Humic Acids Resistant Gene Quantification Assay in Soils

Abstract

Humic acids are the most commonly reported group of inhibitors in environmental samples. Due to the ubiquity and abundance of humic acids in the environment, they are often co-extracted along with the nucleic acids and interfere with gene amplification required for the real-time PCR assay. In order to overcome the adverse effects of humic acids on typical gene quantification methods, a new method, NanoGene assay, has been developed to quantify the bacterial gene. NanoGene assay differs from current methods by using a combination of magnetic beads (MB), dual quantum dot labels (QD565 and QD655), and a DNA hybridization. In this research, it was demonstrated that the NanoGene assay was more resistant than the real-time PCR assay to inhibition caused by humic acids spiked. The gene quantification by both assays was targeted at functional eaeA gene for E. coli O157:H7. The range of the humic acids tested was from 0.001 ng/μL to 100 ng/μL, which is the common concentration range of humic acids in the environment. At 10 ng/μL humic acid, real time PCR was inhibited to 0% of its quantification capability whereas NanoGene assay was able to maintain more than 70% of its quantification capability. Subsequently, the inhibitor resistant ability of the NanoGene assay for the humic acdis from soil samples was demonstrated. Three types of soil samples containing different amounts of humic acids were tested. The results showed the successful quantification of eaeA gene with the linear (R2 = 0.90) range of 4 × 104 through 4 × 108 CFU/g soil for both soils as well as the control soil. However, the real-time PCR assay showed complete inhibition for the two soils containing 0.4% and 1.5% humic acids. Interestingly, the real-time PCR assay failed even after additional purification methods were performed. The study demonstrated that the presented gene quantification method is suitable for the quantitative bacteria monitoring in the humic acid laden soils.