Another alternative approach applied to solution-phase highly mul

Another alternative approach applied to solution-phase highly multiplex PCR has been the replacement of target-specific primers with universal ones. However, this process involves multiple steps starting with enzymatic digestion of the template DNA, ligation to adapters, primer extension and finally two subsequent PCR reactions [30, 31]. Such multi-step approaches are time consuming and prone to contamination

[25] and therefore have not been recommended for bacteriological routine diagnostics. The coupling of a pre-processing multiplex PCR to a medium-density microarray format, displaying hundreds of probes for identification and virulence profile typing of several pathogenic species, SB431542 requires an unbiased multi-target amplification corresponding to several dozens of specific capture probes characterizing a certain pathogen. Since the presence GSK2126458 in vitro and concentration of the particular pathogen in a microbiological laboratory is unknown, the multiplex reaction should include as many primer pairs as capture probes are present on the microarray. Moreover, SRT1720 purchase the reaction has to cope with femtograms of pathogen template DNA whose GC-content can

range between 30 and 70% and which is mixed with nanograms of human DNA. We have shown high fidelity amplification of specific DNA targets using pools of species-specific mixes of up to 800 primer pairs, which improves the sensitivity of the microarray detection of pathogens by a factor of 2 to 3-logs. By using S. aureus DNA (strain ATCC 29213) as template for amplification, we demonstrated that LSplex tolerates the increase in primer mix complexity until at least 800 primer pairs, without significant reduction filipin in the profiling fidelity. LSplex products amplified from 10 and even 1 ng of template generated fluorescent signals as strong as those produced by micrograms of genomic DNA. Nevertheless, the comparison between LSplex hybridization profiles and the ones obtained with 2 μg of S. aureus showed that some probes were poorly amplified with the high

complexity primer mixes. These probes produced a strong fluorescent signal when hybridized with genomic DNA but upon the LSplex protocol they were not considered as positive since their fluorescence difference was less then 2 times SD to the mean fluorescence intensity of the whole microarray. This problem of under-amplification of some targets might be circumvented by a specific increase in the concentration of primer pairs amplifying these specific targets [32]. Such a balancing strategy for individual primer pairs could be applied on the whole set of primers, following a broad comparison between hybridization profiles generated by genomic DNA of many reference strains of all species of interest and the LSplex amplified products.

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