Method for rapidly identifying benthonic animals

Abstract

The invention discloses a benthonic animal rapid identification method, and relates to the technical field of benthonic animal identification, and the benthonic animal rapid identification method comprises the following steps: collecting sediments or a water sample from a benthonic environment, mixing the sediments with a buffer solution or filtering the water sample to obtain an eDNA crude extract; extracting nucleic acid from the eDNA crude extract by adopting a CTAB (Cetyltrimethyl Ammonium Bromide) extraction method, carrying out agarose gel electrophoresis detection, and diluting to a proper concentration; and carrying out high-fidelity PCR (Polymerase Chain Reaction) amplification by using a specific primer pair aiming at the 12SrRNA gene V05 region of the benthonic animal by taking the eDNA crude extract as a template. According to the invention, a benthonic animal specific 12S rRNA primer system and a standardized eDNA crude extract detection process are established, the benthonic animal DNA can be rapidly and stably identified in multiple environments, the annotation accuracy rate is more than 90%, the detection limit is as low as 1 individual / 10g sediment, the whole-process analysis can be completed in 3-5 days, and the monitoring efficiency and reliability are significantly improved.

Description

Technical Field

[0001] This invention relates to the field of benthic animal identification technology, and specifically to a rapid identification method for benthic animals. Background Technology

[0002] Benthic animals are a vital functional group in aquatic ecosystems. Their species composition and community structure are sensitive indicators of nutrient status, pollution gradients, and habitat integrity, and are therefore often used as core indicators for ecological assessment and environmental monitoring. Traditional monitoring relies primarily on morphological identification processes, requiring sorting, fixation, and laboratory microscopic observation after on-site sampling. Species identification is then performed by taxonomists with extensive experience based on morphological characteristics such as body segments, setae, and shells. In recent years, environmental DNA (crude eDNA extract) technology has provided a new pathway for non-destructive or minimally disturbed species detection: by extracting exfoliated DNA from water samples or sediments, combined with molecular amplification and high-throughput sequencing, community information can be obtained in a shorter timeframe, potentially expanding monitoring frequency and coverage.

[0003] The existing technology has the following shortcomings:

[0004] However, existing methods still have significant shortcomings when applied to benthic animals. On the one hand, morphological methods are cumbersome and time-consuming, and prone to misidentification or omission of juveniles, incomplete organisms, and morphologically similar species, heavily relying on scarce taxonomic talent. On the other hand, directly applying general animal barcodes (such as COIs) for eDNA crude extract metabolic barcode analysis often amplifies signals from non-target groups due to insufficient primer specificity. This, coupled with chimeras and non-specific amplification caused by the limited fidelity of conventional PCR enzymes, affects sequencing quality and interpretation accuracy. Existing bioinformatics processing also lacks standardized procedures and dedicated reference databases for benthic animals, and is insufficient in effectively removing contaminating sequences such as mitochondria and chloroplasts and in determining unified thresholds, further contributing to species annotation bias and poor reproducibility. Therefore, there is an urgent need to develop a rapid and accurate method for identifying benthic animals with highly specific primer systems, high-fidelity amplification strategies, and standardized data processing and contamination control rules.

[0005] The information disclosed in the background section is only intended to enhance the understanding of the background of this disclosure, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0006] The purpose of this invention is to provide a rapid identification method for benthic animals to solve the problems mentioned in the background art.

[0007] To achieve the above objectives, the present invention provides the following technical solution: a rapid identification method for benthic animals, comprising the following steps:

[0008] (1) Sample collection and pretreatment: Collect sediment or water samples from the benthic environment, mix the sediment with buffer solution or filter the water sample to obtain crude eDNA extract;

[0009] (2) Extraction and quality detection of crude eDNA extract: Nucleic acid was extracted from the crude eDNA extract using the CTAB extraction method, and then diluted to a suitable concentration after detection by agarose gel electrophoresis;

[0010] (3) Specific PCR amplification: Using the crude eDNA extract as a template, high-fidelity PCR amplification was performed using specific primer pairs targeting the V05 region of the benthic animal 12S rRNA gene to obtain the target amplified fragment.

[0011] (4) PCR product purification and mixing: The amplification products were subjected to electrophoresis detection and purification, and the sequencing samples were prepared by mixing them in equal amounts;

[0012] (5) Library construction and high-throughput sequencing: Sequencing libraries were prepared using a high-throughput sequencing library construction kit. After quality control and quantification, paired-end sequencing was performed on a next-generation sequencing platform.

[0013] (6) Standardized information analysis and species annotation: Import sequencing data into the analysis platform, perform noise reduction, classification, removal of contaminating sequences and comparison with benthic animal-specific 12S rRNA database, and output benthic animal species list and relative abundance information.

[0014] Preferably, the pretreatment method for sediment samples is as follows: take 5-10g of sediment, add buffer solution, shake to mix, centrifuge, and take the supernatant as the crude eDNA extract; the pretreatment method for water samples is as follows: collect water samples from a depth of 10-20cm, filter through a 0.22μm filter membrane, and then wash the filter membrane with buffer solution and sonicate, and take the resulting solution as the crude eDNA extract.

[0015] Preferably, the CTAB extraction method includes: adding an extraction buffer containing 2% CTAB, 100 mM Tris-HCl (pH 8.0), 20 mM EDTA, and 1.4 M NaCl, incubating at 65°C, and then extracting with chloroform-isoamyl alcohol, precipitating with isopropanol, and washing with ethanol to obtain crude eDNA extract.

[0016] Preferably, the specific primer pair has the following sequences: forward primer Benthos-12SV05-F: 5'-TAGAACAGGCTCCTCTAGT-3', reverse primer Benthos-12SV05-R: 5'-TTAGATACCCCACTATGCA-3', and the 5' end of the primer has a 6-12 bp barcode tag.

[0017] Preferably, the PCR reaction system is 25-35 μL, containing high-fidelity DNA polymerase, primers, template DNA and sterile water; the PCR cycling conditions include pre-denaturation, 30±5 cycles (annealing temperature 48-60℃), and final extension.

[0018] Preferably, the high-throughput sequencing platform is Illumina or an equivalent second-generation sequencing platform, the sequencing mode is paired-end sequencing, the read length is 2×150bp or its equivalent length, and the number of effective sequences per sample is not less than 50,000.

[0019] Preferably, the benthic animal-specific 12S rRNA database is established by screening 12S rRNA gene sequences from the phyla Annelida, Mollusca, Arthropoda, and Echinodermata from public databases and clustering them according to 99% sequence similarity.

[0020] Preferably, the information analysis is based on QIIME2 or a software platform with equivalent functionality, using a denoising plugin for sequence quality control and truncation, and a classification plugin for species annotation, with a confidence threshold of 0.8 to 0.9.

[0021] Preferably, contamination removal includes removing mitochondrial sequences, chloroplast sequences, and sequences detected in negative controls.

[0022] Preferably, the detection cycle is 3 to 5 days, the species annotation accuracy is not less than 90%, and the detection limit for low biomass species is not higher than 1 individual / 10g sediment.

[0023] The technical effects and advantages provided by the present invention in the above technical solution are as follows:

[0024] This invention achieves highly specific molecular identification of benthic animals by constructing a benthic animal-specific 12S rRNA primer system and an optimized eDNA crude extract amplification process. The primer design, optimized based on conserved fragments in the V05 region, accurately identifies benthic animal DNA in complex sample backgrounds, significantly reduces interference from non-target organisms, and produces single, highly reproducible amplified bands. This specific primer system stably obtains the target fragment in various environmental samples, with an annotation accuracy exceeding 90%, effectively improving the accuracy and reliability of benthic animal species identification.

[0025] This invention utilizes a standardized analysis workflow combining a high-fidelity enzyme system with the DADA2 denoising algorithm to optimize the entire process from sample extraction and amplification to sequencing annotation. The optimized high-fidelity amplification system significantly reduces the proportion of chimeras and false positives, and combined with standardized bioinformatics parameters, it can stably output high-quality sequence data. This workflow ensures minimal variability and high reproducibility between results, is suitable for analyzing various sample types, and can rapidly and accurately generate benthic animal community structure information, providing technical support for ecological assessment.

[0026] This invention significantly improves detection sensitivity and timeliness by optimizing the extraction method and detection process. The improved cDNA crude extract extraction step effectively removes sediment inhibitors, enabling efficient DNA recovery from low biomass samples, with a detection limit as low as 1 individual / 10g sediment. The entire process from sampling to species annotation can be completed within 3 to 5 days, significantly shortening the detection cycle compared to traditional methods. This provides rapid response capabilities for ecological monitoring and environmental management, and has significant application value. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0028] Figure 1 This is a flowchart of a rapid identification method for benthic animals according to the present invention. Detailed Implementation

[0029] Exemplary embodiments will now be described more fully with reference to the accompanying drawings. However, these exemplary embodiments can be implemented in many forms and should not be construed as limited to the examples set forth herein; rather, they are provided so that the description of this disclosure will be more complete and fully convey the concept of the exemplary embodiments to those skilled in the art.

[0030] This invention provides, for example Figure 1 The method for rapid identification of benthic animals shown includes the following steps:

[0031] Sample collection and pretreatment: Sediment or water samples were collected from the benthic environment. The sediment was mixed with buffer solution or the water sample was filtered to obtain crude eDNA extract.

[0032] eDNA crude extract extraction and quality detection: Nucleic acid was extracted from the eDNA crude extract using the CTAB extraction method, and after detection by agarose gel electrophoresis, it was diluted to a suitable concentration;

[0033] Specific PCR amplification: Using the crude eDNA extract as a template, high-fidelity PCR amplification was performed using specific primer pairs targeting the V05 region of the benthic animal 12SrRNA gene to obtain the target amplified fragment.

[0034] PCR product purification and mixing: The amplification products were subjected to electrophoretic detection and purification, and then mixed in equal volumes to prepare sequencing samples;

[0035] Library construction and high-throughput sequencing: Sequencing libraries were prepared using a high-throughput sequencing library construction kit, and after quality control and quantification, paired-end sequencing was performed on a next-generation sequencing platform;

[0036] Standardized information analysis and species annotation: Sequencing data is imported into the analysis platform, and noise reduction, classification, contamination sequence removal and comparison with benthic animal-specific 12S rRNA database are performed to output a list of benthic animal species and relative abundance information.

[0037] Example 1: Identification of benthic animals in freshwater sediments;

[0038] This embodiment aims to verify the applicability and accuracy of the rapid benthic animal identification method of the present invention in freshwater sediment samples. Environmental DNA (crude eDNA extract) was extracted from typical lake sediments, and amplified using benthic animal-specific 12S rRNA primers followed by high-throughput sequencing analysis to achieve rapid and accurate species annotation and diversity assessment. The experimental site was a typical freshwater lake (30°15'N, 120°30'E), which is significantly affected by surrounding agricultural non-point source pollution and seasonal eutrophication. Benthic animal diversity reflects the intensity of environmental stress and the health of the ecosystem. Sampling was conducted during the spring dry season when the lake surface was calm and the bottom water had high transparency, which was beneficial for sample stability control. Three surface (0-10cm) sediment samples were collected using a Petersen sediment sampler, each weighing approximately 5g, and immediately sealed in sterile 50mL centrifuge tubes. DNA-free gloves were worn during all operations, and sampling instruments were thoroughly cleaned with 10% bleach and sterile water, then wiped with ethanol to prevent exogenous DNA contamination. Samples were transported to the laboratory in a 4°C incubator within 24 hours. A negative control sample (empty tubes treated similarly) was included during the experiment to monitor for contamination risk.

[0039] In the sample processing stage, 5g of each sediment sample was added to 10mL of sterile PBS (pH 7.4) buffer. The mixture was vortexed for 10 minutes to fully release adsorbed or embedded free DNA fragments in the sediment. The supernatant was then collected by centrifugation at 8000rpm for 5 minutes to obtain crude eDNA extract. To ensure extraction efficiency, different combinations of PBS volume ratio, vortexing time, and centrifugation speed were compared. The results showed that 10mL PBS, 10 minutes of vortexing, and 8000rpm centrifugation yielded the highest concentration of supernatant DNA while maintaining DNA integrity. CTAB extraction was used to remove common inhibitors such as polysaccharides and humic acids from the sediment. The supernatant was mixed with an equal volume of 2×CTAB extraction buffer (containing 2% CTAB, 100mM Tris-HCl, pH 8.0, 20mM EDTA, and 1.4M NaCl), and incubated in a 65℃ water bath for 30 minutes, gently inverting every 10 minutes to promote complete cell wall lysis and the formation of a complex with CTAB. Then, an equal volume of chloroform-isoamyl alcohol (24:1 v / v) was added, and the mixture was gently inverted for 10 minutes to denature the protein and remove impurities. After centrifugation at 12,000 rpm for 10 minutes, the supernatant was collected. To further purify the DNA, 0.8 v / v of isopropanol was added to the supernatant, and the mixture was incubated at −20°C for 30 minutes to promote DNA precipitation. The precipitate was then recovered by centrifugation at 12,000 rpm for 15 minutes. The precipitate was washed twice with 75% ethanol to remove salts and residual CTAB, air-dried at room temperature, and then dissolved in 50 μL of sterile water. The purity of the extracted crude eDNA was determined using a NanoDrop 2000 spectrophotometer (A260 / A280 value 1.78–1.82). 1% agarose gel electrophoresis showed a single main band with no significant degradation or extraneous bands, indicating good DNA integrity. To meet the concentration requirements for downstream PCR reactions, the crude eDNA extract was diluted to 1 ng / μL and stored at 4°C for short-term use.

[0040] In the specific amplification stage, PCR was performed targeting the V05 variable region of the evolutionarily conserved 12S rRNA gene in benthic animals using specific primer pairs designed in this invention. The forward primer sequence was 5'-TAGAACAGGCTCCTCTAGT-3' (Benthos-12SV05-F), and the reverse primer was 5'-TTAGATACCCCACTATGCA-3' (Benthos-12SV05-R). Both primers had an 8bp barcode tag (e.g., ACGTTGAT) at the 5' end for sample differentiation and subsequent sequence splitting. The total volume of the PCR reaction system was 30 μL, including 15 μL of 2×Phusion High-Fidelity PCR Master Mix with GC Buffer, 1 μL each of the forward and reverse primers (1 μM / μL), 10 μL of crude eDNA template extract (1 ng / μL), and 2 μL of sterile water. A Bio-Rad T100 PCR instrument was used. The program was set as follows: 98℃ pre-denaturation for 1 minute, followed by 30 cycles (98℃ denaturation for 10 seconds, 50℃ annealing for 30 seconds, 72℃ extension for 30 seconds), and a final extension at 72℃ for 5 minutes. After PCR, 5 μL of the product was electrophoresed on a 2% agarose gel (120V, 25 minutes). The target band was clear, approximately 200 bp in size, corresponding to the benthic animal 12S rRNA V05 region, with no obvious non-specific amplification or primer dimers. Repeat experiments (n=3) showed good consistency of the amplified products, indicating high primer specificity and system stability.

[0041] PCR products were purified using the TianGen Universal DNA Purification and Recovery Kit, following the manufacturer's instructions: the mixed product was added to 5 volumes of binding buffer, transferred to an adsorption column, centrifuged, and the solution discarded. After two washes, the DNA was finally eluted with 30 μL of sterile water. The resulting product concentration was approximately 20 ng / μL after quantification using Qubit. 100 ng of PCR product from each sample was mixed in equal volumes to ensure balanced representativeness in the subsequent library. Library construction was performed using the NEBNext UltraDNA Library Prep Kit, including end repair, A-tailing, adapter ligation, and library amplification. The library amplification reaction was performed in a 25 μL system, with 12 cycles. The library fragment size was determined to be 260-280 bp using an Agilent 5400 Bioanalyzer, and the concentration was 15 nM, meeting the requirements for the Illumina platform.

[0042] Sequencing was performed on the Illumina MiSeq platform using 2×150bp paired-end sequencing mode, generating raw FastQ data files. An average of 53,000 valid sequences were obtained per sample, for a total of approximately 159,000 sequences. To prevent cross-contamination and bias, independent tagging and PhiX control contamination were used throughout the sequencing process. No amplification signals were detected in the negative control samples.

[0043] The obtained data underwent standardized analysis on the QIIME2 platform. First, the fastq file was imported into QZA format using the qiimetoolsimport plugin. Then, denoising and splicing were performed using the qiimedada2denoise-paired plugin with parameters set to --p-trim-left-f10, --p-trim-left-r10, --p-trunc-len-f140, and --p-trunc-len-r140 to remove low-quality bases and chimeric sequences. The representative ASV sequences were aligned to the benthic 12S rRNA database constructed in this invention (clustered at 99% similarity, including reference sequences from annelids, mollusks, arthropods, and echinoderms) using the qiimefeature-classifierclassify-sklearn plugin, with a confidence threshold of 0.8. Finally, sequences detected in mitochondria, chloroplasts, and negative controls were removed using the qiimefeature-tablefilter-features plugin. After standardization and contamination removal, a total of 128 ASVs were obtained, with an average length of 198 bp.

[0044] Species annotation results show that the main taxa in the sample include Annelida (42%), with Oligochaeta (e.g., *Tubifex*) and Hirudo (e.g., *Hirudo*) being the dominant groups; Molluscaria (35%), mainly Gastropoda (*Cipangopaludina*) and Bivalvia (*Anodonta*); and Arthropoda (23%), mainly Crustacea (*Gammarus*) and a small number of aquatic insect larvae (e.g., Chironomidae). Compared with the morphological identification results (28 species detected), this method additionally detected 6 species with low abundance or larval stages, including morphologically difficult-to-identify oligochaetes and small snails, indicating that the method of this invention has significant advantages in the comprehensive identification of benthic communities and the detection of low-abundance species. Statistical analysis shows that the species annotation accuracy rate reaches 92%, the results are highly consistent with the morphological classification results, and the deviation of the biodiversity index (Shannon index) is less than 5%, indicating that the method of the present invention has good accuracy and repeatability.

[0045] The entire process from sampling to result output took 4 days, including approximately 6 hours for sample pretreatment and DNA extraction, 5 hours for PCR amplification and purification, 10 hours for library construction and sequencing preparation, and 1 day for bioinformatics analysis and result annotation. Compared to traditional morphological methods (which typically take 7-14 days), the detection cycle is shortened by approximately 60-70%. Furthermore, this method uses a non-destructive cDNA crude extract, eliminating the need for collecting intact organisms and significantly reducing ecological disturbance. Overall, this embodiment fully verifies the feasibility, accuracy, and stability of the rapid benthic animal identification method described in this invention in freshwater environments, demonstrating that this method possesses high efficiency, sensitivity, and standardization advantages in ecological monitoring and biodiversity assessment, and can replace or supplement traditional morphological methods for benthic animal community monitoring.

[0046] Example 2: Identification of benthic animals in seawater sediment;

[0047] This embodiment was used to verify the applicability, accuracy, and sensitivity of the rapid identification method for benthic animals of the present invention in marine sediment samples. The focus was on examining the method's performance in high-salinity, high-organic-matter environments, including the efficiency of crude eDNA extraction, PCR amplification stability, and species annotation accuracy. The experimental site was a typical intertidal zone along the coast (29°50′N, 122°10′E), an area strongly influenced by tidal changes and land-sea interactions, resulting in a complex benthic animal community comprising various annelids, mollusks, arthropods, and echinoderms. To minimize seasonal and human interference, samples were collected during low tide (mid-May) at an ambient temperature of approximately 21°C, a surface seawater salinity of 28‰, and a dissolved oxygen concentration of 7.3 mg / L.

[0048] Three samples of surface sediment (0-10 cm) were collected from the intertidal zone using a Petersen sediment sampler, each approximately 10 g in size. The samples were immediately transferred to sterile 50 mL centrifuge tubes, sealed, and pre-cooled in a portable liquid nitrogen container before being transported to the laboratory at −20°C (storage time less than 72 hours). To avoid cross-contamination, the sampling equipment was immersed in a 10% sodium hypochlorite solution for 10 minutes and repeatedly rinsed with sterile seawater after each use. The upper layer of sediment from each sample was used for DNA extraction, while the lower coarse-particle portion was discarded to minimize the impact of inorganic impurities on extraction efficiency.

[0049] In the sample pretreatment stage, 10 mL of sterile seawater buffer (containing 0.9% NaCl, pH 7.8) was added to each sediment sample, and the mixture was vortexed for 10 minutes to ensure thorough mixing and release of cell debris and free DNA into the solution. The solid and liquid phases were then separated by centrifugation at 8000 rpm for 5 minutes, and the supernatant was used as the crude eDNA extract. To improve DNA recovery, PBS and sterile seawater buffer systems were compared. The results showed that seawater buffer effectively maintained DNA stability and reduced precipitation and adsorption losses under high-salt conditions. The obtained crude eDNA extract was clear and colorless, with no obvious suspended matter, indicating sufficient pretreatment.

[0050] The DNA extraction procedure follows the CTAB extraction method described in Example 1 exactly. A brief description is as follows:

[0051] Add an equal volume of 2×CTAB extraction buffer (containing 2% CTAB, 100 mM Tris-HCl pH 8.0, 20 mM EDTA, and 1.4 M NaCl) to the supernatant, and incubate at 65°C for 30 minutes to lyse cell membranes and bind impurities. Add an equal volume of chloroform-isoamyl alcohol (24:1), gently invert for 10 minutes, centrifuge at 12000 rpm for 10 minutes, collect the supernatant, add 0.8 volumes of isopropanol to precipitate DNA, incubate at −20°C for 30 minutes, and then centrifuge to recover the DNA precipitate. Wash twice with 75% ethanol, air dry, and dissolve in 50 μL of sterile water. The purity of the obtained crude eDNA extract was determined by NanoDrop (A260 / A280 value 1.79-1.85), with a concentration between 8-10 ng / μL. 1% agarose gel electrophoresis showed a single master band with no obvious degradation bands, indicating good DNA quality suitable for downstream PCR amplification. The crude eDNA extract was diluted to 1 ng / μL and stored at 4°C for later use. No DNA bands were detected in the negative control sample (containing only sterile seawater buffer) after the same procedure, indicating that the experiment was uncontaminated.

[0052] The PCR amplification stage used the benthic animal-specific 12S rRNA gene V05 region primer pair designed in this invention, with the forward primer Benthos-12SV05-F (5'-TAGAACAGGCTCCTCTAGT-3') and the reverse primer Benthos-12SV05-R (5'-TTAGATACCCCACTATGCA-3'). The 5' ends of the primers each carried different barcode tags (CGTACGCT, GATCAGCT, etc.) for sample differentiation. The total volume of the PCR reaction system was 30 μL, including 15 μL of 2×Phusion High-Fidelity PCR Master Mix, 1 μL of each primer (1 μM / μL), 10 μL of crude eDNA extract template (1 ng / μL), and 2 μL of sterile water. Amplification was performed using a Bio-Rad T100 PCR instrument. The program was as follows: 98℃ pre-denaturation for 1 minute, 30 cycles (98℃ denaturation for 10 seconds, 50℃ annealing for 30 seconds, 72℃ extension for 30 seconds), and a final extension at 72℃ for 5 minutes. After PCR, 5 μL of the reaction product was electrophoresed on a 2% agarose gel (120V, 25 minutes). The results showed that the target band was located at approximately 200 bp, with no obvious non-specific amplification or primer dimers. Compared with the universal COI primer control group, the primers of this invention have higher amplification efficiency and more concentrated bands.

[0053] PCR products were purified using the TianGen Universal DNA Purification and Recovery Kit: After mixing, the PCR products were added to 5 volumes of binding buffer, loaded onto a column, centrifuged at 12000 rpm for 30 seconds, and the waste liquid was discarded. After two rinses, the empty column was centrifuged for 2 minutes to remove residual liquid, and then eluted with 30 μL of sterile water. The resulting product concentration was approximately 22 ng / μL, quantified using Qubit. 100 ng of product was taken from each sample and mixed in equal volumes to reduce sequencing bias. Library construction was performed using the NEBNext UltraDNA Library Prep Kit, including end repair, A-tailing, adapter ligation, and library amplification. The amplification cycle was 12 times. Analysis using an Agilent 5400 Bioanalyzer showed that the library fragments were concentrated around 270 bp, and the Q-PCR quantification concentration was 20 nM, meeting the requirements for sequencing.

[0054] Sequencing was performed using the Illumina NovaSeq platform in 2×150bp paired-end sequencing mode with a single lane loading of 10Gbp, yielding approximately 100,000 valid sequences per sample. Sequencing quality control showed an average Q30 value of 94.2%, indicating high data quality. To prevent cross-contamination, sample barcodes were split before sequencing to verify the absence of duplicate tags. The resulting FASTQ files were analyzed on the QIIME2 (2019.1) platform. First, the data was imported using the qiimetoolsimport plugin and converted to QZA format. Then, the qiimedada2denoise-paired plugin was used for denoising, splicing, and quality trimming, with parameters set to --p-trim-left-f10, --p-trim-left-r10, --p-trunc-len-f140, and --p-trunc-len-r140 to obtain high-quality signature sequences (ASVs). Species annotation was performed using the qiimefeature-classifierclassify-sklearn plugin, aligning representative sequences to the benthic 12S rRNA database established in this invention. This database is based on the NCBInt dataset, and 12S rRNA sequences from annelids, mollusks, arthropods, and echinoderms were screened and clustered according to 99% similarity. The classification confidence threshold was set to 0.8, and contaminating sequences detected in mitochondria, chloroplasts, and negative controls were removed.

[0055] The analysis revealed 156 ASVs with an average sequence length of 198 bp. Annotation results showed that the main species composition of the samples included: 28% echinoderms (primarily *Apostichopus* and *Strongylocentrotus*); 32% arthropods (primarily *Eriocheir* and *Penaeus*); 30% mollusks (e.g., *Ruditapes* and *Sinonovacula*); and 10% annelids (primarily *Nereis*). Compared with traditional morphological identification results conducted at adjacent sites during the same period (detecting 52 benthic animal species), this method detected 68 species, an increase of 16 species compared to traditional methods. These were mainly concentrated in microscopic or larval stages of arthropods and mollusks, demonstrating a significant sensitivity advantage.

[0056] Further statistical analysis showed that the species annotation accuracy of this method reached 93%, with good repeatability (similarity index between samples > 0.9), indicating that the method can work stably even in complex marine sedimentary environments. In addition to accuracy and sensitivity, this method also has a significant advantage in terms of operation time: from sample collection to species catalog output, it only takes about 5 days, including about 7 hours for DNA extraction and purification, about 5 hours for PCR amplification and purification, about 10 hours for library construction and sequencing preparation, and about 36 hours for sequencing and bioinformatics analysis. Compared with traditional morphological methods that take 10–14 days, this method is nearly three times more efficient.

[0057] Furthermore, this embodiment evaluated the inhibition resistance of sediment samples to high salinity and high organic matter content. Comparison of amplification performance between high-fidelity Phusion enzyme and conventional Taq enzyme revealed that Phusion enzyme exhibits strong tolerance to the inhibitory effects of residual salt and humic acid in seawater samples, demonstrating high amplification efficiency and good product purity. The DADA2 denoising algorithm effectively eliminated chimeras (chimerism rate <2%), ensuring sequencing accuracy. These results demonstrate that the rapid benthic animal identification method provided by this invention possesses excellent operability, accuracy, and inhibition resistance in both intertidal and high-salinity sediment samples. Its highly standardized and reproducible workflow makes it suitable for various applications, including marine ecological surveys, nearshore pollution monitoring, and long-term benthic community change studies, providing a reliable molecular tool for establishing a unified marine biodiversity monitoring system.

[0058] Example 3: Validation and comparative analysis of benthic animal-specific primers;

[0059] This embodiment aims to verify the specificity, coverage, and superior application of the benthic animal-specific primer pair (Benthos-12SV05-F / R) designed in this invention for the detection of metabolic barcoding in benthic environmental DNA (crude eDNA extract). It also systematically compares the primers with the commonly used international animal DNA barcoding primers, specifically the COI gene primers (LCO1490 / HCO2198), evaluating their performance differences in terms of molecular amplification characteristics, sequencing accuracy, species annotation efficiency, and anti-interference ability. This further demonstrates the innovation and reliability of the primer system of this invention in the rapid identification of benthic animals.

[0060] The experimental samples were obtained from benthic sediment samples from a typical freshwater-estuary transition zone. This area has a complex habitat, a significant salinity gradient, and a rich diversity of benthic animals, covering major groups such as annelids, mollusks, arthropods, and echinoderms, which can comprehensively reflect the species coverage performance of the primers. Sampling methods followed the standard procedures of Examples 1 and 2: five 10g samples of surface (0-10cm) sediment were collected using a Petersen sediment sampler, placed in sterile 50mL centrifuge tubes on-site, pre-cooled in liquid nitrogen, and then transported to the laboratory for storage at −20℃ (storage time not exceeding 48 hours). All samples included negative controls (empty tubes) to detect environmental or experimental contamination. Each sample was pretreated with PBS and extracted using the CTAB method to obtain a high-quality crude eDNA extract. Electrophoresis showed a clear main DNA band with an A260 / A280 ratio between 1.78 and 1.82, meeting the requirements for PCR amplification.

[0061] To ensure the reproducibility and fairness of the comparative experiments, the PCR system and reaction conditions were kept completely consistent between the COI universal primer set and the Benthos-12SV05 specific primer set, with only the primer sequences being changed. The Benthos-12SV05-F sequence is 5'-TAGAACAGGCTCCTCTAGT-3', and the Benthos-12SV05-R sequence is 5'-TTAGATACCCCACTATGCA-3'; the COI universal primer LCO1490 sequence is 5'-GGTCAACAAATCATAAAGATATTGG-3', and the HCO2198 sequence is 5'-TAAACTTCAGGGTGACCAAAAAATCA-3'. The PCR reaction volume was 30 μL, containing 15 μL of Phusion® High-Fidelity PCR Master Mix, 1 μL of each primer (1 μM / μL), 10 μL of template DNA (1 ng / μL), and 2 μL of sterile water. The reaction program was set as follows: 98℃ pre-denaturation for 1 min, 30 cycles (98℃ denaturation for 10 s, 50℃ annealing for 30 s, 72℃ extension for 30 s), and 72℃ extension for 5 min. All PCR reactions were performed on the same model of PCR instrument (Bio-RadT100) to ensure consistent temperature control.

[0062] Amplification results were detected by 2% agarose gel electrophoresis (120V, 25min). The results showed that the Benthos-12SV05 primer amplification product exhibited a single, uniform band with a target fragment size of approximately 200bp, corresponding to the V05 region of the 12S rRNA gene, and showed no obvious non-specific bands or primer dimers. In contrast, the COI universal primer product displayed multiple non-specific bands, with some amplified fragments ranging from 400-700bp in length, indicating a significant amount of non-target amplification in complex benthic samples. Further replicate experiments (n=5) yielded consistent results: the Benthos-12SV05 primer amplification showed high reproducibility (CV < 5%), while the COI primer amplification efficiency fluctuated significantly (CV ≈ 15%), indicating that the Benthos-12SV05 system had better stability.

[0063] The amplified products from both groups were then purified using the TianGen Universal DNA Purification and Recovery Kit. Library construction was performed using the NEBNext UltraDNA Library Prep Kit. Analysis using an Agilent 5400 microarray showed that the fragments in the Benthos-12SV05 group were concentrated in the 260-280 bp range, while the fragments in the COI group were more widely distributed (250-700 bp). Sequencing on the Illumina MiSeq platform (2 × 150 bp) yielded 163,000 original sequences for the Benthos-12SV05 group and 148,000 for the COI group. The sequencing data were uniformly processed using the QIIME2 platform (version 2019.1), with noise reduction and assembly performed using the DADA2 plugin, with consistent parameters (--p-trim-left-f10, --p-trim-left-r10, --p-trunc-len-f140, --p-trunc-len-r140). The number of valid ASVs obtained were 147 for the Benthos-12SV05 group and 118 for the COI group. Species annotation was performed using the feature-classifierclassify-sklearn plugin. The Benthos-12SV05 group used the benthic animal-specific 12S database constructed in this invention, while the COI group used the internationally recognized COI database (BOLD System Reference Set).

[0064] Analysis showed that in the Benthos-12SV05 annotated sequences, benthic animal sequences accounted for 94.7% of the total effective sequences, with annelids accounting for 39.8%, mollusks for 31.4%, arthropods for 20.7%, and echinoderms for 8.1%; non-target groups (such as fish, zooplankton, and protozoa) accounted for only 5.3%. In contrast, in the COI primer set, benthic animal sequences accounted for only 61.2%, while non-target groups accounted for as much as 38.8%, with fish DNA accounting for approximately 15% and planktonic DNA accounting for approximately 12%, indicating that universal primers have significant non-specific amplification problems in the context of complex environmental DNA. Furthermore, at the species annotation level, the Benthos-12SV05 group can accurately identify 67 species at the species level, while the COI group only identified 45 species. Among the newly detected species in the Benthos-12SV05 group are several oligochaetes (such as Tubifex tubifex, Limnodrilushoffmeisteri), mollusks (such as Cipangopaludinachinensis, Bellamyapurificata), and echinoderms (such as Asterinapectinifera), which are difficult to distinguish by traditional morphology.

[0065] To further verify the primer coverage, the Benthos-12SV05 primers designed in this invention showed high conservation of their annealed regions in annelids, mollusks, arthropods, and echinoderms in database comparisons, matching sequences of over 95% of common benthic species, while the COI primers only matched approximately 78%. Especially in small or cryptic benthic animals (such as small oligochaetes and microcrustaceans), the Benthos-12SV05 primers exhibited a higher amplification success rate. Combined with GC content analysis, the average GC content of the Benthos-12SV05 amplified fragment was 45.2%, which is closer to the physicochemical characteristics of the benthic mitochondrial 12S gene region than the COI amplified fragment (38.7%), thus demonstrating better amplification compatibility and thermostability.

[0066] Non-target amplification analysis showed that the non-target sequences amplified by the Benthos-12SV05 primers mainly originated from residual DNA from planktonic organisms in the water, while the non-target sequences amplified by the COI primers included not only planktonic DNA but also a large amount of fish and insect DNA, indicating that universal primers lack sufficient taxonomic constraints in benthic environments. The primers of this invention, through systematic alignment and annealing site optimization of specific regions in the 12S rRNA gene V05 region, achieve a balance between high specificity and broad coverage while considering conservation and diversity, avoiding cross-phylum non-specific amplification.

[0067] In addition, to evaluate the anti-inhibition performance and accuracy of the two primers, a spiked validation experiment was conducted in this embodiment: known numbers (1, 5, and 10 individuals) of Tubifex oligochaete DNA standards were added to sediment samples to test the detection limit. The results showed that the Benthos-12SV05 primer could stably detect the target sequence at the lowest spiked concentration (equivalent to 1 individual / 10g sediment), with a high signal-to-noise ratio and a detection limit ≤ 1 individual / 10g; while the COI primer had a minimum detection limit of 5 individuals / 10g under the same conditions, indicating significantly lower sensitivity.

[0068] Statistical analysis of species composition similarity indices among different samples revealed high species composition consistency in the Benthos-12SV05 group (Bray-Curtis similarity > 0.9), while the COI group showed a similarity of 0.74, indicating better reproducibility and less background interference in the amplification results obtained by the primers of this invention. Further calculation of amplification efficiency (product concentration / reaction cycle ratio) showed an efficiency of 0.85 for the Benthos-12SV05 group and 0.63 for the COI group, demonstrating that the primers of this invention can obtain high-quality products with fewer cycles, saving reaction time and reducing the risk of chimera formation. After denoising using the DADA2 algorithm, the chimera ratio of the primer amplification products of this invention was less than 2%, while it exceeded 7% in the COI group, showing a significant advantage in data quality for the system of this invention.

[0069] In summary, the results of this embodiment fully demonstrate that the Benthos-12SV05 primer pair designed in this invention has the following significant advantages compared with traditional universal COI primers: (1) Higher specificity: the proportion of target group sequences is increased by about 35%, and non-target amplification is significantly reduced; (2) Stronger coverage: it can cover more than 95% of common species in the four major phyla of benthic animals; (3) Higher sensitivity: the detection limit is as low as 1 individual / 10g sediment; (4) Better data quality: low chimera ratio and high ASV stability; (5) Better reproducibility and applicability: high consistency of results among different samples, applicable to various environments such as freshwater, estuaries and oceans. Therefore, the primer system of this invention not only overcomes the defects of universal COI primers in benthic environments, which are easily affected by non-target interference and have serious amplification bias, but also achieves high specificity, high accuracy and rapid identification of benthic animal species through standardized PCR and bioinformatics processing procedures, providing an efficient and reliable molecular tool for ecological monitoring, pollution assessment and biodiversity research.

[0070] Example 4: Verification of adaptability and sensitivity in multiple environments;

[0071] This embodiment aims to systematically evaluate the universality, sensitivity, repeatability, and environmental adaptability of the proposed rapid identification method for benthic animals in different ecological environments. Parallel comparative experiments were conducted in three typical ecosystems: freshwater lakes, estuarine wetlands, and nearshore marine areas. These experiments verified the detection performance and stability of the method under conditions of significant differences in water physicochemical properties, substrate composition, and microbial background. Furthermore, the detection sensitivity was quantitatively evaluated through artificial spiked experiments. The experimental design fully considered environmental gradient differences to demonstrate that the method of this invention can maintain high specificity, high accuracy, and high repeatability under various environmental conditions.

[0072] The experimental area selected three representative environmental types: ① Freshwater lake environment – ​​a medium-sized freshwater lake in western China (30°18′N, 119°53′E), with high water transparency, moderate eutrophication, and a sedimentary bed dominated by silt; ② Estuarine wetland environment – ​​the mouth of the Qiantang River (30°02′N, 121°36′E), characterized by a significant salinity gradient, strong tidal dynamics, and complex sediment particle composition; ③ Nearshore marine environment – ​​the intertidal zone off the Zhoushan Islands (29°45′N, 122°06′E), with a seawater salinity of approximately 30‰ and a sedimentary bed dominated by a mixture of sand and mud. Three sediment samples and three overlying water samples were collected from each environmental type, for a total of 18 samples. All sampling was conducted at low tide to ensure sufficient exposure of the benthic habitat. Sampling tools included a Petersen sediment sampler and sterile water sampling bottles. To prevent cross-contamination, all sampling instruments were soaked in 10% bleach for 10 minutes and rinsed with sterile water after each sampling. All samples were transported to the laboratory under refrigeration on-site. Sediment samples were stored at 4°C, and water samples were filtered through a 0.22 μm filter membrane and then frozen at −20°C.

[0073] Sample pretreatment was performed according to the steps of this invention. For sediment samples, 5-10g of each sample was added to 10mL of sterile PBS buffer, vortexed for 10 minutes, and then centrifuged at 8000rpm for 5 minutes. The supernatant was used as the crude eDNA extract. For water samples, 5mL of PBS buffer was added to the filter membrane, and the samples were sonicated for 5 minutes (300W power, 3s interval). The supernatant was collected as the crude extract. The crude eDNA extract was extracted using the CTAB extraction method. An equal volume of 2×CTAB buffer (containing 2% CTAB, 100mM Tris-HCl pH 8.0, 20mM EDTA, and 1.4M NaCl) was added, and the samples were incubated at 65℃ for 30 minutes. Then, chloroform-isoamyl alcohol extraction was performed, followed by isopropanol precipitation. After washing twice with 75% ethanol, the eDNA was dissolved in 50μL of sterile water. The DNA purity was determined by NanoDrop, with an A260 / A280 ratio between 1.78 and 1.85 and a concentration of 5-9ng / μL, meeting the PCR amplification standards. No DNA bands were detected in the negative control (empty tube), indicating that there was no contamination during the experiment.

[0074] PCR amplification used the benthic animal-specific primers Benthos-12SV05-F (5'-TAGAACAGGCTCCTCTAGT-3') and Benthos-12SV05-R (5'-TTAGATACCCCACTATGCA-3') designed in this invention. The 5' ends of the primers were marked with different 8bp barcode tags to distinguish samples. The reaction system consisted of 30 μL, containing 15 μL of Phusion® High-Fidelity PCR Master Mix, 1 μL of each primer (1 μM / μL), 10 μL of template DNA (1 ng / μL), and 2 μL of sterile water. The reaction program was: 98℃ pre-denaturation for 1 minute, 30 cycles (98℃ for 10 seconds, 50℃ for 30 seconds, 72℃ for 30 seconds), and extension at 72℃ for 5 minutes. The PCR products were detected by 2% agarose gel electrophoresis. All three environmental samples yielded a single, clear target band, approximately 200 bp in size, indicating good primer specificity and no non-specific amplification. The results of five repeated amplification experiments were consistent, with a coefficient of variation of less than 5%, indicating that the system has excellent repeatability.

[0075] PCR products were purified using the TianGen kit and libraries were constructed using the NEBNext UltraDNA Library Prep Kit. Analysis using an Agilent 5400 Bioanalyzer showed that the library fragments were concentrated between 260-280 bp, with Q-PCR quantification concentrations between 15-20 nM. Sequencing was performed on the Illumina MiSeq platform using 2×150 bp paired-end sequencing mode, yielding 50,000-120,000 valid sequences per sample. Sequencing quality analysis showed that the Q30 ratio was greater than 94%, indicating excellent data quality.

[0076] Data processing was performed on the QIIME2 (2019.1) platform. First, the fastq file was imported using the qiimetoolsimport plugin. DADA2 was used for quality control and denoising with the parameters --p-trim-left-f10, --p-trim-left-r10, --p-trunc-len-f140, and --p-trunc-len-r140. The obtained ASV sequences were aligned to the benthic 12S rRNA database established in this invention (clustered at 99% similarity, covering four major groups: annelids, mollusks, arthropods, and echinoderms) using the feature-classifierclassify-sklearn plugin, with a confidence threshold set to 0.8. Chloroplast, mitochondrial, and negative control sequences were removed using qiimefeature-tablefilter-features.

[0077] The analysis results show that high-quality target amplification fragments and effective sequences can be stably obtained in all three types of environments, with the number of effective sequences ranging from 50,000 to 120,000 across samples. In freshwater lake samples, annelids accounted for approximately 45%, mollusks 35%, and arthropods 20%; in estuarine wetland samples, arthropods increased to 33%, mollusks 29%, annelids 28%, and echinoderms 10%; in nearshore marine samples, echinoderms increased to 26%, arthropods 32%, mollusks 30%, and annelids 12%. The average species annotation accuracy reached 92%-95% in all three environments, demonstrating that the method maintains high stability and specificity in different salinity and substrate environments. Compared with traditional morphological identification results, the number of benthic animal species detected by this method increased by an average of 27%, especially in low-abundance groups (such as micro-annelids and juvenile crustaceans), indicating that the method has high sensitivity.

[0078] To further verify the sensitivity and quantitative capability of the method, artificial spiking experiments were conducted in a freshwater lake environment. One, five, and ten individuals of the oligochaete Tubifex were added to sterilized sediment samples with a known absence of benthic fauna, with each group repeated in triplicate. Crude eDNA was extracted and amplified using the procedures outlined in this invention. Results showed that even at the lowest spiking level (1 individual / 10g sediment), the Benthos-12SV05 primers could stably detect the target sequence, with consistent results in replicate samples and a signal-to-noise ratio >10. The detection limit was ≤1 individual / 10g sediment, an improvement of 1-2 orders of magnitude compared to traditional morphological methods. The concentration of the amplified product showed a good linear relationship with the spiking amount (R²=0.983), indicating that the method of this invention has high sensitivity and quantitative potential under low biomass conditions.

[0079] Furthermore, to evaluate the reproducibility and cross-environmental stability of the method, five independent replicate tests (independent extraction, amplification, and sequencing) were performed on three types of environmental samples. The calculated coefficients of variation (CV) were all less than 5%, indicating that the method has excellent consistency in terms of operation and results. Differential analysis showed that environmental salinity, pH, and organic matter content had no significant effect on amplification efficiency (P>0.05), indicating that the system has strong resistance to environmental interference and broad applicability.

[0080] By comparing the sample structure and diversity indices of the three environmental types, it was found that the species diversity index (Shannon-Wiener index) detected by the method of this invention was highly correlated with the morphological results (r=0.91), verifying the reliability of its ecological interpretation. Furthermore, based on the results of principal component analysis (PCA), the community structure among samples from different environments can be clearly distinguished, indicating that this method can be used for benthic community difference analysis and ecosystem health assessment under environmental gradients.

[0081] In summary, the rapid identification method for benthic animals of this invention exhibits high stability, versatility, and sensitivity in three typical environments: freshwater, estuary, and marine. This method can complete the entire process from sampling to species annotation in a short time (3-5 days), offering significant advantages such as ease of operation, short timeframe, high sensitivity, and strong reproducibility. Regardless of whether in freshwater or high-salinity environments, this method can effectively amplify target fragments and accurately annotate major benthic animal groups, providing efficient and reliable technical support for diversity monitoring, pollution assessment, and ecological restoration research.

[0082] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. A rapid identification method for benthic animals, characterized in that, Includes the following steps: (1) Collect sediments or water samples in a benthic environment, mix the sediments with buffer solution or filter the water samples to obtain crude eDNA extract; (2) Nucleic acid was extracted from the crude eDNA extract using the CTAB extraction method, and then diluted to a suitable concentration after detection by agarose gel electrophoresis; (3) Using the crude eDNA extract as a template, high-fidelity PCR amplification was performed using a specific primer pair targeting the V05 region of the benthic animal 12S rRNA gene to obtain the target amplified fragment; (4) The amplification products were subjected to electrophoretic detection and purification, and the sequencing samples were prepared by mixing them in equal amounts; (5) Use a high-throughput sequencing library construction kit to prepare sequencing libraries. After quality control and quantification, perform paired-end sequencing on a next-generation sequencing platform. (6) Import the sequencing data into the analysis platform, perform noise reduction, classification, removal of contaminating sequences and comparison with the benthic animal-specific 12S rRNA database, and output the benthic animal species list and relative abundance information.

2. The rapid identification method for benthic animals according to claim 1, characterized in that, In step (1), the pretreatment method for sediment samples is as follows: take 5-10g of sediment, add buffer solution, shake to mix, centrifuge, and take the supernatant as the crude eDNA extract. The water sample pretreatment method is as follows: Water samples were collected from a depth of 10–20 cm, filtered through a 0.22 μm filter membrane, and then the filter membrane was eluted with buffer and sonicated. The resulting solution was used as the crude eDNA extract.

3. The rapid identification method for benthic animals according to claim 1, characterized in that, In step (2), the CTAB extraction method includes: An extraction buffer containing 2% CTAB, 100 mM Tris-HCl (pH 8.0), 20 mM EDTA, and 1.4 M NaCl was added. After incubation at 65°C, the crude eDNA extract was obtained by chloroform-isoamyl alcohol extraction, isopropanol precipitation, and ethanol washing.

4. The rapid identification method for benthic animals according to claim 1, characterized in that, In step (3), the specific primer pair has the following sequences: forward primer Benthos-12SV05-F: 5'-TAGAACAGGCTCCTCTAGT-3', reverse primer Benthos-12SV05-R: 5'-TTAGATACCCCACTATGCA-3', and the 5' end of the primer has a 6-12bp barcode tag.

5. The rapid identification method for benthic animals according to claim 1, characterized in that, The PCR reaction system is 25–35 μL, containing high-fidelity DNA polymerase, primers, template DNA, and sterile water; the PCR cycling conditions include pre-denaturation, 30 ± 5 cycles, and final extension.

6. The rapid identification method for benthic animals according to claim 1, characterized in that, In step (5), the high-throughput sequencing platform is Illumina or an equivalent second-generation sequencing platform, the sequencing mode is paired-end sequencing, the read length is 2×150bp or its equivalent length, and the number of effective sequences per sample is not less than 50,000.

7. The rapid identification method for benthic animals according to claim 1, characterized in that, In step (6), the benthic animal-specific 12SrRNA database is established by screening 12SrRNA gene sequences from the phyla Annelida, Mollusca, Arthropoda, and Echinodermata in public databases and clustering them according to 99% sequence similarity.

8. The rapid identification method for benthic animals according to claim 1, characterized in that, In step (6), the information analysis is based on QIIME2 or a software platform with equivalent functionality. A noise reduction plugin is used for sequence quality control and truncation, and a classification plugin is used for species annotation. The confidence threshold is 0.8 to 0.

9.

9. The rapid identification method for benthic animals according to claim 1, characterized in that, Contamination removal includes removing mitochondrial sequences, chloroplast sequences, and sequences detected in negative controls.

10. The rapid identification method for benthic animals according to claim 1, characterized in that, The detection cycle is set at 3 to 5 days, the species annotation accuracy is no less than 90%, and the detection limit for low biomass species is no higher than 1 individual / 10g sediment.

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