Conventional Polymerase Chain Reaction Assays

As an alternative to DNA hybridization methods, PCR procedures were proved to be rapid and highly specific for the detection of a number of pathogens. Polymerase chain reaction assays based on the flagellin A encoding gene (faA), the 16S/23S intergenic spacer region of the rRNA operon, and the iap gene have been developed for the identification of the Listeria genus.[23-25] Continuous efforts for the specific identification of L. monocytogenes using polymerase chain reaction assays were made. Apart from PCR identification procedures targeting the 16S rRNA gene, the 16S/23S spacer region, and the amino-peptidase C gene (pepC), these assays amplified DNA sequences derived from genes proved or suspected to be implicated in the virulence mechanism of this species, such as hlyA, plcB, actA, prfA, inlA, inlB, iap, the gene coding for LmA antigen (lmaA or Dth18 gene), or the fibronectin-binding protein (ffbp) gene.[17,23,24,26-36] The most frequently chosen target has been the hlyA gene from which amplification fragment of different sizes has been generated with specific primers designed by several authors.[37] As L. monocytogenes strains with mutations or deletions in one or more virulence determinants can be encountered, a multiplex PCR for the simultaneous detection of multiple genes in a single step has also been developed.[6-9,26] Whereas vast number of protocols for the PCR identification of L. monocytogenes have been published, only few studies have considered the identification of the other members of the genus Listeria.[37] Nevertheless, the identification of non-L. monocytogenes species can also be achieved by the specific amplification of part of the 16S rRNA gene of L. ivanovii, internal iap sequences of L. grayi, L. seeligeri, L. welshimeri, and L. innocua, part of the L. welshimeri fbp gene, and part of the L. innocua gene encoding the lin046 putative transcriptional regulator.[16,27,38,39] One of the above PCR assay is a multiplex PCR that allows the simultaneous detection of all Listeria species and the differentiation between some (but not all) of them, in a single step.[38] The comparative analysis of genome sequences of L. monocytogenes and L. innocua revealed also the presence of 270 L. monocytogenes and 149 L. innocua strain-specific genes.[40] These genes are putative targets for the further development of PCR assays for the specific identification of both species.

The introduction of commercial PCR kits such as the Probelia L. monocytogenes system which targets the hlyA gene (Bio-Rad Lab., Richmond, CA) and the Bax system for L. monocytogenes and also for Listeria genus (Qualicon-Dupont, Willington, DE) has made easier the routine use of the PCR method for the identification of Listeria. PCR products are detected by agarose gel electrophoresis for the Bax system or in a microplate postamplification capture hybridization assay for the Probelia system. These automatized methods, developed to screen food and environmental samples, need a pre-enrichment step of the samples before the PCR assays to increase the number of target bacteria and to avoid the detection of dead cells by diluting out any nonviable cells that may be present.

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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