Primer and method for monitoring biomass of chlamys nobilis based on environmental DNA amplification
By using primers MN-F/MN-R and qPCR technology, the problem of biomass monitoring of the noble scallop *Ctenopharynx spp.* was solved, achieving efficient, low-cost, and non-destructive marine biomass monitoring and overcoming the shortcomings of traditional methods.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SOUTH CHINA SEA INST OF OCEANOLOGY CHINESE ACAD OF SCI
- Filing Date
- 2026-02-04
- Publication Date
- 2026-04-14
AI Technical Summary
Existing technologies lack effective methods for monitoring the biomass of the noble scallop, especially in complex marine environments, where traditional survey methods are inefficient, costly, and fail to accurately reflect its true population.
Environmental DNA was amplified using specific primers MN-F/MN-R, and combined with qPCR technology. The biomass of the noble scallop was monitored by extracting seawater eDNA. PCR amplification and qPCR detection were performed using primers MN-F/MN-R, and a standard curve was constructed to estimate the biomass.
It enables rapid, low-cost, and non-destructive monitoring of marine biomass without the need for diving operations, accurately reflecting the true number of noble scallops and avoiding the limitations of traditional methods.
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Figure CN121852557A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of molecular biology, specifically to primers and methods for monitoring the biomass of the noble scallop *Ctenopharynx chinensis* based on environmental DNA amplification. Background Technology
[0002] The noble scallop (Mimachlamys nobilis) belongs to the phylum Mollusca, class Bivalvia, order Pectinida, family Pectinidae, and genus Mimachlamys. It is widely distributed in the South my country Sea, coastal cities of Fujian and Guangdong provinces, and is one of the important aquaculture species in southern China. It is a shallow-sea filter-feeding bivalve with high bioaccumulation characteristics, exhibiting advantages such as rapid growth and high nutritional content, with a protein content higher than other shellfish. The noble scallop has suffered large-scale mortality due to germplasm degradation and poor aquaculture management, resulting in losses for the industry. Understanding its distribution and biomass in the ocean is a crucial foundation for resource conservation and ecological aquaculture.
[0003] Environmental DNA (eDNA) technology originated from environmental microbiology research, initially primarily used for the isolation and purification of microbial DNA from sediments. Its application saw a breakthrough at the beginning of this century, with its first successful application in 2008 to the detection of the invasive species, the American bullfrog, in aquatic bodies, marking eDNA technology as an effective tool for aquatic organism monitoring. With the development of molecular technology, eDNA is currently applied to biodiversity research mainly through two pathways: community composition analysis based on metabarcoding and high-throughput sequencing, and single-species quantitative monitoring relying on real-time quantitative PCR (qPCR) or digital PCR (ddPCR). Compared with traditional survey methods, eDNA technology has advantages such as high efficiency, high sensitivity, and non-invasiveness to organisms and the environment, making it particularly suitable for large-scale waters with complex topography where traditional methods are difficult to implement. In the field of marine biological monitoring, eDNA technology has been successfully applied to fish diversity assessment and rare species tracking, demonstrating its enormous potential in marine biological resource surveys.
[0004] To date, there is no widely accepted scientific methodology for investigating the biomass of the noble scallop (Ctenophora spp.) both domestically and internationally. The limited literature primarily relies on traditional field surveys, such as benthic trawls, underwater video observation, and sampling using culture traps. Since the noble scallop is a benthic marine animal, primarily inhabiting shallow sea areas below the low tide line to depths exceeding 100 meters, and particularly favoring clear, fast-flowing waters with hard bottoms, this presents a significant challenge for artificial diving surveys of its biomass. Advances in modern molecular technology have provided an opportunity to develop novel methods for investigating the biomass of the noble scallop. Summary of the Invention
[0005] The first objective of this invention is to provide a primer for monitoring the biomass of the noble scallop *Ctenopharynx* based on environmental DNA amplification, comprising a forward primer (MN-F) and a reverse primer (MN-R), the sequences of which are as follows:
[0006] MN-F: 5'-CACCTTTCGTTTGGGCGTTGG-3' (SEQ ID NO. 1);
[0007] MN-R: 5'-ACACCAGTAGGGACAGCAATCA-3' (SEQ ID NO. 2).
[0008] A second objective of this invention is to provide a method for testing the sensitivity and specificity of the aforementioned primers, comprising the following steps:
[0009] (1) Sensitivity detection: The extracted genomic DNA of the noble scallop was serially diluted. Using the serially diluted genomic DNA of the noble scallop as a template, qPCR amplification was performed using primers MN-F / MN-R. The amplification curve and melting curve were observed to determine the sensitivity of the primers.
[0010] (2) Specificity detection: Closely related mollusks of the noble scallop class, including the bay scallop (Argopectenirradians) and the scallop (Patinopecten yessoensis), as well as common marine species, including the pearl oyster (Pinctada imbricata), the giant oyster (Crassostrea hongkongensis), the sea cucumber (Holothuria leucospilota), and the Litopenaeus vannamei, were selected. Genomic DNA was extracted from them and used as a template. Conventional PCR amplification was performed using primers MN-F / MN-R. Agarose gel electrophoresis was performed and the bands were observed to verify the specificity of the primers.
[0011] Preferably, the concentrations of genomic DNA after gradient dilution in step (1) are 2.1 × 10⁻⁶. 1 ng / μL, 2.1×10 0 ng / μL, 2.1×10 -1 ng / μL, 2.1×10 -2 ng / μL, 2.1×10 -3 ng / μL, 2.1×10 -4 ng / μL, 2.1×10 -5 ng / μL, 2.1×10 -6 ng / μL.
[0012] Preferably, the qPCR reaction system in step (1) is as follows: 0.5 μL plasmid DNA, 10 μL 2×SYBR Green Pro TaqHS Premix, 0.4 μL 10-20 μM / μL forward primer MN-F, 0.4 μL 10-20 μM / μL reverse primer MN-R, 8.7 μL ddH2O, for a total of 20 μL; the reaction conditions are: 95℃ for 30 sec; 95℃ for 5 sec, 60℃ for 30 sec, for 40 cycles.
[0013] Preferably, the PCR reaction system in step (2) is as follows: 1 μL of extracted tissue DNA, 10 μL of 2× plus Taq HiFiPCR mix (with blue dye), 1 μL of 10-20 μM / μL forward primer MN-F, 1 μL of 10-20 μM / μL reverse primer MN-R, 7 μL of ddH2O, for a total of 20 μL; the reaction conditions are: 95℃ for 3 min; 94℃ for 25 sec, 57℃ for 25 sec, 72℃ for 60 sec, for 35 cycles; 72℃ for 5 min.
[0014] A third objective of this invention is to provide the application of the above-mentioned primers in the preparation of a kit for monitoring the biomass of the noble scallop *Ctenopharynx chinensis* based on environmental DNA amplification.
[0015] A fourth objective of this invention is to provide a kit for monitoring the biomass of the noble scallop *Ctenopharynx chinensis* based on environmental DNA amplification, which contains the aforementioned primers.
[0016] The fifth objective of this invention is to provide a method for monitoring the biomass of the noble scallop *Ctenopharynx scallop* based on environmental DNA amplification, comprising the following steps:
[0017] (1) Using the genomic DNA of the noble scallop as a template, PCR amplification was performed using primers MN-F / MN-R; the PCR product was ligated to the plasmid vector and cultured; the plasmid was extracted; the plasmid copy number was calculated and serially diluted; using the serially diluted plasmid as a template, qPCR amplification was performed using primers MN-F / MN-R, a standard curve of the fragment copy number and Ct value of the noble scallop was prepared, and the amplification curve and melting curve were observed to determine the sensitivity and specificity of the primers;
[0018] (2) Collect bottom seawater in a certain marine environment, collect and extract eDNA from the water body using filtration method, use the water eDNA as a template, use primers MN-F / MN-R for qPCR detection, and record the Ct value;
[0019] (3) Based on the standard curve of fragment copy number and Ct value of the order Sterculiaceae obtained in step (1) and the Ct value of the seawater eDNA sample obtained in step (2), calculate the eDNA copy number of Sterculiaceae in the water body; then compare the eDNA copy number of Sterculiaceae in the water body with the standard curve of fragment copy number and Ct value of the corresponding order Sterculiaceae, and estimate the biomass of Sterculiaceae.
[0020] Preferably, the copy number of the target fragment after gradient dilution of the plasmid in step (1) is 10. 8 copies / μL, 10 7 copies / μL, 10 6 copies / μL, 10 5 copies / μL, 10 4 copies / μL, 10 3 copies / μL, 10 2 copies / μL.
[0021] Preferably, the PCR reaction system in step (1) is as follows: 0.5 μL of *Scallop genomic DNA*, 10 μL of 2×SYBR Green Pro Taq HS Premix, 0.4 μL of 10-20 μM / μL forward primer MN-F, 0.4 μL of 10-20 μM / μL reverse primer MN-R, 8.7 μL of ddH2O, for a total of 20 μL; the reaction conditions are: 95℃ for 30 sec; 95℃ for 5 sec, 60℃ for 30 sec, for 40 cycles.
[0022] Preferably, the qPCR reaction system in step (1) is as follows: 0.5 μL plasmid DNA, 10 μL 2×SYBR Green Pro TaqHS Premix, 0.4 μL 10-20 μM / μL forward primer MN-F, 0.4 μL 10-20 μM / μL reverse primer MN-R, 8.7 μL ddH2O, for a total of 20 μL; the reaction conditions are: 95℃ for 30 sec; 95℃ for 5 sec, 60℃ for 30 sec, for 40 cycles.
[0023] Preferably, the qPCR reaction system in step (2) is as follows: 0.5 μL of water eDNA, 10 μL of 2×SYBR Green ProTaq HS Premix, 0.4 μL of 10-20 μM / μL forward primer MN-F, 0.4 μL of 10-20 μM / μL reverse primer MN-R, 8.7 μL of ddH2O, for a total of 20 μL; the reaction conditions are: 95℃ for 30 sec; 95℃ for 5 sec, 60℃ for 30 sec, for 40 cycles.
[0024] Preferably, the filtration method described in step (2) is to use a filter membrane with a pore size of 0.8 μm.
[0025] A sixth objective of this invention is to provide the application of the above-described primers, kits, or methods in monitoring the biomass of the noble scallop.
[0026] Compared with traditional methods for surveying noble scallops using underwater transects, this invention has the following advantages and positive effects:
[0027] (1) No need for divers to go underwater for large-scale operations, water samples are easy to obtain quickly and require a small volume of water samples, thus greatly improving work efficiency and reducing costs.
[0028] (2) Only water samples are taken, so there is no damage to the noble scallop and its environment, and there are no strict requirements on the seabed of the survey area;
[0029] (3) Since the noble scallop has reef-dwelling and hiding characteristics and is widely distributed, the number of noble scallops observed by diving is limited and the observation range is effective. Therefore, the data obtained by traditional artificial diving often cannot accurately reflect the true number of noble scallops. The eDNA amplification method of the present invention is not affected by the reef-dwelling and hiding characteristics and distribution range of noble scallops. Attached Figure Description
[0030] Figure 1 The amplification and melting curves of qPCR were obtained using the genomic DNA of the noble scallop as a template.
[0031] Figure 2 The amplification and melting curves of qPCR were obtained using standard plasmid DNA from the noble scallop as a template.
[0032] Figure 3 This is the result of primer specificity verification for this invention.
[0033] Figure 4 Standard curves were constructed for the standard sample of the noble scallop.
[0034] Figure 5 This is a graph showing the species abundance variation of the noble scallop. Detailed Implementation
[0035] The following embodiments are further illustrations of the present invention, but not limitations thereof.
[0036] Specific experimental conditions and methods are not specified in the following examples, and the technical means used are generally conventional means well known to those skilled in the art.
[0037] The manufacturers of the instruments, reagents, and materials used in the examples are as follows: PCR instrument (Bio-Rad, USA), real-time fluorescence PCR instrument (ROCGENE, China), NanoDrop 2000 fluorescence spectrophotometer (Thermo), gel imaging system (Bio-Rad, USA), marine animal tissue genomic DNA extraction kit (Tiangen, China), universal DNA purification and recovery kit (Tiangen, China), pMDTM19-T Vector Cloning Kit (TaKaRa, Japan), DH5α competent cells (Tiangen, China), endotoxin-free plasmid small-volume extraction kit (Tiangen, China), AP-9950 vacuum pump (Automatic Science, China), filter membrane (0.8 μm) purchased from Sangon Biotech (Shanghai) Co., Ltd., 2 ×plus Taq HiFi PCR mix purchased from Shenzhen Maikes Biotechnology Co., Ltd., and SYBR Green Pro Taq HS premixed qPCR kit purchased from Hunan Aikerui Biotechnology Co., Ltd.
[0038] Example 1: Verification of primer sensitivity and specificity
[0039] 1. Take body wall tissue samples from the noble scallop (Ctenophora indica) and extract genomic DNA from it according to the instructions of the marine animal tissue genomic DNA extraction kit. The extracted genomic DNA was then serially diluted to concentrations of 2.1 × 10⁻⁶. 1ng / μL, 2.1×10 0 ng / μL, 2.1×10 -1 ng / μL, 2.1×10 -2 ng / μL, 2.1×10 -3 ng / μL, 2.1×10 -4 ng / μL, 2.1×10 -5 ng / μL, 2.1×10 -6 ng / μL. Using serially diluted genomic DNA of the noble scallop *Ctenopharynx chinensis* as a template, qPCR amplification was performed using primers MN-F / MN-R. The qPCR reaction system consisted of: 0.5 μL plasmid DNA, 10 μL 2×SYBR Green Pro Taq HS Premix, 0.4 μL 10-20 μM / μL forward primer MN-F, 0.4 μL 10-20 μM / μL reverse primer MN-R, and 8.7 μL ddH2O, for a total of 20 μL. The reaction conditions were: 95℃ for 30 sec; 95℃ for 5 sec, 60℃ for 30 sec, for 40 cycles. Amplification and melting curves were observed to determine the sensitivity and interference resistance of the primers.
[0040] The results are shown below. Figure 1 The amplification curve shows a gradient peak, while the melting curve shows a single peak, indicating that the primers have high sensitivity and good specificity.
[0041] 2. Tissue samples were collected from bay scallops, scallops, pinnatifida, giant oysters, sea cucumbers, and Litopenaeus vannamei. Genomic DNA was extracted from each sample according to the instructions of the marine animal tissue genomic DNA extraction kit. Using the extracted genomic DNA as templates, PCR amplification was performed using primers (MN-F / MN-R). The PCR reaction mixture consisted of: 1 μL of each genomic DNA sample, 10 μL of 2× plus Taq HiFi PCR mix, 1 μL of forward primer MN-F (10 μM / μL), 1 μL of reverse primer MN-R (10 μM / μL), and 7 μL of ddH2O, for a total of 20 μL. The reaction conditions were: 95℃ for 3 min; 94℃ for 25 sec, 57℃ for 25 sec, 72℃ for 1 min, 35 cycles; 72℃ for 5 min. Gel electrophoresis was then performed to observe the banding. The blank control group consisted of environmental DNA samples without *Ctenopharynx chinensis*. The results showed… Figure 3 Only the product amplified using the genomic DNA of the noble scallop showed a single and bright band, indicating that the primer had high specificity.
[0042] Example 2: Preparation of plasmid standards and construction of standard curves for the noble scallop *Ctenopharynx*.
[0043] (1) Take a sample of the body wall tissue of the noble scallop and extract the genomic DNA of the noble scallop according to the instructions of the marine animal tissue genomic DNA extraction kit.
[0044] (2) Using the genomic DNA of the noble scallop *Ctenopharynx glabripennis* as a template, PCR amplification was performed using specific primers (MN-F / MN-R). The PCR reaction system was as follows: 1 μL of *Ctenopharynx glabripennis* genomic DNA, 25 μL of 2 × plus Taq HiFi PCR mix, 2 μL of forward primer MN-F (10 μM / μL), 2 μL of reverse primer MN-R (10 μM / μL), and 20 μL of ddH2O, for a total of 50 μL. The reaction conditions were: 95℃ for 3 min; 94℃ for 25 sec, 57℃ for 25 sec, 72℃ for 1 min, for 40 cycles; 72℃ for 5 min. Gel electrophoresis was then performed to obtain a single band of the target fragment.
[0045] (3) The fragments of the order Ctenopharynx were recovered according to the instructions of the general DNA purification and recovery kit.
[0046] (4) Follow the instructions of the pMDTM19-T Vector Cloning Kit to ligate the target fragment with the vector, and then follow the instructions of the DH5α competent cells to transduce the plasmid into the DH5α competent cells for expansion culture.
[0047] (5) The plasmid was extracted according to the instructions of the endotoxin-free plasmid small-scale extraction kit. The concentration of the recovered product was measured to be 25.4 ng / μL using a NanoDrop2000 fluorescence spectrophotometer.
[0048] (6) The copy number of the plasmid calculated according to the formula is: 7.97791969 × 10⁻⁶ 9 The plasmid was then serially diluted to 7.97791969 × 10⁻⁶ copies / μL; 8 copies / μL, 7.97791969×10 7 copies / μL, 7.97791969×10 6 copies / μL, 7.97791969×10 5 copies / μL, 7.97791969×10 4 copies / μL, 7.97791969×10 3 copies / μL, 7.97791969×102 copies / μL.
[0049] (7) Using sequentially diluted plasmids as templates for qPCR, each gradient was replicated in triplicate. The qPCR reaction mixture consisted of: 0.5 μL plasmid DNA, 10 μL 2×SYBR Green Pro Taq HS Premix, 0.4 μL forward primer MN-F (10 μM / μL), 0.4 μL reverse primer MN-R (10 μM / μL), and 8.7 μL ddH2O, for a total of 20 μL. The reaction conditions were: 95℃ for 30 sec; 95℃ for 5 sec, 60℃ for 30 sec, for 40 cycles. The amplification and melting curves of the qPCR standard of the noble scallop are shown in the figure. Figure 2 The amplification curve showed a gradient peak, while the melting curve showed a single peak, indicating high primer sensitivity and good specificity. The qPCR data were then processed to obtain a copy number versus Ct standard curve. The results showed that the copy number of the fragment from the order *Ctenophora* (noble scallops) was correlated with the Ct value, with the equation: y = 46.608 - 3.7462x; R² = 0.9942. See details... Figure 4 As shown.
[0050] The primers used for PCR and qPCR amplification reactions are shown below:
[0051] Forward primer (MN-F): 5'-CACCTTTCGTTTGGGCGTTGG-3';
[0052] Reverse primer (MN-R): 5'-ACACCAGTAGGGACAGCAATCA-3'.
[0053] Example 3: Biomass assessment of the noble scallop *Ctenopharynx* in the Wanshan sea area of Guangdong
[0054] (1) Filtration and collection of bottom seawater (eDNA) samples from the Wanshan Islands, Guangdong
[0055] Bottom seawater samples were taken from the Wanshan Islands in September and December. Bottom seawater samples were collected from each point in the Wanshan Islands using a water sampler and stored in 1L sterile disposable plastic bottles. Each point was sampled three times. The collected seawater was then filtered using a filtration device and a vacuum pump. The filter membrane had a pore size of 0.8 μm. After the seawater was filtered, the filter membrane was picked up from the edge with sterile tweezers and sprayed with 95% alcohol. It was then stored in a sterile centrifuge tube and subsequently frozen for preservation.
[0056] (2) Extraction of bottom seawater (eDNA) samples from the Wanshan Islands, Guangdong
[0057] Remove the filter membrane from step (1) with sterile tweezers, cut the filter membrane into pieces with sterile scissors, and use the marine animal tissue genomic DNA extraction kit instructions together with the filter membrane to extract eDNA from the bottom seawater of the Wanshan Islands in Guangdong Province. Then store it at -20℃ for later use.
[0058] (3) qPCR amplification of bottom seawater (eDNA) samples from the Wanshan Islands in Guangdong Province
[0059] Seawater eDNA samples from the bottom of the Wanshan Islands and two standard plasmids with different copy numbers (plasmid copy number concentration: 7.97791969 × 10⁻⁶) were collected. 5 copies / μL and 7.97791969×10 3 qPCR amplification was performed using copies / μL of the sample as template. Each sample and plasmid standard were subjected to 3 replicates of qPCR amplification. The qPCR reaction system was as follows: 0.5 μL of eDNA sample, 10 μL of 2×SYBR Green Pro Taq HS Premix, 0.4 μL of forward primer MN-F (20 μM / μL), 0.4 μL of reverse primer MN-R (20 μM / μL), and 8.7 μL of ddH2O; the reaction conditions were: 95℃ for 30 sec; 95℃ for 5 sec, 60℃ for 30 sec, for 40 cycles.
[0060] The primers used for qPCR amplification are shown below:
[0061] Forward primer (MN-F): 5'-CACCTTTCGTTTGGGCGTTGG-3';
[0062] Reverse primer (MN-R): 5'-ACACCAGTAGGGACAGCAATCA-3'.
[0063] (4) Assessment of the biomass of the noble scallop in the Wanshan Islands waters of Guangdong
[0064] First, the Ct values generated from three technical replicates of each sample and plasmid standard were averaged to obtain the Ct value for each sample and plasmid standard. Second, the Ct values of two plasmid standards with different copy concentrations were compared with the Ct values of the corresponding copy concentrations of plasmids in the standard curve constructed in Example 2. If the difference between the two values was ≤1, the samples amplified by qPCR in this study could be used for subsequent analysis. Third, the Ct value of each sample was substituted as the y value into the standard curve (y=46.608-3.7462x) to calculate x, and then x was substituted into 10. xIn the next step, the copy number of the *Ctenophora* order fragment from each qPCR reaction was obtained. Fourth, the copy number of the *Ctenophora* order fragment from each qPCR reaction was multiplied by 100 to obtain the copy number of the *Ctenophora* order fragment from 1 L of seawater collected from the bottom of the Wanshan Islands. See Table 1 for details. Figure 5 As shown.
[0065] Table 1. Copy numbers of fragments from the order Ctenophora in bottom water samples from the Wanshan Islands in September and December.
[0066] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A primer for monitoring the biomass of the noble scallop *Ctenopharynx* based on environmental DNA amplification, characterized in that... The primers are: MN-F: 5'-CACCTTTCGTTTGGGCGTTGG-3', MN-R: 5'-ACACCAGTAGGGACAGCAATCA-3'.
2. The application of the primers described in claim 1 in the preparation of a kit for monitoring the biomass of the noble scallop *Ctenopharynx chinensis* based on environmental DNA amplification.
3. A kit for monitoring the biomass of the noble scallop *Ctenopharynx* based on environmental DNA amplification, characterized in that... It contains the primers as described in claim 1.
4. A method for monitoring the biomass of the noble scallop *Ctenopharynx* based on environmental DNA amplification, characterized in that, Includes the following steps: (1) Using the genomic DNA of the noble scallop as a template, PCR amplification was performed using the primers MN-F / MN-R described in claim 1; the PCR product was ligated to the plasmid vector and cultured; the plasmid was extracted; the plasmid copy number was calculated and serially diluted; using the serially diluted plasmid as a template, qPCR amplification was performed using the primers MN-F / MN-R described in claim 1 to generate a standard curve of the copy number of the noble scallop fragment versus the Ct value; (2) Collect bottom seawater in a certain marine environment, collect and extract eDNA from the water body by filtration, use the water eDNA as a template, use the primers MN-F / MN-R described in claim 1 to perform qPCR detection, and record the Ct value. (3) Based on the standard curve of fragment copy number and Ct value of the order Stelleria scallops obtained in step (1) and the Ct value of the seawater eDNA sample obtained in step (2), calculate the copy number of Stelleria scallop eDNA in the water. Then, the cDNA copy number of the noble scallop in the water was compared with the standard curve of the copy number of the corresponding noble scallop fragment and Ct value, and the biomass of the noble scallop was estimated accordingly.
5. The method for monitoring the biomass of the noble scallop *Ctenopharynx* based on environmental DNA amplification according to claim 4, characterized in that, In step (1), the copy number of the target fragment after serial dilution of the plasmid was 10. 8 copies / μL, 10 7 copies / μL, 10 6 copies / μL, 10 5 copies / μL, 10 4 copies / μL, 10 3 copies / μL, 10 2 copies / μL.
6. The method for monitoring the biomass of the noble scallop *Ctenopharynx* based on environmental DNA amplification according to claim 4, characterized in that, The PCR reaction system in step (1) was as follows: 0.5 μL of genomic DNA from the noble scallop, 10 μL of 2×SYBR GreenPro Taq HS Premix, 0.4 μL of 10-20 μM / μL forward primer MN-F, 0.4 μL of 10-20 μM / μL reverse primer MN-R, 8.7 μL of ddH2O, for a total of 20 μL; the reaction conditions were: 95℃ for 30 sec; 95℃ for 5 sec, 60℃ for 30 sec, for 40 cycles.
7. The method for monitoring the biomass of the noble scallop *Ctenopharynx* based on environmental DNA amplification according to claim 4, characterized in that, The reaction system for qPCR in step (1) is as follows: 0.5 μL plasmid DNA, 10 μL 2×SYBR Green Pro Taq HS Premix, 0.4 μL 10-20 μM / μL forward primer MN-F, 0.4 μL 10-20 μM / μL reverse primer MN-R, 8.7 μL ddH2O, for a total of 20 μL; the reaction conditions are: 95℃ for 30 sec; 95℃ for 5 sec, 60℃ for 30 sec, for 40 cycles.
8. The method for monitoring the biomass of the noble scallop *Ctenopharynx* based on environmental DNA amplification according to claim 4, characterized in that, The reaction system for qPCR in step (2) is as follows: 0.5 μL of water eDNA, 10 μL of 2×SYBR Green Pro Taq HSPremix, 0.4 μL of 10-20 μM / μL forward primer MN-F, 0.4 μL of 10-20 μM / μL reverse primer MN-R, 8.7 μL of ddH2O, for a total of 20 μL; the reaction conditions are: 95℃ for 30 sec; 95℃ for 5 sec, 60℃ for 30 sec, for 40 cycles.
9. The method for monitoring the biomass of the noble scallop *Ctenopharynx* based on environmental DNA amplification according to claim 4, characterized in that, The filtration method described in step (2) is to use a filter membrane with a pore size of 0.8 μm.
10. The application of the primers of claim 1, the kit of claim 3, or the method of claim 4 in monitoring the biomass of the noble scallop.