Quantitative PCR

Recently, a number of investigators have described semiquantitative PCR methods that allow to identify and to know the number of bacteria present in the sample, and that simplify the need to perform postamplification techniques. An enzyme-linked immunosorbent assay-mediated PCR technique was described to detect and quantify L. monocytogenes in food products. Using this method, a fragment of the iap gene and of an internal standard are amplified in the presence of fluorescein dUTP. PCR products were then hybridized in streptavidin-coated microtiter plate prepared with biotinylated specific DNA probes. After the addition of an alkaline phospha-tase-conjugated antibody to fluorescein, the PCR products are quantitated based on an optical density reading.[4] Bassler et al.[5] described a 5' nuclease PCR-based assay for L. monocytogenes that uses the hydrolysis of a dually labeled internal fluorogenic probe (TaqMan® PCR detection, Applied Biosystems, Foster City, CA) to monitor the amplification of the target (a region of the hly A gene). Quantification was found to be linear over a range of 5 x 10 to 5 x 105 CFU. A variation of the above method was described by Koo and Jaykus[6] for the detection of L. monocytogenes using hly A or iap as a target, and an inexpensive asymmetric probe set in place of the TaqMan probe. The above methods are endpoint detection assays and the PCR products have to be detected by fluorescence or optical density reading immediately following termination of the PCR.

Strategies based on the detection of PCR products simultaneously as the PCR is progressing (real-time PCR) and avoiding further postprocessing were also developed for the detection and quantification of L. monocytogenes and Listeria innocua. Those methods exploit also the 5' nuclease activity of the Taq DNA polymerase and a dually labeled fluorogenic TaqMan hybridization probe, but use the ABI Prism® 7700 sequence detection system of Perkin-Elmer (Foster City,

CA) and either the hlyA or iap genes as target.[7-9] A protocol enabling the simultaneous detection and quantification of Escherichia coli O157:H7, L. monocytogenes, and Salmonella strains was also formatted using commercial Bax PCR kits (for Bax, see Listeria spp. entry: DNA probes and standard PCR assays) and by monitoring the PCR in real time with the iCycler of BioRad Laboratories (Hearles, CA) after the addition of the nonspecific fluorescent dye SYBR Green I. A melting curve analysis of PCR amplicons serves as a confirmatory test, with the Tm of each amplicon being dependent on the length as well as the G+C content of the sequence.[10]

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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