Pathogen analysis and genetic predisposition testing using microelectronic arrays and isothermal amplification.

Abstract

BACKGROUND: A simple yet powerful tool for providing for rapid gene identification in the clinic would be the combination of isothermal gene amplification with electronic microchip analysis. This is a first report of such a union of these technologies. METHODS: The first assay demonstrates discrimination between four bacterial pathogens. For this, one portion of the bacterial 16S rRNA gene encompassing a microheterogeneous region was isothermally amplified using Strand Displacement Amplification (SDA). Type identification was then made by "sandwich" assay format either using selective electronic hybridization of amplicons to sequence-specific capture oligonucleotides and a universal, fluorescently 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 and either the corresponding antisense wild type or mutant primer, multiple patient amplicons were targeted to specified locations on the microarray and visualized using a fluorescently labeled reporter oligonucleotide. Positive 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% agreement was observed between PAGE analysis, microarray results, and clinical 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.