The Implications of Fragmented Genomic DNA Size Range on the Hybridization Efficiency in NanoGene Assay

DNA hybridization-based assays are well known for their ability to detect and quantify specific bacteria. Assays that employ DNA hybridization include a NanoGene assay, fluorescence in situ hybridization, and microarrays. Involved in DNA hybridization, fragmentation of genomic DNA (gDNA) is necessary to increase the accessibility of the probe DNA to the target gDNA. However, there has been no thorough and systematic characterization of different fragmented gDNA sizes and their effects on hybridization efficiency. An optimum fragmented size range of gDNA for the NanoGene assay is hypothesized in this study. Bacterial gDNA is fragmented via sonication into different size ranges prior to the NanoGene assay. The optimum size range of gDNA is determined via the comparison of respective hybridization efficiencies (in the form of quantification capabilities). Different incubation durations are also investigated. Finally, the quantification capability of the fragmented (at optimum size range) and unfragmented gDNA is compared

Detection of Cyanobacteria in Eutrophic Water Using a Portable Electrocoagulator and NanoGene Assay

We have demonstrated the detection of cyanobacteria in eutrophic water samples using a portable electrocoagulator and NanoGene assay. The electrocoagulator is designed to preconcentrate cyanobacteria from water samples prior to analysis via NanoGene assay. Using <i>Microcystis aeruginosa</i> laboratory culture and environmental samples (cell densities ranging from 1.7 × 10⁵ to 4.1 × 10⁶ and 6.5 × 10³ to 6.6 × 10⁷ cells·mL^–1, respectively), the electrocoagulator was evaluated and compared with a conventional centrifuge. Varying the operation duration from 0 to 300 s with different cell densities was first investigated. Preconcentration efficiencies (obtained via absorbance measurement) and dry cell weight of preconcentrated cyanobacteria were then obtained and compared. For laboratory samples at cell densities from 3.2 × 10⁵ to 4.1 × 10⁶ cells·mL^–1, the preconcentration efficiencies of electrocoagulator appeared to be stable at ∼60%. At lower cell densities (1.7 and 2.2 × 10⁵ cells·mL^–1), the preconcentration efficiencies decreased to 33.9 ± 0.2 and 40.4 ± 5.4%, respectively. For environmental samples at cell densities of 2.7 × 10⁵ and 6.6 × 10⁷ cells·mL^–1, the electrocoagulator maintained its preconcentration efficiency at ∼60%. On the other hand, the centrifuge’s preconcentration efficiencies decreased to nondetectable and below 40%, respectively. This shows that the electrocoagulator outperformed the centrifuge when using eutrophic water samples. Finally, the compatibility of the electrocoagulator with the NanoGene assay was verified via the successful detection of the microcystin synthetase D (<i>mcyD</i>) gene in environmental samples. The viability of the electrocoagulator as an in situ compatible alternative to the centrifuge is also discussed