Pathogen analysis and genetic predisposition testing using microelectronicarrays and isothermal amplification

Background: A simple yet powerful tool for providing for rapid gene identif ication in the clinic would be the combination of isothermal gene amplifica tion with electronic microchip analysis, This is a first report of such a u nion of these technologies. Methods: The first assay demonstrates discrimination between four bacterial pathogens. For this, one portion of the bacterial 16S rRNA gene encompassi ng a microheterogeneous region was isothermally amplified using Strand Disp lacement Amplification (SDA), Type identification was then made by "sandwic h" assay format either using selective electronic hybridization of amplicon s to sequence-specific capture oligonucleotides and a universal, fluorescen tly labeled reporter oligonucleotide, or, alternatively, sequence-specific reporters and a universal capture oligonucleotide. The second assay tested for the presence or absence of the Factor V Leiden point mutation using DNA obtained from 18 patients in a blind assay. For this, allele-specific SDA was developed. Following amplification using a sense-biotinylated primer an d either the corresponding antisense wild type or mutant primer, multiple p atient amplicons were targeted to specified locations on the microarray and visualized using a fluorescently labeled reporter oligonucleotide. Positiv e signals were scored as greater than or equal to two times the background. Results: Bacterial type-specific signals were between 3- to 10-fold greater than nonspecific in both assay formats. Using allele-specific SDA, 100% ag reement was observed between PAGE analysis, microarray results, and clinica l diagnosis in Factor V mutation analysis. Conclusions: We demonstrated two model clinical assays combining amplified materials and microelectronic arrays, one potentially suitable for pathogen screening and the other for a deleterious genetic mutation.