Optimization and Validation of Concentration-based Standard Curve for Quantification of Alexandrium spp. using eDNA

Abstract

In recent years, environmental DNA (eDNA) has emerged as a promising tool for detecting microorganisms in the natural environment. One specific application of eDNA technology is detecting and monitoring Harmful Algal Blooms (HABs), which can cause significant ecological and economic damage. In addition, quantitative analysis using eDNA is limited by the lack of optimized and validated Standard Curves. In this study, we aimed to optimize and validate a concentration-based Standard Curve for the quantitative analysis of HABs using eDNA. Instead of using DNA dilutions, which are commonly used in quantitative PCR (qPCR), the toxic red tide Alexandrium catenella was cultured and counted under a microscope, and Standard Curves were prepared by DNA extraction after dilution. By adjusting experimental conditions such as concentration, PCR amplification cycle, and specific primer design, we obtained an optimal Standard Curve with an R2 value close to 1. This Standard Curve showed similar results to those obtained using DNA dilution-based methods. Simultaneous quantification of eDNA from clonal Alexandrium catenella cultured in the laboratory and Alexandrium species from field samples showed a higher correlation with cell dilution than with DNA dilution. These results provide evidence that each Alexandrium spp. cells have a different amount of DNA. Consequently, when using a cell dilution-based Standard Curve to quantify Alexandrium spp. the Standard Curve is better determined by a dilution method based on cell counting. Overall, this study provides a useful method to optimize and validate concentration-based Standard Curves for eDNA-based quantitative analysis of HABs.