to as Hot Start.6 Another technique, referred to
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