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

onocytogenes. Primer pair 341F with GC-clamp and 534 R yielded a single band corresponding to a 250 bp DNA fragment when the PCR reaction was carried out with Pfu polymerase in the presence of DNA from L. monocytogenes. That a single band was obtained with each primer pair indicates that only the target gene was amplified, with no non-specific primer attachment or hetroduplexes formed.

PCR of DNA extracted from different dilutions of the three Listeria spp. was undertaken to determine the minimal number of cells required for successful amplification of the conserved region of their 16S rRNA gene using PfuTurbo polymerase. A detectable PCR product was obtained using either set of primer pairs with as little as 1.3 ng DNA from 1.3 x 104 colony forming units (cfu) of L. innocua. A detectable PCR product was obtained with as little as 1.6 ng DNA from 5.2 x 103 cfu of L. monocytogenes using primer pair 1055F and 1406R with GC-clamp. Ten times this amount of L. monocytogenes DNA was required to obtain a detectable PCR product using the primer pair 341F with GC-clamp and 534R. A detectable PCR product was obtained with as little as 7.7 ng DNA from 6.9 x 103 cfu of L. seeligeri using primer pair 1055F and 1406R with GC-clamp, whereas, the other primer pair required ten times this amount of L. seeligeri DNA for a detectable product. The minimum quantity of DNA from these Listeria spp. that was required for successful amplification of fragments by PCR was generally less than that (25500 ng) recommended by the suppliers of the polymerase.

L. monocytogenes was distinguished from L. innocua and L. seeligeri when fragments of their 16S rRNA gene(s), amplified by PCR using PfuTurbo polym
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