Dimeric oligonucleotide probes enhance diagnostic macroarray performance

Disease management can be improved with rapid and accurate pathogen detection and identification techniques. Here we describe the development of a macroarray diagnostic technique with enhanced detection sensitivity and only small reduction in specificity. With probes designed based on the internal transcribed spacer sequences of the rRNA genes of fungal and oomycete strains, we produced a macroarray, which included five types of oligonucleotide probes: monomers (20-24nt), dimers (40-48nt), dimers with a poly-A spacer of 10 bases between the two repeats (50-58nt), monomers with a poly-A tail of 10 (30-34nt) and 20 (40-44nt) bases. The use of repeat sequence probes (dimers) greatly improved the sensitivity of the macroarray. The dimeric probes could reliably detect 0.01fg target genomic DNA, which is lower than the detection limits of most currently available molecular diagnostic methods, such as the conventional PCR and real-time PCR. Dimer probes also had lower signal variability, thereby increasing the macroarray signal uniformity. However, in a few cases, specificity was reduced in the dimer probes. Cross-hybridization occurred in highly similar sequences where the mismatched base was located near the end or in a chain of the same base, but this should be prevented in future array probe design.