Probe primer and method for monitoring holothuria scabra based on environmental DNA technology

Abstract

The application discloses a kind of probe primer and method based on environmental DNA technology to the probe of rough sea cucumber monitoring.The sequence of the primer is: Hs-F: 5'-GGAGTAAACTTAACCTTCTTC-3', Hs-R: 5'-GTCTGGGTAGTCTGAATAC-3';The sequence of the probe is: Hs-P: 5'-FAM-ATC(C)CC(G)CT(A)AG(C)CTA-BHQ1-3'.The method of the application determines the copy number of rough sea cucumber in water body under different sea areas environment, compares it with the standard curve of corresponding rough sea cucumber DNA copy number and Ct value and detection lower limit LOD, provides the existence condition of field rough sea cucumber, realizes the purpose of monitoring rough sea cucumber, and provides technical support for field distribution investigation of rough sea cucumber and relevant policy making.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, specifically to a probe primer and method for monitoring sea cucumbers based on environmental DNA technology. Background Technology

[0002] Environmental DNA (eDNA) is a molecular technique developed in the 1980s to study microbial diversity. With its increasing sophistication, it was first applied to aquatic environments at the beginning of this century. Environmental DNA refers to DNA extracted directly from environmental samples (water, air, soil, feces, etc.). Environmental DNA technology involves using high-throughput sequencing or real-time quantitative PCR (qPCR) to analyze population composition or quantitatively and qualitatively identify individual species. In aquatic animal surveys, environmental DNA technology offers advantages over traditional methods, including high efficiency, cost-effectiveness, and non-invasiveness. Therefore, it has been widely applied in monitoring invasive and endangered species, and assessing biodiversity and biomass.

[0003] As a commercially valuable sea cucumber species, the rough sea cucumber (Cucumis melo var. rubra) is highly sought after by people in the Asia-Pacific region. However, due to huge market demand and the impact of human activities, wild resources of the rough sea cucumber have declined sharply. In 2013, it was listed as an endangered species on the IUCN Red List of Threatened Species. Because of the rough sea cucumber's unique nocturnal and sand-dwelling habits, traditional fisheries resource surveys cannot be used to conduct resource surveys. Therefore, a rapid and accurate technical means is needed to determine the distribution and population size of wild rough sea cucumbers, providing support for resource conservation policies. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of existing technologies by providing specific primers, probes, and methods for monitoring sea cucumbers based on environmental DNA technology. By utilizing these specific primers and probes and employing real-time quantitative PCR technology, the distribution of sea cucumbers in different sea areas can be monitored, providing a research foundation and technical support for sea cucumber resource surveys.

[0005] The first objective of this invention is to provide a primer-probe set for monitoring sea cucumbers based on environmental DNA technology. The primers comprise one forward primer (Hs-F) and one reverse primer (Hs-R), with the following sequences:

[0006] Hs-F: 5'-GGAGTAAACTTAACCTTCTTC-3',

[0007] Hs-R: 5'-GTTCTGGGTAGTCTGAATAC-3'.

[0008] The probe sequence is: Hs-P: 5'-ATC(C)CC(G)CT(A)AG(C)CTA-3', with the 5' end modified with FAM and the 3' end modified with BHQ1. Note: () indicates that the base is an LNA-modified base.

[0009] A second objective of this invention is to provide the application of the above-described primer-probe set in the preparation of a kit for monitoring sea cucumbers based on environmental DNA technology.

[0010] A third objective of this invention is to provide a kit for monitoring sea cucumbers based on environmental DNA technology, which includes the aforementioned primer and probe set.

[0011] The fourth objective of this invention is to provide a method for monitoring sea cucumbers based on environmental DNA technology, comprising the following steps:

[0012] (1) Using the genomic DNA of sea cucumber as a template, PCR amplification was performed using the primer and probe set 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 primer and probe set described in claim 1, a standard curve of the copy number of the target fragment of sea cucumber and the Ct value was prepared, and the lower limit of detection (LOD) of qPCR was determined.

[0013] (2) Collect water bodies under different marine environments, collect and extract eDNA from the water bodies using a filtration method, use the water body eDNA as a template, perform qPCR detection using the primer and probe set described in claim 1, and record the Ct value.

[0014] (3) Based on the standard curve of copy number and Ct value of the target fragment of sea cucumber obtained in step (1) and the limit of detection (LOD) of qPCR, and the Ct value of the water eDNA sample obtained in step (2), calculate the copy number of sea cucumber eDNA in the water; then compare the copy number of sea cucumber eDNA in the water with the standard curve of copy number and Ct value of the target fragment of sea cucumber and the limit of detection (LOD) of qPCR, and determine the distribution and presence of sea cucumber based on this.

[0015] Preferably, the copy number of the target fragment after the plasmid gradient dilution in step (1) is 10. 9 copies / μL, 10 8 copies / μL, 10 7 copies / μL, 10 6 copies / μL, 10 5copies / μL, 10 4 copies / μL, 10 3 copies / μL, 10 2 copies / μL, 10 1 copies / μL.

[0016] Preferably, the PCR reaction system in step (1) is as follows: 1 μL of sea cucumber genomic DNA, 25 μL of 2×HiFi Taq PCRStarMix with Loading Dye (GenStear), 1 μL of 10 μM forward primer Hs-F, 1 μL of 10 μM reverse primer Hs-R, 22 μL of ddH2O, for a total of 50 μL; the reaction conditions are: 94℃ pre-denaturation for 2 min; 94℃ denaturation for 30 sec, 53℃ annealing for 30 sec, 72℃ extension for 30 sec, for 40 cycles; 72℃ final extension for 5 min.

[0017] Preferably, the qPCR reaction system in step (1) is as follows: 3 μL plasmid DNA, 10 μL 2×Pro Taq HS ProbePremix, 0.6 μL 10 μM forward primer Hs-F, 0.6 μL 10 μM reverse primer Hs-R, 0.6 μL 10 μM probe Hs-P, 5.2 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 50 cycles.

[0018] Preferably, the qPCR reaction system in step (2) is as follows: 3 μL of water eDNA, 10 μL of 2×Pro Taq HS ProbePremix, 0.6 μL of 10 μM forward primer Hs-F, 0.6 μL of 10 μM reverse primer Hs-R, 0.6 μL of 10 μM probe Hs-P, 5.2 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 50 cycles.

[0019] Preferably, the filtration method described in step (2) is to use a filter membrane with a pore size of 0.22 μm.

[0020] Preferably, in step (2), the R package “ednar” is used to determine the lower limit of detection (LOD) for qPCR.

[0021] A fifth objective of this invention is to provide the application of the above-described primer-probe set, the above-described kit, or the above-described method in the monitoring of rough sea cucumbers.

[0022] The method provided by this invention can quickly identify sea cucumbers in the wild by collecting water samples, and can provide information on the presence of sea cucumbers based on the eDNA copy number in the water sample, providing technical support for field distribution surveys of sea cucumbers and the formulation of related policies. Attached Figure Description

[0023] Figure 1 The standard curve and limit of detection (LOD) for the target fragment copy number versus Ct value in *Cuora esculenta* are shown in the figure. The equation of the standard curve in this figure is: y = 41.09716 - 2.86884x; the limit of detection (LOD) is 13.13572 copies / μL.

[0024] Figure 2 To collect location information of bottom seawater in the ocean area.

[0025] Figure 3 This is the amplification curve for qPCR detection of eDNA samples from water bodies. Detailed Implementation

[0026] The following embodiments are further illustrations of the present invention, but not limitations thereof. Specific experimental conditions and methods are not specified in the following embodiments, and the techniques used are generally conventional methods well known to those skilled in the art.

[0027] The manufacturers of the instruments, reagents, and materials used in the following examples are: PCR instrument (Bio-Rad), Real-time Fluorescence PCR Instrument (TaKaRa), NanoDrop 2000 Fluorescence Spectrophotometer (Thermo), Gel Imaging System (Bio-Rad, USA), Marine Animal Tissue Genomic DNA Extraction Kit (Tiangen), Universal DNA Purification and Recovery Kit (Tiangen), pMD TM The 19-T Vector Cloning Kit (TaKaRa), DH5α competent cells (Tiangen), endotoxin-free plasmid small-scale extraction kit (Tiangen), AP-9950 vacuum pump (Automatic Science), and filter membrane (0.22μm) were purchased from Shanghai Sangon Biotech Co., Ltd., and the Pro Taq HS premixed probe qPCR kit was purchased from Hunan Aikerui Biotechnology Co., Ltd.

[0028] Example 1

[0029] (1) Design of specific primers and probes for sea cucumber.

[0030] Based on the cox1 sequence of the mitochondrial DNA gene in sea cucumber, qPCR primers and probes were designed as follows:

[0031] Forward primer (Hs-F): 5'-GGAGTAAACTTAACCTTCTTC-3';

[0032] Reverse primer (Hs-R): 5'-GTCTGGGTAGTCTGAATAC-3';

[0033] Probe (Hs-P): 5'-FAM-ATC(C)CC(G)CT(A)AG(C)CTA-BHQ1-3', Note: () indicates that the base is an LNA-modified base.

[0034] (2) DNA extraction from sea cucumber, plasmid construction, preparation of standard curve of target fragment copy number and Ct value from sea cucumber and determination of limit of detection (LOD)

[0035] DNA extraction from rough sea cucumber: Take gonadal tissue samples from rough sea cucumbers and extract gDNA from them according to the instructions of the marine animal tissue genomic DNA extraction kit.

[0036] Plasmid construction: First, using sea cucumber gDNA as a template, PCR amplification was performed using specific primers, followed by gel electrophoresis to obtain a single target fragment. The PCR reaction system was as follows: sea cucumber genomic DNA 1 μL, 2×HiFiTaq PCR StarMix with Loading Dye (GenStear) 25 μL, 10 μM forward primer Hs-F 1 μL, 10 μM reverse primer Hs-R 1 μL, ddH2O 22 μL, total 50 μL; the reaction conditions were: 94℃ pre-denaturation for 2 min; 94℃ denaturation for 30 sec, 53℃ annealing for 30 sec, 72℃ extension for 30 sec, 40 cycles; 72℃ final extension for 5 min. Second, the target fragment was recovered according to the instructions of the universal DNA purification and recovery kit. Third, the target fragment was ligated to the vector according to the instructions of the pMDTM19-T Vector Cloning Kit, and then the recombinant plasmid was transduced into DH5α competent cells for amplification culture according to the instructions of the DH5α competent cell kit. Fourth, the plasmid was extracted according to the instructions of the endotoxin-free plasmid small-volume extraction kit, and the concentration of the recovered product was measured to be 27.6 ng / μL using a NanoDrop 2000 fluorescence spectrophotometer. Fifth, the copy number of the plasmid was calculated according to the formula as: 9.08828355 × 10⁻⁶. 9 The plasmid was then serially diluted to 9.08828355 × 10⁻⁶ copies / μL; 9 copies / μL, 9.08828355×10 8 copies / μL, 9.08828355×10 7copies / μL, 9.08828355×10 6 copies / μL, 9.08828355×10 5 copies / μL, 9.08828355×10 4 copies / μL, 9.08828355×10 3 copies / μL, 9.08828355×10 2 copies / μL, 9.08828355×10 1 copies / μL.

[0037] Construction of standard curves for copy number and Ct values ​​of the target fragment from *Cuora esculenta* and determination of the limit of detection (LOD): Quantitatively diluted plasmids were used as templates for qPCR reactions. Each gradient was repeated in quadruplicates. The qPCR reaction mixture consisted of: 3 μL plasmid DNA, 10 μL 2×Pro Taq HS Probe Premix, 0.6 μL 10 μM forward primer Hs-F, 0.6 μL 10 μM reverse primer Hs-R, 0.6 μL 10 μM probe Hs-P, and 5.2 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 50 cycles. After the reactions, the results were imported into the R package "ednar" for processing to obtain the copy number and Ct standard curves and the limit of detection (LOD).

[0038] The results showed that the copy number of the target fragment from *Diplodocus acutissima* correlated with the Ct value in a curve, with the equation: y = 41.09716 - 2.86884x. The limit of detection (LOD) was 13.13572 copies / μL. See details... Figure 1 As shown.

[0039] (3) Extraction of eDNA from water

[0040] For information on water sample collection points, please see [link / reference]. Figure 2 As shown. Bottom seawater was collected at each point using a water sampler and stored in a 1L sterile disposable plastic bottle. Each point was sampled three times. The collected seawater was then filtered using a filter bottle and a vacuum pump. The filter membrane had a pore size of 0.22μm. 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 stored at -20℃.

[0041] Remove the filter membrane with sterile forceps, cut it into pieces with sterile scissors, and use the marine animal tissue genomic DNA extraction kit along with the filter membrane to extract seawater eDNA. The eDNA concentration was measured using a NanoDrop 2000 fluorescence spectrophotometer.

[0042] (4) Relationship between cDNA copy number and presence in sea cucumber

[0043] Seawater eDNA samples and two standard quality granules with different copy concentrations (9.08828355×10⁻⁶) were used to analyze the eDNA in the seawater. 6 copies / μL and 9.08828355×10 3 Using copies / μL) as templates, qPCR detection was performed. Each sample and plasmid standard was amplified three times in duplicate, and the Ct values ​​of each sample and plasmid standard were recorded. See details... Figure 3 As shown in Table 1, the Ct values ​​and copy numbers of the target fragment from *Diplostomum sibiricum* were compared with the standard curve and limit of detection (LOD) of Ct values ​​to determine the positive amplification status of the loci. The qPCR reaction system was as follows: 3 μL of aquatic eDNA, 10 μL of 2×Pro Taq HS Probe Premix, 0.6 μL of 10 μM forward primer Hs-F, 0.6 μL of 10 μM reverse primer Hs-R, 0.6 μL of 10 μM probe Hs-P, and 5.2 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 50 cycles.

[0044] Table 1. Positive results of sea cucumber (Cuora esculenta) in seawater eDNA samples from Xuwen sea area.

[0045]

[0046] 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 and probe set for monitoring sea cucumbers based on environmental DNA technology, characterized in that, It includes primers Hs-F / Hs-R and probe Hs-P; the primer sequences are: Hs-F: 5'-GGAGTAAACTTAACCTTCTTC-3', Hs-R: 5'-GTCTGGGTAGTCTGAATAC-3'; the probe sequence is: Hs-P: 5'-ATCCCCGCTAAGCCT The probe is A-3', with the 5' end modified with FAM and the 3' end modified with BHQ1. The C bases at positions 4, 7, 10, and 13 in the probe sequence are LNA-modified bases.

2. The application of the primer and probe set according to claim 1 in the preparation of a kit for monitoring sea cucumbers based on environmental DNA technology.

3. A kit for monitoring sea cucumbers based on environmental DNA technology, characterized in that, It includes the primer and probe set as described in claim 1.

4. A method for monitoring sea cucumbers based on environmental DNA technology, characterized in that, Includes the following steps: (1) Using the genomic DNA of sea cucumber as a template, PCR amplification was performed using the primer and probe set 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 primer and probe set described in claim 1, a standard curve of copy number of the target fragment and Ct value of sea cucumber was generated, and the lower limit of detection (LOD) of qPCR was determined. (2) Collect water bodies under different marine environments, collect and extract eDNA from the water bodies using a filtration method, use the water body eDNA as a template, perform qPCR detection using the primer and probe set described in claim 1, and record the Ct value. (3) Based on the standard curve of copy number and Ct value of the target fragment of sea cucumber obtained in step (1) and the limit of detection (LOD) of qPCR, and the Ct value of the water eDNA sample obtained in step (2), calculate the copy number of sea cucumber eDNA in the water; then compare the copy number of sea cucumber eDNA in the water with the standard curve of copy number and Ct value of the target fragment of sea cucumber and the limit of detection (LOD) of qPCR, and determine the distribution and presence of sea cucumber.

5. The method for monitoring sea cucumbers based on environmental DNA technology 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. 9 copies / μL, 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, 10 1 copies / μL.

6. The method for monitoring sea cucumbers based on environmental DNA technology according to claim 4, characterized in that, The PCR reaction system in step (1) was as follows: 1 μL of sea cucumber genomic DNA, 25 μL of 2× HiFi Taq PCR StarMix with LoadingDye, 1 μL of 10 μM forward primer Hs-F, 1 μL of 10 μM reverse primer Hs-R, 22 μL of ddH2O, for a total of 50 μL; the reaction conditions were: 94℃ pre-denaturation for 2 min; 94℃ denaturation for 30 sec, 53℃ annealing for 30 sec, 72℃ extension for 30 sec, for 40 cycles; and 72℃ final extension for 5 min.

7. The method for monitoring sea cucumbers based on environmental DNA technology according to claim 4, characterized in that, The reaction system for qPCR in step (1) is as follows: plasmid DNA 3 μL, 2×Pro Taq HS Probe Premix 10 μL, 10 μM forward primer Hs-F 0.6 μL, 10 μM reverse primer Hs-R 0.6 μL, 10 μM probe Hs-P 0.6 μL, ddH2O 5.2 μL, total 20 μL; reaction conditions are: 95℃ for 30 sec; 95℃ for 5 sec, 60℃ for 30 sec, 50 cycles.

8. The method for monitoring sea cucumbers based on environmental DNA technology according to claim 4, characterized in that, The reaction system for qPCR in step (2) is as follows: 3 μL of water eDNA, 10 μL of 2×Pro Taq HS Probe Premix, 0.6 μL of 10 μM forward primer Hs-F, 0.6 μL of 10 μM reverse primer Hs-R, 0.6 μL of 10 μM probe Hs-P, 5.2 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 50 cycles.

9. The method for monitoring sea cucumbers based on environmental DNA technology 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 0.22 μm.

10. The application of the primer and probe set of claim 1, the kit of claim 3, or the method of claim 4 in the monitoring of rough sea cucumber.

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