Sequence specific detection of DNA using nicking endonuclease signal amplification (NESA).

We have developed a new method for identifying specific single- or double-stranded DNA sequences called nicking endonuclease signal amplification (NESA). A probe and target DNA anneal to create a restriction site that is recognized by a strand-specific endonuclease that cleaves the probe into two pieces leaving the target DNA intact. The target DNA can then act as a template for fresh probe and the process of hybridization, cleavage and dissociation repeats. Laser-induced fluorescence coupled with capillary electrophoresis was used to measure the probe cleavage products. The reaction is rapid; full cleavage of probe occurs within one minute under ideal conditions. The reaction is specific since it requires complete complementarity between the oligonucleotide and the template at the restriction site and sufficient complementarity overall to allow hybridization. We show that both Bacillus subtilis and B. anthracis genomic DNA can be detected and specifically differentiated from DNA of other Bacillus species. When combined with multiple displacement amplification, detection of a single copy target from less than 30 cfu is possible. This method should be applicable whenever there is a requirement to detect a specific DNA sequence. Other applications include SNP analysis and genotyping. The reaction is inherently simple to multiplex and is amenable to automation.

Luminex detection of fecal indicators in river samples, marine recreational water, and beach sand.

Research to understand and remediate coastal pollution is moving toward a multitiered approach in which traditional enumeration of fecal indicators is accompanied by molecular analysis of a variety of targets. Technology that rapidly detects multiple microbial contaminants would benefit from such an approach. The Luminex 100 system is a suspension array that assays multiple analytes rapidly in a single well of a microtiter plate. The ability of the system to simultaneously detect multiple fecal indicating bacteria in environmental samples was tested. Primer/probe sets were designed to simultaneously detect the following fecal indicators: the Bacteroides fragilis group, Enterococcus spp., Escherichia coli and Shigella spp., Bacteroides distasonis, and Ent. faecalis. Specificity and sensitivity of the Luminex probes was tested against laboratory cultures. In addition, sequencing, culture plate testing, and specificity testing with environmental isolates were steps taken to validate the function of the assay with environmental samples. Luminex response to cultures and to environmental samples was consistent with sequencing results, suggesting that the technology has the potential to simultaneously detect multiple targets for coastal water quality applications, particularly as progress is made to efficiently extract DNA from water and sediment matrices.

Rapid identification of adult and naupliar stages of copepods using DNA hybridization methodology.

Larval stages of common marine invertebrates and their ecological roles within their respective communities are frequently ignored because they are hard to identify. Morphological characters are often insufficient to differentiate between genera, much less species. To overcome the obstacles associated with species identification of copepod larvae, we developed a microtiter plate-based hybridization assay. Species-specific probes based on rDNA sequences were bound to microplates and used to capture target DNA. A novel method of linking the probes to the plate with poly-T tail ensured the probes were positioned above the plate surface and available for hybridization; this significantly increased the sensitivity of the assay. Target DNA extracted from individual copepods was amplified with biotin-labeled primers. The labeled target DNA bound to the probe specific for that species and produced a colorimetric change in the assay. The assay can be rapidly performed on freshly caught or ethanol preserved samples and the results visually interpreted.

Comparative analysis of the intergenic spacer regions and population structure of the species complex of the pathogenic yeast Cryptococcus neoformans.

Cryptococcus neoformans is an opportunistic basidiomycete responsible for the high incidence of cryptococcosis in patients with AIDS and in other immune-compromised individuals. This study, which focused on the molecular structure and genetic variability of the two varieties in the C. neoformans and Cryptococcus gattii species complex, employed sequence analysis of the intergenic spacer regions, IGSI and IGSII. The IGS region is the most rapidly evolving region of the rDNA families. The IGSI displayed the most genetic variability represented by nucleotide base substitutions and the presence of long insertions/deletions (indels). In contrast, the IGSII region exhibited less heterogeneity and the indels were not as extensive as those displayed in the IGSI region. Both intergenic spacers contained short, interspersed repeat motifs, which can be related to length polymorphisms observed between sequences. Phylogenetic analysis undertaken in the IGSI, IGSII and IGSI +5S rRNA + IGSII regions revealed the presence of six major phylogenetic lineages, some of which segregated into subgroups. The major lineages are represented by genotypes 1 (C. neoformans var. grubii), genotype 2 (C. neoformans var. neoformans), and genotypes 3, 4, 5 and 6 represented by C. gattii. Genotype 6 is a newly described IGS genotypic group within the C. neoformans species complex. With the inclusion of IGS subgenotypic groups, our sequence analysis distinguished 12 different lineages. Sequencing of clones, which was performed to determine the presence of multiple alleles at the IGS locus in several hybrid strains, yielded a single IGS sequence type per isolate, thus suggesting that the selected group of cloned strains was mono-allelic at this locus. IGS sequence analyses proved to be a powerful technique for the delineation of the varieties of C. neoformans and C. gattii at genotypic and subgenotypic levels.