Large scale multiplex PCR improves pathogen detection by DNA microarrays

To increase the detection power of a fluorescence-based prototype-microarray designed to identify pathogenic microorganisms involved in sepsis, we propose a large scale multiplex PCR (LSplex PCR) for amplification of several dozens of gene-segments of 9 pathogenic species. This protocol employs a large set of primer pairs, potentially able to amplify 800 different gene segments that correspond to the capture probes spotted on the microarray. The LSplex protocol is shown to selectively amplify only the gene segments corresponding to the specific pathogen present in the analyte. Application of LSplex increases the microarray detection of target templates by a factor of 100 to 1000.Our data provide a proof of principle for the improvement of detection of pathogen DNA by microarray hybridization by using LSplex PCR.Clinical microbiological diagnostics, environmental survey, food quality control and biodefence strategies have a common keystone: accurate and rapid identification of pathogenic microorganisms. Several molecular biology-based methods have been recently developed for microbial diagnostics and offer noticeable advantages over conventional techniques in microbiology. Among the molecular biology-based methods, DNA microarray technology presents the potential of direct and rapid identification of multiple DNA sequences [1-7]. A microarray displaying DNA probes corresponding to a collection of genes of a broad spectrum of pathogens is a powerful tool for simultaneous species identification, detection of virulence factors and antimicrobial resistance determinants [2]. Major drawbacks in using DNA microarrays as a standard technique for pathogen detection are linked to the low representation of pathogen DNA in the analytes, but also to the relatively low sensitivity of fluorescence-based microarrays. The amount of specific pathogen DNA present in clinical, environmental, and food samples is sometimes as low as few femtograms [8-14], while the detection limit for gen