A primer and probe set for the specific detection of neon damselfish based on environmental DNA

Abstract

This invention provides a primer and probe set for the specific detection of neon damselfish based on environmental DNA, including primer pairs SEQ ID NO.1 and SEQ ID NO.2 for PCR amplification, and / or probe SEQ ID NO.3 for qPCR, targeting the Cytb gene fragment for detection in neon damselfish. This invention establishes a real-time fluorescent PCR detection method for the environmental DNA of neon damselfish, which only requires collecting water samples from the waters where neon damselfish live to accurately and rapidly detect the environmental DNA of neon damselfish in seawater.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a primer and probe set for the specific detection of neon damselfish based on environmental DNA. Background Technology

[0002] The periodic outbreaks of crown-of-thorns starfish are one of the main reasons for the continuous decline in coral cover in the Indo-Pacific region. This has led to a sustained decrease in coral cover and continuous degradation of coral reef habitats, resulting in decreased biodiversity, ecosystem imbalance, and further coastal erosion, threatening island safety. Neon damselfish can feed heavily on crown-of-thorns starfish larvae. Intervention with neon damselfish at an early stage in the crown-of-thorns starfish life cycle can regulate populations, delaying or reducing outbreaks, thus contributing to maintaining ecological balance and protecting coral reef ecosystems.

[0003] Neon damselfish are typical coral reef fish. They can consume large numbers of crown-of-thorns starfish larvae in a short period of time, which helps to regulate the population size during crown-of-thorns starfish outbreaks. This helps to delay or reduce the scale of outbreaks, providing a buffer space for the coral reef ecosystem to cope with crown-of-thorns starfish outbreaks, and thus helps to maintain ecological balance and protect the coral reef ecosystem.

[0004] *Pomacentrus coelestis*, belonging to the phylum Chordata, class Actinopterygii, order Perciformes, family Pomacentridae, and genus *Pomacentrus*, has an elongated, laterally compressed body, with a standard body length 2.5–2.6 times its body depth. The snout is short and blunt. The mouth is medium-sized; the jaw teeth are in two rows, small and conical. The suborbital bone is exposed with a smooth lower margin, and there is no notch between the preorbital and suborbital bones; the posterior margin of the preopercle is serrated. The body is covered with ctenoid scales; the nasal region has scales; there are 17–18 perforated scales along the lateral line. The dorsal fin is single, with the soft rays not elongated but slightly pointed, with XIII spines and 13–15 soft rays; the anal fin has II spines and 14–15 soft rays; the pectoral fin has 17–18 rays; the caudal fin is forked, with the upper and lower lobes pointed at the ends. Its body color varies greatly. When alive, its back is bright blue, and its belly, anal fin, caudal peduncle, and caudal fin are bright yellow. When frightened, its body color becomes dull or grayish-white. After death, its body turns dark brown.

[0005] Because the neon damselfish is extremely similar in appearance to the golden-bellied damselfish (Pomacentrus auriventris) and Allen's damselfish, both of which are also distributed in the South China Sea, and because its DNA barcode sequence and the 12S gene sequence fragment commonly used in marine fish environmental DNA are highly similar, it is difficult to accurately distinguish these species of damselfish. Therefore, it is urgent to design new environmental DNA primers for the neon damselfish to distinguish various similar species, in order to explore the spatiotemporal distribution and resource characteristics of the neon damselfish in the ocean. This is of great significance for the stock enhancement and release or other fisheries management measures taken in response to the outbreak of crown-of-thorns starfish. Therefore, it is essential to establish a rapid and accurate method for detecting the neon damselfish. Summary of the Invention

[0006] To address the aforementioned issues, this invention provides a primer-probe set for the specific detection of neon damselfish based on environmental DNA. This primer-probe set can specifically identify the environmental DNA of neon damselfish and achieve rapid and accurate identification of neon damselfish using real-time fluorescent PCR detection, providing technical support for the survey of neon damselfish distribution and resource abundance in the ocean.

[0007] To achieve the objectives of this invention, the present invention provides a nucleotide composition for detecting neon damselfish based on environmental DNA technology, comprising primer pairs SEQ ID NO.1 and SEQ ID NO.2 for PCR amplification, and / or probe SEQ ID NO.3 for qPCR.

[0008] Preferably, the primer pair and the probe are designed for the Cytb gene fragment of the neon damselfish, and the Cytb gene fragment is a Genbank sequence: MW631122.1.

[0009] To achieve the objectives of this invention, this invention also provides a kit comprising the above-described nucleotide composition.

[0010] Preferably, the environmental DNA of the neon damselfish in seawater is used to assess the species’ distribution and resource abundance in the ocean, thereby providing data support for evaluating the effects of stock enhancement and release of the neon damselfish, a natural enemy of the crown-of-thorns starfish, fisheries management, and related resource surveys.

[0011] Preferably, it is used to control or respond to outbreaks of crown-of-thorns starfish in the ocean.

[0012] This invention provides a method for detecting neon damselfish using the above-described nucleotide composition, comprising the following steps:

[0013] Filter the water sample and extract DNA from the filter membrane;

[0014] PCR amplification can be performed using primer pairs SEQ ID NO.1 and SEQ ID NO.2, and / or probe SEQ ID NO.3;

[0015] If there are obvious bands in the range of 100~200 bp, it indicates the presence of neon damselfish.

[0016] This invention designs a highly specific primer-probe set based on the Cytb sequence of the neon damselfish (Genbank: MW631122.1), wherein the Cytb gene fragment is used as a molecular marker to identify the neon damselfish.

[0017] The primer (Cytb-729-F / Cytb-882-R) and probe (Cytb-779-Probe) sequences used in the PCR amplification program are as follows:

[0018] Cytb-729-F: 5'-TCTGTTTCTCCCCGAATCTCTAA-3', SEQ ID NO.1;

[0019] Cytb-882-R: 5'-CAAGGCCAGGACTCCTCCTA-3', SEQ ID NO.2;

[0020] Cytb-779-Probe: 5'-FAM-ACCCATAGTAACTCCGCCCCACA-BHQ2-3', SEQ ID NO.3.

[0021] Preferably, the PCR amplification reaction system comprises:

[0022] 2×Taqmix........................15μl;

[0023] DNA template............................1μl;

[0024] Cytb-729-F (10pmol / μL)............1μl;

[0025] Cytb-882-R (10pmol / μL)............1μl;

[0026] Add ddH2O to a final volume of 30 μl.

[0027] Preferably, the PCR amplification reaction conditions consist of: 95°C pre-denaturation for 30 s, 95°C denaturation for 10 s, 55°C annealing for 30 s, 72°C extension for 30 s, 40 cycles, 72°C final extension for 5 min, and storage at 4°C.

[0028] Preferably, the qPCR reaction system comprises:

[0029] 2×AceQ qPCR Probe Master Mix......5μl;

[0030] Cytb-729-F (10pmol / μL)............0.2μl;

[0031] Cytb-882-R (10pmol / μL)............0.2μl;

[0032] Cytb-779-Probe (10pmol / μL)............0.1μl;

[0033] Water sample DNA template........................2μl;

[0034] ROX........................0.2μl;

[0035] Add ddH2O to a final volume of 10 μl.

[0036] Preferably, the qPCR reaction conditions consist of: 95°C pre-denaturation for 5 min, 95°C denaturation for 10 s, 60°C annealing for 30 s, 72°C extension for 30 s, for 45 cycles.

[0037] The beneficial effects of this invention are:

[0038] Neon damselfish and its relatives share similar body size and minimal DNA barcode differences, making accurate differentiation difficult with conventional primers. This invention utilizes primers designed based on the Cytb fragment of the neon damselfish to rapidly and specifically identify the target sequence in seawater, improving identification efficiency. Furthermore, the real-time fluorescent PCR detection method for neon damselfish environmental DNA, established based on the designed primer and probe sets, enables more precise identification of the species, helping to reflect its distribution and resource abundance characteristics in water bodies. This provides data support for evaluating the effectiveness of restocking and releasing neon damselfish, a natural enemy of crown-of-thorns starfish, as well as for fisheries management and related resource surveys. Attached Figure Description

[0039] Figure 1 shows the results of the 154 bp comparison of the Ctyb gene between Neon Damselfish, Golden-bellied Damselfish (Genbank No.: KM198808.1), and Allen's Damselfish (Genbank No.: KM198778.1) in this invention.

[0040] Figure 2 shows the gel electrophoresis images of PCR products from three species of damselfish fish. M: DNAMaker; 1: Negative control; 2: Mixed sample of DNA from *Pteranodon gargarizans*; 3: Mixed sample of DNA from *Pteranodon argenti*; 4: DNA from *Pteranodon nigra*.

[0041] Mixed samples.

[0042] Figure 3 Gel electrophoresis images of PCR products from a mixed sample of eDNA: 1: Neon damselfish eDNA mixture; 2: Negative control; M: DNAMaker.

[0043] Figure 4 Amplification curves were generated for a neon damselfish eDNA sample.

[0044] Figure 5 Standard curves were prepared based on neon damselfish eDNA samples.

[0045] Figure 6 The changes in eDNA copy number and biomass of neon damselfish over time.

[0046] Figure 7 This shows the fit between the eDNA copy number of neon damselfish and the culture density.

[0047] Figure 8 The fitting relationship between the eDNA copy number of neon damselfish and the culture density. Detailed Implementation

[0048] The technical solution of the present invention will be further explained below through embodiments, but the scope of protection of the present invention is not limited in any way by the embodiments. Example 1

[0049] The specificity test for the neon damselfish primers Cytb-729-F / Cytb-882-R includes the following steps:

[0050] 1. Design primers

[0051] The following primers were designed based on the Cytb fragment of the neon damselfish: Cytb-729-F / Cytb-882-R (primers and probes were synthesized by Shanghai Paisenno Biotechnology Co., Ltd.):

[0052] Cytb-729-F: 5'-TCTGTTTCTCCCCGAATCTCTAA-3';

[0053] Cytb-882-R: 5'-CAAGGCCAGGACTCCTCCTA-3'.

[0054] 2. Extracting DNA from Neon Damselfish

[0055] Muscles from Neon Damselfish, Golden Damselfish, and Allen's Damselfish were placed in 1.5ml centrifuge tubes, with three fish of each species. The muscle was minced with scissors and DNA was extracted using the TIANGEN Marine Animals DNA Kit. All test samples were collected from island and reef areas in the South China Sea.

[0056] 3. PCR amplification

[0057] After DNA extraction, equal amounts of DNA were taken from three DNA samples of the same fish species and mixed as templates for PCR amplification. The amplification results were then purified, sequenced, and compared with the sequencing results.

[0058] The reaction system for PCR amplification consists of:

[0059] 2×Taqmix............15μl;

[0060] Muscle DNA template..............1μl;

[0061] Cytb-729-F (10pmol / μL)......1μl;

[0062] Cytb-882-R (10pmol / μL)......1μl;

[0063] Add ddH2O to a final volume of 30 μl.

[0064] The PCR amplification reaction conditions were as follows: 95°C pre-denaturation for 30 s, 95°C denaturation for 10 s, 55°C annealing for 30 s, 72°C extension for 30 s, 40 cycles, 72°C final extension for 5 min, and storage at 4°C.

[0065] Gel electrophoresis results are as follows Figure 2 As shown, the Neon Damselfish DNA pool showed a clear band at 100-200 bp after amplification, while the negative control, the Golden-bellied Damselfish DNA pool, and the Allen's Damselfish DNA pool showed no bands. This indicates that the primer has good specificity and can accurately identify the target species. Example 2

[0066] A real-time fluorescent PCR detection method for environmental DNA of neon damselfish includes the following steps:

[0067] 1. Design primers and probes

[0068] Based on the Cytb fragment of the neon damselfish, the following primers and probes were designed: Cytb-729-F / Cytb-882-R and Cytb-779-Probe (primers and probes were synthesized by Shanghai Paisennuo Biotechnology Co., Ltd.):

[0069] Cytb-729-F: 5'-TCTGTTTCTCCCCGAATCTCTAA-3',

[0070] Cytb-882-R: 5'-CAAGCCCAGGACTCCTCCTA-3',

[0071] Cytb-779-Probe: 5'-ACCCATAGTAACTCCGCCCCACA-BHQ2-3'.

[0072] 2. Extraction of environmental DNA from Neon Damselfish

[0073] Several neon damselfish caught in the South China Sea island and reef area were selected and placed in nine tanks. Each tank contained 15L of filtered seawater. The experimental conditions were 28.0±0.3°C, salinity 33-34‰, and pH 8.1-8.2. Neon damselfish of similar size were placed in three tanks at groups of 1, 2, and 4, with two replicates per group. At 0, 12, 24, 48, 72, 96, 120, 144, and 168 hours after fish addition, 250ml of water samples were collected and filtered through a 0.2μm membrane. An additional 250ml of filtered ultrapure water served as a negative control. DNA was extracted from the filtered water samples using a DNeasy Blood & Tissue Kit (Qiagen, Germany).

[0074] 3. Real-time fluorescent PCR detection

[0075] Five DNA samples were randomly selected and mixed in equal amounts as templates for PCR amplification. The amplification results were detected by electrophoresis. The target fragment was recovered and purified, ligated into the p-MD18-T vector (Takara, Japan), and transformed into competent cells. Single colonies were selected and sequenced to verify positive strains. After overnight culture, plasmids were extracted using a plasmid mini-extraction kit (Jerex, China). Standard plasmids were serially diluted to six concentration gradients, and Ct values ​​were measured to construct a standard curve. Subsequently, qPCR was used to quantify the eDNA samples.

[0076] The reaction system for PCR amplification consists of:

[0077] 2×Taqmix............15μl;

[0078] Water sample DNA template...................1μl;

[0079] Cytb-729-F (10pmol / μL)..........1μl;

[0080] Cytb-882-R (10pmol / μL)..........1μl;

[0081] Add ddH2O to a final volume of 30 μl.

[0082] The PCR amplification reaction conditions were as follows: 95°C pre-denaturation for 30 s, 95°C denaturation for 10 s, 55°C annealing for 30 s, 72°C extension for 30 s, 40 cycles, 72°C final extension for 5 min, and storage at 4°C.

[0083] The qPCR reaction system consists of:

[0084] 2×AceQ qPCR Probe Master Mix....5μl;

[0085] Cytb-729-F (10pmol / μL)............0.2μl;

[0086] Cytb-882-R (10pmol / μL)............0.2μl;

[0087] Cytb-779-Probe (10pmol / μL)..........0.1μl;

[0088] Water sample DNA template......................2μl;

[0089] ROX........................0.2μl;

[0090] Add ddH2O to a final volume of 10 μl.

[0091] The qPCR reaction conditions were as follows: 95°C pre-denaturation for 5 min, 95°C denaturation for 10 s, 60°C annealing for 30 s, 72°C extension for 30 s, for 45 cycles.

[0092] PCR results as follows Figure 3 As shown, the DNA mixture exhibits a relatively clear band at 100-200 bp, while the negative control shows no band, indicating that the primer has good specificity and can accurately identify the DNA of neon damselfish in the water sample. The Ctyb amplification curve and standard curve are shown below. Figure 4 , 5 As shown, the amplification curve exhibited a good S-shape, the standard curve showed good linearity (R²>0.995), and the amplification efficiency was good (E=85%~105%). The obtained standard curve equation can be used for subsequent gene copy number analysis. Water samples from indoor-cultured neon damselfish at various time points were tested, and the results are shown in Figure 6. Figure 6 shows a significant positive correlation between the culture density of neon damselfish and the eDNA concentration. The eDNA concentration of neon damselfish reached a relatively stable stage after 96 hours of culture. Regression analysis was performed on the eDNA copy numbers of neon damselfish in the four time groups from 96h to 168h, and the results are shown in Figures 7 and 8. Figure 8 As shown, the linear function (Y=267120000 X -234320000) has a good fitting effect, with a correlation coefficient R2=0.985. Under normal circumstances, it can be used well for biomass assessment of neon damselfish in stable water bodies.

[0093] In summary, the designed primers and probes can specifically bind to and amplify the Cytb sequence of the target species (neon damselfish), which helps to reflect the distribution characteristics and resource quantity characteristics of this species in water bodies, thus providing data support for the evaluation of the propagation and release effect of neon damselfish, a natural enemy of crown-of-thorns starfish, fisheries management, and related resource surveys.

[0094] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A nucleotide composition for detecting neon damselfish based on environmental DNA technology, characterized in that: This includes primer pairs as shown in SEQ ID NO.1 and SEQ ID NO.

2.

2. The nucleotide composition according to claim 1, characterized in that: It also includes probes such as those shown in SEQ ID NO.

3.

3. A kit comprising the nucleotide composition of claim 1 or 2.

4. A method for detecting neon damselfish using the nucleotide composition according to claim 1 or 2, characterized in that: Includes the following steps: DNA extraction from the sample to be tested; The extracted DNA was amplified by PCR or qPCR using the nucleotide composition of claim 1 or 2. If PCR produces an amplification band or qPCR produces an S-shaped amplification curve, it indicates that neon damselfish is present in the sample being tested.

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