[Protocol optimization of eDNA analysis workflow for detecting Hucho bleekeri.]

Area of Science:

• Aquatic ecology
• Molecular biology
• Conservation genetics

Background:

• Environmental DNA (eDNA) analysis is crucial for detecting rare aquatic species and mapping their distribution.
• Optimizing eDNA molecular workflows is essential to reduce uncertainty in species detection, particularly for rare species.

Purpose of the Study:

• To investigate the impact of filtering method, water sample volume, and DNA extraction protocols on the detection rate of Sichuan taimen (Hucho bleekeri) eDNA.
• To establish an optimized eDNA analysis protocol for detecting Sichuan taimen.

Main Methods:

• Collected water samples ranging from 250 mL to 2 L from a Sichuan taimen culture pond using filtration.
• Extracted and purified eDNA using either the DNeasy Tissue and Blood DNA extraction kit or the MoBio Power Water DNA extraction kit.
• Amplified the target gene from the mtDNA D-loop region of Sichuan taimen using PCR with specific primers.

Main Results:

• The DNeasy Tissue and Blood DNA extraction kit achieved a 100% detection rate, while the MoBio Power Water DNA extraction kit yielded a 0% detection rate.
• Larger water sample volumes improved PCR product yield, with 2 L being the most effective volume.
• Successful amplification of the target sequence from the mtDNA D-loop region confirmed the presence of Sichuan taimen DNA.

Conclusions:

• DNA extraction method and water sample volume significantly influence eDNA detection rates.
• A protocol combining filtration, a 2 L water sample volume, and the DNeasy Tissue and Blood DNA extraction kit is suitable for eDNA analysis.
• The mtDNA D-loop region is recommended for detecting Sichuan taimen DNA in water samples.