Cut-off values supporting the decision between
positive or negative signals are determined empirically and should be specifically adapted to different experimental setups. Although several calculation methods are described Go6983 molecular weight in the literature, they basically represent subjective evaluation of the signal to noise ratio. Some authors consider a signal positive when it is only two or three times higher than the assay background [33, 16], while others take only signals ten times higher . The fact that the LSplex protocol could allow concomitant amplification and labelling represents a valuable feature for selleckchem future application in diagnostics since it should reduce the total time required for providing the identification of the pathogen. The optimized LSplex protocol using Vent exo- performed reliable amplification and efficient incorporation Wortmannin chemical structure of amino-allyl modified nucleotides, allowing indirect labelling of PCR products. However, direct incorporation of fluorescent nucleotides
during the multiplex PCR under the same amplification conditions led to weak label incorporation making the separate labelling step necessary to achieve a good profiling fidelity. Alternatively, the use of labelled primers can be employed for obtaining fluorescent multiplex PCR products . LSplex successfully amplified less than 10 nanograms of DNA from several different pathogens (Gram-positive, Gram-negative and fungi) generating signals in general stronger and more specific than the ones generated with 2–5 micrograms of DNA. LSplex improved the specificity
of the hybridization assay and enriched the sample for the target sequences present in the template. Interestingly, Candida albicans produced non-detectable signals when 2 μg of genomic DNA are used for hybridization. After amplification of 10 ng of C. albicans DNA by LSplex protocol resulted in the clear hybridization pattern (Fig. 4). We would like to emphasize that a reduction in the limit of sensitivity of the LSplex protocol to picograms or to femtograms would be desirable in order to detected pathogens directly from every clinical, food or environmental samples. In the last two years the publication of several reports referring Carbohydrate to rapid identification of bacterial species by multiplex PCR coupled to microarrays detection [5, 35, 6, 17, 16, 36–38, 17, 3, 37, 3, 4, 23, 7] demonstrated the usefulness of this approach and the growing interest in implementing it in routine diagnostics. It also underlines the necessity of finding robust protocols for amplifying the target DNA before microarray analysis. Whole genome amplification (WGA) is a powerful technique for the amplification of total genomic DNA (e.g. for comparative hybridization ). However, the random priming employed in WGA will amplify every DNA in the sample. Therefore, the application of WGA is difficult if the DNA of interest is contaminated by unwanted DNA.