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

hat of the bands from the other two species may be due to a difference in %G+C of the fragments arising from the possible base differences cited above. This remains to be confirmed, however.

The general bacterial primers used in this study amplify target sequences within the 16S rRNA gene of many different bacteria. Competition for primer, enzyme and dNTPs often occurs with DNA from the different bacteria in the sample during PCR amplification. When the copy number of a gene from one type of bacterium is considerably higher than the copy number of the same gene from another type of bacterium (i.e., an abundance of cells of one species over cells of another), the gene with the greater copy number will be amplified preferentially over the gene present in lower copy number. An experiment was conducted to determine the minimal ratio of L. monocytogenes DNA to L. innocua and L. seeligeri DNA that still enabled detection of the unique band from L. monocytogenes in DGGE gels after fragment amplification by PCR using PfuTurbo polymerase and primer pair 1055F and 1406R with GC-clamp. The results indicate that L. monocytogenes-specific band could still be detected in the presence of 23 times its weight of DNA contributed by the other two species. When the difference in relative amounts were above this, the L. monocytogenes-specific band was undetectable.

In summary, DGGE using the DCode system facilitates detection of variations in 16S rDNA sequence that result in different G+C content among closely-related bacterial species as well as among multiple alleles of the same gene in a single species. Separation based on subtle differences in G+C content by DGGE into discrete visible bands facilitates subsequent isolatio
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