Environment rna-based detection method for ocellated river fish and primer combination thereof

Abstract

This invention discloses a method for detecting *Echinochloa serrata* based on environmental RNA and its primer combination, solving the technical problems of existing environmental RNA detection technologies in benthic fish applications, such as lack of molecular markers, insufficient detection specificity, and inability to reflect physiological states. Based on environmental RNA (eRNA) technology, this invention designs primer pairs targeting the *Ptger4b* gene and combines them with quantitative real-time PCR (qPCR) technology to achieve rapid and sensitive detection of *Echinochloa serrata* in isolated rearing systems. This method is simple to operate, highly sensitive, and has minimal impact on the ecological environment. It is suitable for the detection and quantitative verification of target species in isolated rearing systems and has the potential to be extended to natural marine areas for fish population monitoring, fisheries resource assessment, and species conservation in the future.

Description

Technical Field

[0001] This invention belongs to the field of molecular ecology technology, specifically relating to a method for detecting spotted double-sawed fish based on environmental RNA and its primer combination. Background Technology

[0002] Eye-spotted double-sawfish ( Amphiprion ocellaris ), belonging to the order Perciformes, family Pomacentridae, genus Diplodocus ( Amphiprion The spotted clownfish (Pterodon spp.), commonly known as the "Young Master Clownfish," is a typical rocky reef fish, primarily inhabiting nearshore coral reefs, rocky reefs, and mixed sandy bottom areas. It is wary and highly elusive, feeding on benthic invertebrates and small fish, making it a unique mid-to-upper-level carnivorous fish. There is an urgent need to establish efficient and reliable species monitoring and identification technologies to support its resource management and ecological protection efforts.

[0003] Environmental RNA (eRNA) technology is an emerging ecological molecular detection method. By collecting RNA from environmental samples and combining it with reverse transcription, PCR amplification, and bioinformatics analysis, it enables the identification of species composition and gene expression activity. Environmental RNA mainly originates from cellular, vesicle, or free RNA actively released into the environment by organisms, reflecting the "living presence" and "metabolic state" of organisms over short timescales. Compared to environmental DNA (eDNA) technology, which may retain information from deceased individuals and transmit "historical signals," environmental RNA, due to its rapid degradation and low stability, is better able to reflect the dynamic changes of target organisms. Furthermore, environmental RNA technology is non-invasive, highly sensitive, and has minimal ecological interference, making it particularly suitable for benthic fish such as the spotted double-sawed fish, whose ecological behavior is elusive and whose samples are difficult to obtain, enabling rapid identification and dynamic monitoring of their ecological status.

[0004] However, current environmental RNA research often uses mitochondrial genes (such as COI and cyt b) as molecular markers. Although these genes have high amplification efficiency, their genetic information is derived solely from maternal inheritance, lacking information on expression regulation, and is susceptible to interference from non-target organisms. This limits their ability to reflect the physiological state and ecological dynamics of the target species. Furthermore, environmental RNA research on benthic fish is still in its early stages, lacking a functionally relevant and species-specific nuclear gene molecular marker system, which limits the accuracy and applicability of this technology in practical ecological monitoring.

[0005] In view of the above problems, based on publicly available gene sequence data of target species, it is necessary to develop a nuclear gene marker system with specificity and functional relevance, combined with environmental RNA detection technology, to achieve in vivo identification and dynamic monitoring of benthic fish such as the spotted double-sawfish. In particular, verifying the feasibility of this method first in an independent rearing system is expected to fill existing technological gaps and improve the application level of molecular ecology technology in marine biological resource conservation. Summary of the Invention

[0006] To address the shortcomings of existing environmental RNA detection technologies in benthic fish applications, such as lack of molecular markers, insufficient detection specificity, and difficulty in reflecting physiological states, this invention introduces the nuclear gene Ptger4b (encoding the prostaglandin E receptor 4b subtype), which has expression regulatory functions, into the environmental RNA detection system for the first time. A specific primer combination based on this gene and its application method were developed in a single-species artificial rearing system. A standard curve for quantitative real-time PCR of environmental RNA in *Apatis fasciatus* was constructed to identify the presence of *Apatis fasciatus* in the environment and to quantitatively analyze the nucleic acid of *Apatis fasciatus*. This provides technical support for promoting the application of environmental RNA technology in the identification of ecological behavior, monitoring of reproductive activities, and resource conservation of benthic fish.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] This invention provides a primer combination for detecting environmental RNA in the spotted double-sawed fish, the primer combination comprising a forward primer and a reverse primer, the nucleotide sequences of which are shown in SEQ ID NO:7 and SEQ ID NO:8.

[0009] This invention also provides a specific application of the above primer combination in detecting environmental RNA of the spotted double-sawed fish, for rapidly and accurately identifying the presence of this species from environmental samples.

[0010] The present invention further provides a kit for detecting environmental RNA in the spotted double-sawed fish, comprising the above-mentioned specific primers.

[0011] The present invention also provides a real-time fluorescent kit for detecting environmental RNA in the spotted double-sawfish, comprising the primer combination described above.

[0012] In addition, the present invention provides a real-time quantitative PCR method for environmental RNA of the spotted double-sawed fish, specifically: RNA extracted from environmental samples is reverse transcribed and quantitatively amplified using the above primer combination, a standard curve of Ptger4b gene standard plasmid concentration versus Ct value is plotted, and the presence of the spotted double-sawed fish is determined by analyzing the amplification curve and Ct value, and quantitative analysis is performed.

[0013] Preferably, the real-time PCR reaction system is 20 μL, comprising: 10 μL of 2×qPCR Mix, 0.4 μL of forward primer, 0.4 μL of reverse primer, 2 μL of template cDNA, and 7.2 μL of RNase-Free H2O.

[0014] Preferably, the amplification program of real-time PCR adopts a two-step method, specifically including: (1) pre-denaturation at 95℃ for 5 min; (2) denaturation at 95℃ for 10 s, annealing and extension at 60℃ for 30 s, for 40 cycles.

[0015] Based on the above technical solution, the embodiments of the present invention can produce at least the following technical effects:

[0016] (1) The present invention selects the nuclear gene Ptger4b as the environmental RNA detection gene, which overcomes the problems of single genetic information of mitochondrial gene markers and pollution interference, and improves the ability of detection to indicate physiological state.

[0017] (2) The present invention designs a specific primer pair for the spotted double-sawed fish, which has high specificity, sensitivity and repeatability, and can achieve accurate detection of environmental RNA of the target species with stable and reliable results;

[0018] This invention establishes and validates a rapid detection method based on environmental RNA technology in an independent rearing system. This method offers advantages such as non-invasiveness, high sensitivity, strong specificity, ease of operation, and time and labor savings. Under controlled conditions, this method can effectively detect and quantify environmental RNA in *Dichthys oryzae*, providing experimental verification of its technical feasibility. Furthermore, it has the potential to be extended to natural marine areas for population monitoring, breeding season activity identification, and habitat assessment of concealed benthic fish. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0020] Figure 1 The results show the specificity verification of the Ptger4b-1 primer on NCBI Primer-BLAST.

[0021] Figure 2 The results show the specificity verification of the Ptger4b-2 primer on NCBI Primer-BLAST.

[0022] Figure 3 The results show the specificity verification of the Ptger4b-3 primer on NCBI Primer-BLAST.

[0023] Figure 4 The results show the specificity verification of the Ptger4b-4 primer on NCBI Primer-BLAST.

[0024] Figure 5 Melting curve of Ptger4b-1 primer;

[0025] Figure 6 Melting curve of Ptger4b-2 primers;

[0026] Figure 7 Melting curve of Ptger4b-3 primer;

[0027] Figure 8 Melting curve of Ptger4b-4 primer;

[0028] Figure 9 The sequencing peak diagram shows the amplification product of the upstream primer of Ptger4b-4.

[0029] Figure 10 Sequencing peak diagram of the amplification product of the downstream primer of Ptger4b-4;

[0030] Figure 11 The image shows the BLAST alignment results of the sequencing results of the upstream primer of Ptger4b-4.

[0031] Figure 12 The image shows the BLAST alignment results of the sequencing results of the Ptger4b-4 downstream primers.

[0032] Figure 13 Standard curve of environmental RNA concentration versus Ct value for *Eye-spotted Double-sawed Fish*;

[0033] Figure 14 The graphs show the qPCR amplification curves for S1, S2, S3, and NTC. Detailed Implementation

[0034] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. In addition, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0035] The manufacturers of the instruments, reagents and materials used in this embodiment are: QuantStudio™ 5 Real-Time PCR System, Micro Spectrophotometer (DN-100, Hangzhou Mio), a set of precision pipettes (Thermo, USA), Small-volume total RNA extraction kit (extracted from TRIzol® lysate), EZNA® Soil RNA Kit, 5X All-In-One RT MasterMix with AccuRT Genomic DNA Removal Kit, and Hieff® qPCR SYBR Green Master Mix (No Rox).

[0036] Example 1: Primer Design and Screening for the Ptger4b Gene

[0037] This embodiment is for the spotted double-sawed fish ( Amphiprion ocellaris Primer design, in vitro screening, and specificity verification were conducted on the Ptger4b gene to obtain primer pairs suitable for quantitative detection of eRNA in water. First, the Ptger4b gene mRNA sequence (sequence length: 2746 bp, NCBI accession number: XM_023279542.3) from the cell nucleus of *Eriocheir squarrosa* was retrieved from the NCBI database. Four candidate primer pairs were designed using Primer Premier 5.0.

[0038] Subsequently, the specificity, melting temperature (Tm), and dimer structure of the primers were analyzed and evaluated using Oligo7 software to screen out primers with reasonable structure and high specificity. The designed primers were synthesized by Qingdao NIO Biotechnology Co., Ltd., and the four primer pairs were named Ptger4b-1 (F: SEQ ID NO: 1, R: SEQ ID NO: 2), Ptger4b-2 (F: SEQ ID NO: 3, R: SEQ ID NO: 4), Ptger4b-3 (F: SEQ ID NO: 5, R: SEQ ID NO: 6), and Ptger4b-4 (F: SEQ ID NO: 7, R: SEQ ID NO: 8). The specific sequences and expected amplified fragment lengths are shown in Table 1 below.

[0039] Table 1 Primer sequences

[0040]

[0041] To verify the specificity of the primers at the database level, further specificity analysis was performed using the NCBI Primer-BLAST tool. The results showed that, in addition to generating an amplified fragment in the Ptger4b gene of the spotted double-sawed fish, primer Ptger4b-1 also produced potential amplification products in several other fish gene regions in the database, suggesting a risk of non-specific amplification. The Primer-BLAST analysis results are as follows: Figure 1 As shown. Primers Ptger4b-2, Ptger4b-3, and Ptger4b-4 produced a unique and correct amplified fragment only on the Ptger4b gene of *Elaphe squarrosa*. No valid products meeting the amplification conditions were detected in other species in publicly available databases, indicating that these three primer pairs possess good theoretical specificity. The results of Primer-BLAST analysis are shown below. Figures 2 to 4 As shown, the species and gene specificity of the four primer pairs were verified using Primer-BLAST. It should be noted that although currently available public sequence databases of target gene regions are insufficient to establish absolute species specificity, in vitro testing remains the best predictive method at present and provides theoretical support for subsequent in vitro experimental verification.

[0042] Next, in vitro experiments were conducted to validate the four primer pairs. First, muscle tissue from artificially bred *Echinochloa spp.* was extracted using a small-volume total RNA extraction kit (from TRIzol® lysates). The RNA concentration and purity were then determined using a DN-100 ultra-micro spectrophotometer. The results showed an RNA concentration of 161.415 ng / μL and an A260 / A280 value of 2.01, indicating that the RNA purity met the requirements for subsequent reverse transcription and quantitative PCR experiments.

[0043] The obtained RNA samples were reverse transcribed using the 5X All-In-One RT MasterMix with AccuRT Genomic DNARemoval Kit. The procedure included two steps: removal of genomic DNA and cDNA synthesis. The resulting cDNA could be used for subsequent qPCR validation experiments.

[0044] The SYBR Green dye method was used to verify the four primer pairs by qPCR amplification using the obtained cDNA template. The reaction system contained 10 μL of 2×qPCR Mix, 0.4 μL of forward primer, 0.4 μL of reverse primer, 2 μL of template cDNA, 7.2 μL of RNase-Free H2O, and a template-free control was included.

[0045] The quantitative PCR amplification program was set as follows: (1) pre-denaturation at 95℃ for 5 min; (2) denaturation at 95℃ for 10 s, annealing and extension at 60℃ for 30 s, for 40 cycles, followed by melting curve analysis.

[0046] After amplification, the melting curve results were analyzed. Figures 5 to 8 The melting curves of Ptger4b-1, Ptger4b-2, and Ptger4b-3 all showed multiple peaks, indicating non-specific amplification. In contrast, the melting curve of the Ptger4b-4 amplification product showed a single peak, with no primer dimers or non-specific amplification observed, demonstrating good specificity and meeting the target primer design requirements.

[0047] The qPCR amplification product of Ptger4b-4 was purified and then subjected to Sanger sequencing. The electrophoresis peak diagram of the sequencing results is shown below. Figure 9 (Upstream primer) and Figure 10 As shown in the downstream primer, the peak shape is clear and the background noise is low.

[0048] The sequence obtained from sequencing was compared with the target Ptger4b gene sequence using BLAST. The results are shown below. Figure 11 and Figure 12 The comparison results showed that the amplified fragment was highly consistent with the Ptger4b mRNA sequence of the spotted double-sawfish, further verifying the accuracy of the Ptger4b-4 primer amplification product.

[0049] Based on the above results, Ptger4b-4 exhibits superior specificity and amplification efficiency in qPCR amplification compared to the other three primer pairs, making it the optimal primer combination for subsequent standard curve construction and environmental RNA sample detection.

[0050] This embodiment is used for the detection and quantification of Ptger4b in the artificial rearing system of *Pterocarya stenoptera*. Since no other fish species were present in this scenario, no cross-species experiments were conducted for verification. Single-peak melting curve analysis, sequencing consistency confirmation, and negative controls all meeting the accuracy requirements for the detection and quantification of the *Pterocarya stenoptera* gene in this scenario have been achieved. It should be noted that while this method has been validated in the artificial rearing system, further cross-species verification and environmental sample testing are needed in actual marine environments due to the more complex community backgrounds to assess its universality and stability in natural ecosystems.

[0051] Example 2: Establishment of a real-time quantitative PCR detection method based on the eye-spotted double-sawfish standard

[0052] This embodiment aims to construct a standard curve for the quantitative detection of the Ptger4b gene. The standard used is a recombinant plasmid with a concentration of 49.20 ng / μL and a total length of 2797 bp. To obtain the copy number concentration of the standard, the copy number was calculated using the following formula: Copy number (copies / μL) = (Concentration (ng / μL) × 6.022 × 10⁻⁶) 14 )÷(plasmid length (bp)×660).

[0053] Substituting the data into the formula, the calculation is as follows: Copy number = (49.20 × 6.022 × 10) 14 ) ÷ (2797 × 660) ≈ 1.60 × 10 10 Therefore, the initial concentration of the plasmid stock solution is approximately 1.60 × 10⁻⁶ copies / μL. 10 copies / μL.

[0054] Dilute the standard quality grain mother liquor to 1×10 9 Copies / μL were used for subsequent standard curve preparation. Seven different concentrations of standards were prepared sequentially using a 10-fold serial dilution method. The specific procedure is as follows: Using 1×10... 9 The standard solution of copies / μL was used as the starting solution. 10 μL was taken each time and added to 90 μL of sterile, enzyme-free water. After mixing, 1×10⁻⁶ copies / μL of solution was obtained. 8 Standards were obtained in copies / μL. Following this procedure, a series of standards at the following concentrations were obtained: 1×10⁻⁶. 8 copies / μL, 1×10 7 copies / μL, 1×10 6 copies / μL, 1×10 5 copies / μL, 1×10 4 copies / μL, 1×10 3copies / μL, 1×10 2 Copies / μL, a total of 7 concentration gradients.

[0055] The seven standards at different concentrations were used in quantitative real-time PCR (qPCR) to construct a standard curve for the Ptger4b gene.

[0056] The real-time PCR reaction system (20 μL) consisted of: 10 μL of 2×SYBR Green qPCR Mix, 0.4 μL of forward primer, 0.4 μL of reverse primer, 2 μL of template DNA, and 7.2 μL of RNase-Free H2O.

[0057] The amplification program was set as follows: (1) 95℃ pre-denaturation for 5 min; (2) 95℃ denaturation for 10 s, 60℃ annealing extension for 30 s, for 40 cycles.

[0058] The logarithmic concentration of the standard (log 10 Plot a standard curve with copies / μL as the x-axis and Ct as the y-axis, as follows: Figure 13 As shown. The results showed that the Ct value had a good linear relationship with the logarithm of the template concentration. The regression equation of the fitted curve was: y = –3.7385x + 36.775, where x is the logarithmic concentration of the eye-spotted double-sawfish plasmid standard, y is the Ct value of the corresponding concentration of qPCR reaction, the logarithm base is 10, the correlation coefficient of the standard curve was R² = 0.9997, and the amplification efficiency corresponding to the slope was 85.13%.

[0059] The constructed standard curve can serve as an absolute quantitative reference standard for the expression level of the Ptger4b gene in environmental RNA samples, and is suitable for highly sensitive detection and analysis based on qPCR.

[0060] Example 3: Extraction of environmental RNA and detection of target genes

[0061] This embodiment uses the spotted double-sawed fish under artificial breeding conditions as the research object, extracts environmental RNA from its living water, and performs subsequent qPCR detection.

[0062] Three aquariums (S1, S2, and S3) were set up for the experiment. Aquariums S1 and S2 each housed two mature female and two male *Eye-spotted* wrasses. Aquarium S3 served as a control group, containing no fish. 4L of seawater was collected from each aquarium and immediately filtered using a 0.22μm GP (Millipore) filter column and a peristaltic pump. To prevent degradation of environmental RNA, the entire filtration process was conducted at a low temperature of 4–10℃.

[0063] The collected seawater samples were placed in brown sampling bottles, immediately sealed in a foam insulated box, and an appropriate amount of dry ice was added to maintain a low temperature to prevent environmental RNA degradation and water sample freezing.

[0064] After filtration, add 2 mL of RNA preservation solution (RNA Locker) to each filter column and freeze at -80°C for use in subsequent experiments.

[0065] After the filter column was returned to the laboratory, the filter membrane was removed and cut into small pieces under low temperature conditions, and then mechanically ground in a grinder pre-cooled with liquid nitrogen. The ground sample was then quickly transferred to a 2 mL sterile centrifuge tube free of RNase contamination, and environmental RNA was extracted according to the EZNA® Soil RNA Kit manual. RNA concentration and purity were determined using a DN-100 ultra-micro spectrophotometer.

[0066] The extracted environmental RNA samples were reverse transcribed to synthesize complementary DNA (cDNA). The resulting cDNA was used as a template for quantitative real-time PCR (qPCR) amplification and detection according to the method described in Example 2. Three technical replicates were set up for each sample, and a template-free control (NTC) was included to rule out the risk of contamination.

[0067] Amplification results as follows Figure 14 As shown in the table below, the Ct values ​​detected in groups S1 and S2 were 28.53 and 31.43, respectively. No Ct value was detected in group S3 (blank control), and no amplification curve was observed. Similarly, no amplification signal was observed in the NTC group, indicating that the reaction system was free of exogenous nucleic acid contamination. Based on the established Ptger4b standard curve, the environmental RNA concentrations in groups S1 and S2 were 2069.22 copies / μL and 368.997 copies / μL, respectively, while the concentration in group S3 was 0 copies / μL. Specific results are shown in the table below.

[0068] Table 2. Ct values ​​and copy numbers for different samples

[0069]

[0070] The results of this embodiment demonstrate that the method of the present invention can successfully extract environmental RNA from fish-derived RNA in water samples from artificial rearing systems, and achieve the detection and quantification of target genes by combining quantitative real-time PCR and standard curves. Under the experimental conditions, the detection results exhibit good sensitivity and specificity, effectively distinguishing the experimental group from the blank control group, indicating the feasibility of this method in independent rearing systems. In the future, this method has the potential to be further extended to more complex environments for applications in fish population monitoring, breeding season identification, and related ecological research.

[0071] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. The application of primer combinations in detecting *Dystomata occulta* based on environmental RNA in a single-species artificial rearing system, characterized in that... The nucleotide sequences of the primer combination are shown in SEQ ID NO:7 and SEQ ID NO:

8.

2. A method for detecting *Dictyophora sclerotiorum* based on environmental RNA in a single-species artificial rearing system, characterized in that, The primer combination described in claim 1 was used to amplify environmental RNA samples by qPCR, and the presence of *Euphorbia tirucalli* in the water sample was determined by analyzing the qPCR amplification curve and Ct value.

3. The method for detecting *Dictyophora sclerotiorum* based on environmental RNA in a single-species artificial rearing system according to claim 2, characterized in that, qPCR amplification was performed on environmental RNA samples, and a standard curve was plotted between the concentration of the Ptger4b gene plasmid standard and the Ct value. The concentration of environmental RNA was determined based on the standard curve.

4. The method for detecting *Dictyophora sclerotiorum* based on environmental RNA in a single-species artificial rearing system according to claim 2, characterized in that, The qPCR amplification reaction system is 20 μL, including 10 μL of 2×qPCR Mix, 0.4 μL of forward primer, 0.4 μL of reverse primer, 2 μL of template DNA, and 7.2 μL of RNase-free H2O.

5. The method for detecting *Dictyophora sclerotiorum* based on environmental RNA in a single-species artificial rearing system according to claim 2, characterized in that, The qPCR amplification program is a two-step method: (1) pre-denaturation at 95℃ for 5 min; (2) denaturation at 95℃ for 10 s, annealing and extension at 60℃ for 30 s, for 40 cycles.

6. The method for detecting *Dictyophora sclerotiorum* based on environmental RNA in a single-species artificial rearing system according to claim 2, characterized in that, The testing process includes a template-free control to screen for contamination risks. If the water sample to be tested shows an S-shaped amplification curve and has a Ct value, it is determined that the water sample contains *Euphorbia milii*. If the water sample to be tested does not show an amplification curve and has no Ct value, it is determined that the water sample does not contain *Euphorbia milii*.

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