( us PCR protocols have been developed fo...)Use of the DCode System to Detect the Food-Borne Bacterial Pathogen Listeria monocytogenes

us PCR protocols have been developed for detection of L. monocytogenes, but all utilize a primer set that is specific for this species. In such cases the primers typically target a hypervariable region of the 16S rRNA gene (16S rDNA), or a sequence of a gene coding for a virulence factor unique to this bacterium.⁴ There are instances, however, where one may wish to screen for more than one type of bacteria in a sample, or for uncultivable bacteria whose unique sequences have not yet been identified. In such cases one can use a primer set that anneals to a sequence of the 16S rDNA that is common to many different types of bacteria. The PCR products from the different bacteria are the same size, but contain intervening regions where the base sequence is unique. The 16S rRNA of L. monocytogenes has 99.2 % and 98.5% nucleotide homology with the corresponding molecule of the closely-related species L. innocua and L. seeligeri , respectively. This corresponds to an 11 bp difference in the 16S rDNA of L. monocytogenes and L. innocua , and a 22 bp difference in the 16S rRNA molecule of L. monocytogenes and L. seeligeri .⁵

Density gradient gel electrophoresis (DGGE) can be used to separate DNA fragments of the same size when the fragments display differences in the sequence of the base pairs that yield different G+C content. These fragments may be resolved as discrete bands on a gel. This approach has been used to detect bacteria, which have not yet been cultured in the laboratory.⁶ The different fragments responsible for each band in the gel can be sequenced and the sequence used to construct oligonucleotide probes specific to each type of organism present in the sample. In this note, we describe
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