Vulnerability of DNA hybridization in soils is due to Mg²⁺ ion induced DNA aggregation

The NanoGene assay is an inhibitor-resistant gene quantification assay based on magnetic bead and quantum dot nanoparticles. It employs a set of probe and signaling probe DNAs to capture target DNA via hybridization. Using simple DNA preparation that bypasses conventional DNA extraction, it was able to detect and quantify specific bacterial genes in environmental sample. In this study, the vulnerability of the NanoGene assay to the presence of various environmental factors was investigated. A total of 43 soil samples were inoculated with 10⁹ CFU/mL of Pseudomonas putida prior to DNA isolation without purification. Subsequently, the NanoGene assay was performed for quantitative detection of P. putida with respect to 12 soil properties including pH, moisture, humic acids, organic matter, sand, silt, clay, cation exchange capability, sodium, potassium, magnesium, and calcium. Using multiple linear regression, the NanoGene assay was found to be particularly vulnerable to the presence of Mg²⁺, which was selected as a major variable (P = 0.001). The vulnerability of the NanoGene assay to Mg²⁺ was further explored by atomic force microscopy, which indicated significant Mg²⁺-mediated DNA aggregation. The inhibition of the NanoGene assay from some soil samples as a consequence of DNA aggregation could therefore be prevented by the use of Mg²⁺ chelators such as EDTA, enabling application of this method across diverse soil types.