Environmental DNA (eDNA) analysis is effective for non-invasive biodiversity monitoring, revealing species distribution and abundance without ecosystem disruption. Concentration, extraction, and preservation are three essential steps in the eDNA analysis process. Among these, the concentration of eDNA has gained significant research interest, particularly due to the variability of water samples used in studies. To date, various methods for eDNA concentration have been developed, including glass fiber filtration, Sterivex filters, and passive samplers. However, no single method is universally applicable because of the variability in eDNA presence and water characteristics, such as turbidity. Therefore, the development of alternative eDNA concentration methods is crucial for advancing eDNA research. This research introduces QuickConc, a novel nucleic acid capture method that combines benzalkonium chloride (BAC) with dispersed glass fibers. Our results indicate that this approach enhances eDNA capture sensitivity by likely improving the interaction between silica and eDNA. QuickConc was tested in three environments, using metabarcoding and qPCR. Species-specific qPCR results showed that QuickConc detected 2 to 3 times higher copy numbers compared to glass fiber filter and Sterivex methods. Metabarcoding analyses using the MiFish method revealed that the number of fish species detected in river water was higher with QuickConc, compared to other methods, while in sea water, the number of fish species was at a similar level compared to other methods. QuickConc offers new options in eDNA analysis, providing a more sensitive and easily deployable approach to biodiversity monitoring and conservation strategies.

To evaluate the effect of different concentration methods on the number of detected species and community composition, metabarcoding analyses were conducted for fish eDNA. Our sampling sites included river and sea, but notably excluded pond samples from the sequencing process. The decision to omit pond samples was grounded in the site’s unique characteristics; originally a rice field situated in a segregated area, the pond exhibited a limited presence of fish species.

After DNA extraction, subsequent procedures of amplicon library preparation and NGS were performed by Bioengineering Lab. Co., Ltd (Kanagawa, Japan). The DNeasy PowerClean Pro Cleanup Kit (Qiagen) was used to remove PCR-inhibitory substances, and the elution of DNA was performed in a final volume of 50 μL. The library construction began with a two-step tailed PCR method using four primer sets (Table S3 in related publication), which were partially modified fish universal PCR primers, “MiFish,” developed by Miya et al. (2015) to amplify eDNA from fish species.

The first-round PCR reaction was conducted in a 10 μL mixture containing 0.375 U ExTaq HS (TaKaRa), 1× ExTaq Buffer, 0.2 mM of each dNTPs, 0.5 μM of each primer, 1 μL of eDNA template, and ultrapure water. The thermal cycle profile was 94 °C for 2 min, followed by 35 cycles of 20 s at 94 °C, 15 s at 65 °C, and 20 s at 72 °C, and a final extension of 5 min at 72 °C. After purification of the first PCR products using VAHTS DNA Clean Beads (Vazyme), the second-round PCR had a 10 μL total reaction volume, including 0.2 U KOD FX Neo (TOYOBO), 1 × PCR Buffer, 0.4 mM of each dNTPs, 0.25 μM each primer, 2 ng of the first PCR product, and ultrapure water.

The second PCR’s thermal cycles profile consisted of 94 °C for 2 min, 12 cycles at 98 °C for 10 s, 60 °C for 30 s, and 68 °C for 30 s, with a final extension at 68 °C for 2 min. PCR products were purified using VAHTS DNA Clean Beads (Vazyme), and then the paired-end sequence libraries were completed. The quality of the constructed library was verified using a fragment analyzer and the dsDNA 915 Reagent Kit (Agilent Technologies), after which sequencing was conducted on the MiSeq system using the MiSeq Reagent Kit v3 (Illumina) under conditions of 2 × 300 bp.

Only read sequences that were fully matched with primer sequences at the beginning were extracted using the fastq barcode splitter in the FASTX Toolkit (ver. 0.0.14). FASTX-Toolkit's fastx_trimmer was used to remove the primer sequences and the last 120 bases at the 3’ end from the extracted reads. Subsequently, sequences with a quality score below 20 were filtered out using Sickle (ver. 1.33), and any sequences shorter than 40 bases, along with their paired reads, were discarded. Following this, the DADA2 plugin in Qiime2 (ver. 2024.2) was used to remove chimeric and noisy sequences, after which representative sequences and an ASV table were generated. For MiFish analyses, the obtained ASV table was analyzed with USEARCH v 10.0.240 (Edgar 2010), according to the method demonstrated by Wu et al. (2021). The mitochondrial genome database MitoFish (ver. 3.90) was used to assign taxonomic names to ASVs.