A environmental dna detection kit for pseudobagrus eupogastus
By designing a primer-probe composition based on the Cytb gene of the mitochondrial fin of the catfish and using real-time fluorescent PCR technology, the specificity and sensitivity issues of rare fish detection were solved, enabling efficient and accurate monitoring of the catfish, which is suitable for non-destructive detection and continuous monitoring in complex waters.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- PEARL RIVER WATER RESOURCES PROTECTION INST
- Filing Date
- 2026-03-09
- Publication Date
- 2026-06-09
AI Technical Summary
Existing aquatic organism monitoring methods are insufficient to achieve high specificity and sensitivity in detecting rare and endangered fish such as the long-tailed catfish, and traditional fishing monitoring suffers from problems such as high false negative rates and significant habitat limitations.
A kit for detecting environmental DNA from the long-rumped catfish was designed. It contains a primer and probe composition based on the mitochondrial Cytb gene, combined with real-time fluorescence PCR detection technology, using TaqMan-MGB probe and UDG enzyme. The sample is processed by vacuum filtration through a filter membrane and liquid nitrogen grinding to achieve efficient enrichment and qualitative detection of environmental DNA.
It achieves high specificity and high sensitivity detection of catfish, avoids false positive results, simplifies the operation process, is suitable for rapid screening of large batches of samples, adapts to monitoring in complex waters, and supports non-destructive detection and continuous monitoring of rare fish species.
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Figure CN122168763A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of molecular biology detection technology, and in particular to a kit for detecting environmental DNA in the long-rumped catfish. Background Technology
[0002] The long-scrofted catfish is a rare and endangered bottom-dwelling freshwater fish species unique to the Pearl River system and Hainan Island system in my country. It is a key target for the protection of aquatic biological resources and an important species for the release of fish into the water for ecological restoration in related waters. Accurate monitoring of its population distribution and resource quantity is of great significance for the protection and restoration of this species.
[0003] Currently, the main methods for monitoring aquatic organisms are divided into two categories: traditional fishing monitoring and molecular biological detection. Traditional fishing monitoring is a routine method for investigating aquatic fish resources, while molecular biological detection, due to its high specificity and accuracy, has gradually become an important supplement to the monitoring of rare fish species. Environmental DNA detection technology, as a new molecular detection method, does not require capturing biological entities; it can achieve species detection simply by collecting environmental DNA samples from the water. This meets the protection needs of rare and endangered fish species and has been widely used in the field of aquatic organism monitoring.
[0004] The mitochondrial Cytb gene, characterized by maternal inheritance, high intraspecific conservation, and significant interspecific differences, is a classic molecular target for fish species identification. It provides a molecular basis for the specific detection of environmental DNA in the long-tailed catfish. Designing a dedicated primer-probe combination based on this gene and constructing an efficient real-time fluorescent PCR detection system has become a key technological direction for achieving accurate monitoring of the long-tailed catfish. Summary of the Invention
[0005] The purpose of this invention is to provide an environmental DNA detection kit for the long-rumped catfish to solve the problems mentioned in the background art.
[0006] To achieve the above objectives, the present invention adopts the following technical solution:
[0007] A DNA detection kit for the environmental organism *Cyprinus longipedensis* includes a primer and probe composition for real-time fluorescence PCR detection based on the *Cyprinus longipedensis* mitochondrial Cytb gene. The composition includes an upstream primer, a downstream primer, and a TaqMan-MGB probe. The nucleotide sequence of the upstream primer is shown in SEQ ID No. 1, the nucleotide sequence of the downstream primer is shown in SEQ ID No. 2, and the nucleotide sequence of the TaqMan-MGB probe is shown in SEQ ID No. 3. The 5' end of the TaqMan-MGB probe is modified with a fluorescent reporter group, and the 3' end is coupled with a minor groove binder and a non-fluorescent quencher group.
[0008] As a further improvement to this technical solution: the working concentrations of the upstream primer, downstream primer, and TaqMan-MGB probe are all 10 μmol / L.
[0009] As a further improvement to this technical solution: the kit also includes qPCR amplification premix, DNA extraction reagent, positive control and negative control, wherein the qPCR amplification premix is 2×ProbeqPCRSuperMix containing UDG enzyme.
[0010] As a further improvement to this technical solution: the positive control is plasmid DNA containing the amplified fragment between SEQ ID No. 1 and SEQ ID No. 2 of the Cytb gene of the long-tailed catfish or the genomic DNA of the long-tailed catfish, and the negative control is nuclease-free ultrapure water.
[0011] As a further improvement to this technical solution: the primer and probe composition is used to amplify the total DNA of the sample to be tested using real-time fluorescent PCR to complete the qualitative detection of the long-rumped catfish.
[0012] As a further improvement to this technical solution: the sample to be tested is a water sample or a sediment sample from the water area to be tested. The water sample is vacuum filtered through a filter membrane to enrich environmental DNA, and the sediment sample is ground with liquid nitrogen. The total DNA extracted from the treated sample is used as a template for real-time fluorescent PCR amplification.
[0013] As a further improvement to this technical solution: the total volume of the real-time fluorescence PCR amplification system is 20 μL, including 10 μL of 2×ProbeqPCRSuperMix, 0.4 μL of 10 μmol / L upstream primer, 0.4 μL of 10 μmol / L downstream primer, 0.4 μL of 10 μmol / L TaqMan-MGB probe, 0.4 μL of 50×PassiveReferenceDye, 1 μL of DNA template, and the remaining volume is made up with nuclease-free ultrapure water.
[0014] As a further improvement to this technical solution: the reaction program of the real-time fluorescence PCR is as follows: pre-denaturation at 94℃ for 5 min, followed by 94℃ for 5 s and 60℃ for 30 s, for a total of 40 cycles. After amplification, samples with a Ct value ≤ 35 and a typical S-shaped amplification curve are judged as positive, and samples with a Ct value > 35 or no amplification curve are judged as negative.
[0015] Compared with the prior art, the beneficial effects of the present invention are:
[0016] 1. This invention provides a proprietary primer and probe composition and a complete detection system for detecting environmental DNA in *Catfish spp.*, achieving high specificity and sensitivity for *Catfish spp.* The kit uses *Catfish spp.*-specific mitochondrial genes as the detection target, and the accompanying TaqMan-MGB probe has the ability to accurately identify subtle differences in gene sequences, effectively distinguishing *Catfish spp.* from closely related species in the same domain, thus avoiding false positive results caused by cross-amplification. Simultaneously, based on the easily degraded and fragmented characteristics of environmental DNA, the amplification fragments are designed to efficiently enrich and detect trace amounts of target DNA in samples. Combined with the UDG enzyme anti-contamination system and full-process positive and negative control quality control, errors caused by experimental operation and system contamination are significantly reduced, ensuring the accuracy and stability of the detection results.
[0017] 2. This invention employs an environmental DNA detection mode, eliminating the need for physical capture of the long-scroped catfish. This enables non-destructive testing of this rare and endangered fish, highly aligning with the requirements of aquatic biological resource protection and allowing for long-term, continuous monitoring of population dynamics. The reagent kit is a complete, ready-to-use product, requiring no additional core reagents from the user. The standardized operating procedure, combined with real-time fluorescent PCR closed-tube detection technology, eliminates the need for subsequent cumbersome steps such as electrophoresis and sequencing, significantly simplifying the operation process, shortening the detection cycle, and adapting to rapid screening of large batches of samples. Furthermore, it allows for on-site monitoring in various aquatic habitats, effectively overcoming the shortcomings of traditional fishing monitoring methods, such as high false negative rates and significant habitat limitations. This provides convenient and scientific technical support for conservation efforts such as long-scroped catfish resource distribution surveys and evaluation of stock enhancement and release effects.
[0018] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it according to the contents of the specification, the preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings. Specific embodiments of the present invention are given in detail below with reference to the accompanying drawings. Attached Figure Description
[0019] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this application, illustrate exemplary embodiments of the invention and, together with their description, serve to explain the invention and do not constitute an undue limitation thereof. In the drawings:
[0020] Figure 1 Agarose gel electrophoresis image of the primer pair's conventional PCR amplification products;
[0021] Figure 2 Amplification curves for real-time fluorescence PCR specificity verification of *Catfish spp.* and closely related species.
[0022] Figure 3 A bar chart comparing the detection results of *Catfish simonii* in the Hongshui River Basin. Detailed Implementation
[0023] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. The invention is described more specifically in the following paragraphs by way of example with reference to the accompanying drawings. It should be noted that the drawings are in a very simplified form and use non-precise proportions, and are only used to facilitate and clarify the illustration of the embodiments of the present invention.
[0024] Please see Figures 1-3 In this embodiment of the invention, a DNA detection kit for the environment of *Cyprinus longipedensis* includes a primer and probe composition for real-time fluorescence PCR detection designed based on the *Cyprinus longipedensis* mitochondrial Cytb gene. The composition includes an upstream primer, a downstream primer, and a TaqMan-MGB probe. The nucleotide sequence of the upstream primer is shown in SEQ ID No. 1, the nucleotide sequence of the downstream primer is shown in SEQ ID No. 2, and the nucleotide sequence of the TaqMan-MGB probe is shown in SEQ ID No. 3. The 5' end of the TaqMan-MGB probe is modified with a fluorescent reporter group, and the 3' end is coupled with a minor groove binder and a non-fluorescent quencher group.
[0025] Specifically, the target gene is the Cytb gene of the mitochondrial mitochondria of the long-tailed catfish, which forms the basis for the design of this primer-probe composition. Mitochondrial genes are characterized by maternal inheritance, high intraspecific conservation, and significant interspecific differences. The Cytb gene is a classic barcode gene for fish species identification. Compared with nuclear genes, it degrades more slowly in environmental water and has a higher intracellular copy number, making it more suitable for the needs of environmental DNA trace detection and ensuring the accuracy of species identification at the gene level.
[0026] Primer and probe composition: This is the core functional element of the kit for achieving specific detection of catfish. It consists of three parts: upstream primer, downstream primer, and TaqMan-MGB probe. The three work together to achieve specific amplification of the target gene and detection of fluorescence signals.
[0027] Upstream primer: The nucleotide sequence is shown in SEQ ID No. 1, which is the legally recognized sequence identification number. The specific DNA base sequence is 5'-CCCACTACTACACACCTCCAAATTAC-3', a total of 27 bases. The primer is the initiation element for PCR amplification. The upstream primer can specifically bind to the upstream complementary sequence of the target fragment of the Cytb gene of *Cyprinus longissima*, guiding Taq enzyme to initiate DNA chain synthesis. This sequence is a *Cyprinus longissima*-specific sequence obtained through multiple sequence alignment and multiple rounds of specific screening with closely related species. It has no homologous match with other catfish species distributed in the same region.
[0028] Downstream primer: The nucleotide sequence is shown in SEQ ID No. 2. The specific DNA base sequence corresponding to SEQ ID No. 2 is 5'-GTCTGCTACAAGGGCTCAAAATAATA-3', which consists of 26 bases. The downstream primer specifically binds to the downstream complementary sequence of the target fragment and pairs with the upstream primer to form a complete amplicon. In this scheme, the target fragment amplified by the upstream and downstream primer pairing is only 82 bp in length, which is suitable for the characteristics of environmental DNA that is easily degraded and severely fragmented.
[0029] TaqMan-MGB probe: The nucleotide sequence is shown in SEQ ID No. 3. The specific DNA base sequence corresponding to SEQ ID No. 3 is 5'-AAGCCTCACCTTCCGTC-3', which has a total of 17 bases. This probe is the core component for realizing real-time fluorescence PCR quantification and high-specificity detection. The modification groups at both ends of the probe are the key to achieving high-specificity and high-sensitivity detection.
[0030] 5' end modified fluorescent reporter group: FAM fluorescent group is usually selected as the source of fluorescence signal emission; when the probe is intact, the emitted fluorescence signal will be absorbed by the 3' end quencher group, and the real-time PCR instrument cannot detect the effective fluorescence signal; after the probe is degraded by enzyme during PCR amplification, the fluorescent reporter group and the quencher group are separated, and the detectable fluorescence signal can be released, so as to achieve complete synchronization between the fluorescence signal and the amplification of PCR products.
[0031] 3' end-coupled minor groove binder (MGB) and non-fluorescent quencher (NFQ): The MGB group can specifically bind to the minor groove region of the DNA double helix, significantly increasing the melting temperature of the probe without increasing the number of probe bases, allowing short probes to achieve stable double-strand binding. At the same time, it greatly improves the sensitivity to single base mismatch recognition, and can accurately distinguish the gene sequence differences between *Catfish spp.* and closely related species in the same domain. The NFQ is a non-fluorescent quencher group that does not produce background fluorescence. Compared with the traditional TAMRA quencher group, it can significantly reduce the background noise of the reaction system and improve the detection capability of weak fluorescence signals in trace samples.
[0032] The working concentrations of the upstream primer, downstream primer, and TaqMan-MGB probe were all 10 μmol / L;
[0033] Specifically, 10 μmol / L is the working concentration of the primer and probe stock solution. This concentration is the optimal concentration determined through multiple rounds of gradient optimization experiments. At this concentration, the final concentration of primers and probes in the PCR amplification system can be guaranteed to reach the optimal level of 0.2 μmol / L. This avoids insufficient amplification efficiency, weak fluorescence signal, and missed sample detection caused by too low a concentration, as well as non-specific amplification and primer dimer formation caused by too high a concentration, thus ensuring the efficiency and specificity of the amplification reaction.
[0034] The kit also includes qPCR amplification premix, DNA extraction reagent, positive control and negative control. The qPCR amplification premix is 2×ProbeqPCRSuperMix containing UDG enzyme.
[0035] Specifically, the qPCR amplification premix is the core reagent for real-time fluorescence PCR amplification reactions. This protocol specifies 2× ProbeqPCRSuperMix containing UDG enzyme, a ready-to-use premix containing hot-start Taq DNA polymerase, dNTPs, Mg²⁺, PCR buffer, UDG enzyme, reference dye, and other core components. The UDG enzyme can eliminate aerosol contamination caused by residual PCR amplification products, fundamentally reducing false positive results caused by aerosol contamination common in molecular laboratories, ensuring the stability and reliability of test results. The 2× concentration is twice the working concentration, allowing for direct dilution before use, significantly simplifying the operation process and reducing human error.
[0036] DNA extraction reagents: used to extract and purify total DNA from water sample filter membranes, sediment, and fish tissue samples. They are essential reagents for obtaining qualified test templates. The conventional genomic DNA extraction kits are column-based or magnetic bead-based. They can efficiently enrich trace DNA in environmental samples while removing PCR inhibitors such as humic acid and heavy metals from water and sediment, ensuring the smooth progress of subsequent amplification reactions.
[0037] Positive and negative controls are core components of quality control in testing experiments. They are used to monitor the effectiveness of the entire testing process, avoid false positive and false negative results, and ensure the reliability and repeatability of experimental data.
[0038] The positive control was plasmid DNA containing the amplified fragment between SEQ ID No. 1 and SEQ ID No. 2 of the Cytb gene of the long-rumped catfish, or the genomic DNA of the long-rumped catfish; the negative control was nuclease-free ultrapure water.
[0039] Specifically, positive controls are available in two types, both of which are nucleic acid samples known to contain the target amplification fragment. Their core function is to verify the effectiveness of the PCR amplification system, monitor whether the amplification reaction is proceeding normally, and avoid false negative results caused by system failure.
[0040] Plasmid DNA containing the target amplification fragment: This is a standard obtained by constructing the 82bp target fragment between the upstream and downstream primers of the Cytb gene of the long-tailed catfish into a plasmid vector. It has the characteristics of stable concentration, known copy number, and no background interference. It can be used not only for system validity verification, but also for constructing standard curves to achieve absolute quantification of the environmental DNA of the long-tailed catfish in water.
[0041] Genomic DNA of Catfish: The whole genome DNA extracted from the fin rays / muscle tissue of Catfish is the closest positive control to the actual test sample, which can fully verify the effectiveness of the entire process from primer binding, probe matching to amplification;
[0042] Negative control: This is nuclease-free ultrapure water. Its core function is to monitor whether there is exogenous nucleic acid contamination or cross-contamination between samples throughout the entire testing process. During the testing process, the negative control participates synchronously in the entire process of sample pretreatment, DNA extraction, and PCR amplification. If the negative control shows an amplification signal, it indicates that there is contamination in the experimental system, and the results of this batch of experiments are invalid and need to be retested.
[0043] The primer and probe composition was used to amplify the total DNA of the sample in real time using fluorescent PCR to complete the qualitative detection of the long-rumped catfish.
[0044] Specifically, the core purpose of the kit is to achieve qualitative detection of catfish in aquatic environments through real-time fluorescent PCR amplification using primer-probe combinations. Real-time fluorescent PCR amplification is the core technology of this kit. Through the specific amplification of the target gene fragment by primers, the fluorescent signal is released simultaneously by the desorption of the probe enzyme. The instrument can collect the fluorescent signal in real time to achieve qualitative detection of the target nucleic acid without the need for subsequent electrophoresis, sequencing and other operations, which greatly shortens the detection cycle. The entire process from water sample collection to test result output can be completed within 6 hours.
[0045] The samples to be tested are water samples or bottom sediment samples from the water area to be tested. The water samples are vacuum filtered through a filter membrane to enrich environmental DNA, and the bottom sediment samples are ground with liquid nitrogen. The total DNA extracted from the treated samples is used as a template for real-time fluorescent PCR amplification.
[0046] Specifically, the types of samples to be tested are limited to water samples or bottom sediment samples from the water area to be tested, which are perfectly suited to the benthic biological characteristics of the long-rumped catfish; water samples are the preferred samples for environmental DNA testing, as they are easy to collect, do not damage the aquatic habitat, and are suitable for large-scale resource surveys; environmental DNA in bottom sediment samples has a longer retention time and more stable concentration, and is not easily affected by water flow, making it suitable for complex habitats with low DNA concentrations in water samples, such as rapids and rock crevices.
[0047] Water sample pretreatment: Vacuum filtration and enrichment: The water sample is vacuum filtered through a sterile filter membrane with a pore size of 0.45μm to trap nucleic acid-containing tissue fragments such as the epidermis, secretions, and excrement of the long-tailed catfish suspended in the water on the filter membrane, thereby enriching trace amounts of environmental DNA in the water.
[0048] Sediment sample pretreatment: Liquid nitrogen grinding treatment, by rapidly freezing the sediment sample at ultra-low temperature with liquid nitrogen, making the sample brittle, and grinding can fully release the environmental DNA adsorbed in the sediment particles, ensuring efficient nucleic acid extraction;
[0049] Template preparation: Total DNA is extracted from the processed sample and used as an amplification template. This involves purifying the enriched environmental DNA and removing PCR inhibitors to obtain a qualified nucleic acid template that can be used for PCR amplification, which is the basis for ensuring the smooth progress of the amplification reaction.
[0050] The total volume of the real-time fluorescence PCR amplification system was 20 μL, including 10 μL of 2×ProbeqPCRSuperMix, 0.4 μL of 10 μmol / L upstream primer, 0.4 μL of 10 μmol / L downstream primer, 0.4 μL of 10 μmol / L TaqMan-MGB probe, 0.4 μL of 50×PassiveReferenceDye, and 1 μL of DNA template. The remaining volume was made up with nuclease-free ultrapure water.
[0051] Specifically, the total system volume is 20 μL: this is the general standard system volume for real-time fluorescence PCR detection, which is compatible with the detection specifications of conventional 8-tube and 96-well plate fluorescence quantitative PCR instruments. The amount of reagents is moderate, which ensures the stability and repeatability of the amplification reaction while controlling the cost of single sample detection.
[0052] 2×ProbeqPCRSuperMix 10μL: This accounts for 50% of the total volume of the system, providing all the basic components required for PCR amplification and ensuring the smooth progress of the amplification reaction;
[0053] Upstream and downstream primers and probes: Add 0.4 μL of each. A stock solution concentration of 10 μmol / L can ensure the optimal final concentration of 0.2 μmol / L in the system. This concentration ratio has been optimized through multiple rounds to achieve the highest amplification efficiency and the lowest non-specific amplification.
[0054] 50×PassiveReferenceDye 0.4μL: This is the passive reference dye, usually ROX dye. Its core function is to correct for differences in fluorescence signals between wells, instrument detection errors, and system volume errors, eliminate non-PCR-related fluorescence signal fluctuations, and ensure the accuracy of Ct value calculation.
[0055] 1 μL DNA template: This is the core template to be tested. The volume has been optimized to ensure sufficient template quantity while avoiding interference from PCR inhibitors carried in the template.
[0056] Nuclease-free ultrapure water: Used to replenish the total volume of the system. It contains no nuclease, no DNase, and no RNase, avoiding exogenous nucleic acid contamination and degradation, and ensuring the stability of the system.
[0057] The reaction program for real-time fluorescence PCR is as follows: pre-denaturation at 94℃ for 5 min, followed by 94℃ for 5 s and 60℃ for 30 s, for a total of 40 cycles. After amplification, samples with a Ct value ≤35 and a typical S-shaped amplification curve are considered positive, while samples with a Ct value >35 or no amplification curve are considered negative.
[0058] Specifically, the real-time fluorescence PCR reaction program is a two-step amplification program, fully adapted to the amplification characteristics of this primer and probe. The specific functions of each stage are as follows:
[0059] Pre-denaturation at 94℃ for 5 min has two key functions: first, it activates the hot-start Taq enzyme in the system, changing it from an antibody-blocked state to an active state, thus formally initiating PCR amplification; second, it completely denatures and dissociates the template DNA, ensuring that the primers can bind smoothly to the complementary sequence of the template. At the same time, the UDG enzyme in the system completes the elimination of potential aerosol contamination at this stage, reducing the risk of false positives.
[0060] Cyclic phase: 94℃ for 5s (denaturation), 60℃ for 30s (annealing + extension), for a total of 40 cycles, is the core stage of PCR amplification. The 94℃ for 5s denaturation step completely breaks down the double-stranded DNA into single strands, providing a template for primer binding. The 60℃ for 30s annealing + extension step is a combined step. The annealing temperature of both primers and probes is adapted to 60℃. At this temperature, the primers and probes can specifically bind to the complementary sequences of the template single strands. Taq enzyme initiates DNA synthesis, and its 5'-3' exonuclease activity cleaves and degrades the probe bound to the template, separating the fluorescent reporter group from the quencher group and releasing a fluorescent signal. The instrument collects the fluorescent signal at this stage. The 40 cycles are optimized to ensure effective amplification with low template concentrations while avoiding non-specific amplification caused by excessive cycles.
[0061] The result judgment criteria are the sole basis for the qualitative assessment of the test results. They have been verified through numerous specificity and sensitivity experiments to ensure the accuracy of the judgment results.
[0062] Positive criteria: Ct value ≤ 35 and the appearance of a typical S-shaped amplification curve; Ct value, or cycle threshold, is the number of cycles required for the fluorescence signal to reach a set threshold. The smaller the Ct value, the higher the initial concentration of the target nucleic acid in the template; a typical S-shaped amplification curve is the core feature of PCR-specific amplification. Only when both conditions are met can it be determined that there is long-tailed catfish environmental DNA in the sample.
[0063] Negative determination criteria: Ct value > 35 or no amplification curve; when the Ct value exceeds 35, the amplification signal is mostly non-specific amplification or background noise, which has no actual positive significance; no amplification curve means that there is no target nucleic acid template in the system, and it is judged as negative. Detailed Implementation
[0064] Example 1: Preliminary verification of primer specificity (conventional PCR method)
[0065] Mitochondrial genome sequences of *Catfish simonii* and closely related fish were downloaded from the NCBI database. Through multiple sequence alignment, regions with high variability and intraspecific conservation in the *Catfish simonii* Cytb gene were screened. Primers and MGB probes were designed using PrimerExpress 3.0 software, and the target primer-probe combination was finally determined after BLAST comparison verification.
[0066] Fin tissues were collected from the long-rumped catfish and its closely related species. Genomic DNA was extracted from each sample using the Ezup column-based animal genomic DNA extraction kit. The extracted products were used as templates for subsequent PCR reactions after concentration determination.
[0067] High-purity, thermostable DNA polymerase 2×TaqMasterMix (DyePlus) was used as the PCR amplification reagent;
[0068] To construct a 50 μL PCR reaction system, add 25 μL of 2×TaqMasterMix (DyePlus), 2.0 μL of 10 μM upstream primer F, 2.0 μL of 10 μM downstream primer R, 2.0 μL of DNA template, and 19 μL of ddH2O in sequence. After thorough mixing, centrifuge at low speed.
[0069] Set up the PCR reaction program: pre-denaturation at 95℃ for 3 min; then perform 35 cycles, each cycle consisting of denaturation at 95℃ for 15 s, annealing at 54℃ for 15 s, and extension at 72℃ for 30 s; after the cycle, perform a final extension at 72℃ for 5 min.
[0070] Eight μL of the PCR product was analyzed by 2% agarose gel electrophoresis. Only the lane of *Catfish brevicus* showed a bright, single, specific band. No amplified bands were found in the lanes of closely related species and the blank control. This confirmed that the primer set had good specificity and only amplified *Catfish brevicus*, and could be used for subsequent qPCR system construction.
[0071] Example 2: qPCR specificity verification based on genomic DNA of the long-scrofted catfish fin rays
[0072] Fin tissues were collected from *Catfish simonii* and its closely related species. Genomic DNA was extracted from each sample using the Ezup column-based animal genomic DNA extraction kit. The extracted products were then adjusted to an appropriate concentration after concentration determination and used as qPCR templates.
[0073] A 20 μL qPCR reaction system was constructed using PerfectStart® II Probe qPCR SuperMix UDG. 10 μL of 2×PerfectStart® II Probe qPCR SuperMix UDG, 0.4 μL of 10 μM upstream primer F, 0.4 μL of 10 μM downstream primer R, 0.4 μL of 10 μM TaqMan-MGB probe P, 0.4 μL of 50×PassiveReferenceDye, 1.0 μL of DNA template, and 7.4 μL of Nuclease-free Water were added sequentially. After thorough mixing, the mixture was centrifuged at low speed.
[0074] A two-step qPCR reaction program was set up, with pre-denaturation at 94℃ for 5 min to activate the hot-start enzyme; then 40 qPCR cycles were performed, each cycle consisting of denaturation at 94℃ for 5 sec and annealing extension at 60℃ for 30 sec, with fluorescence signals collected during the annealing extension phase.
[0075] After the reaction, the results were determined based on the amplification curve and Ct value. A Ct value ≤ 35 and the appearance of a typical S-shaped amplification curve were considered positive, while a Ct value > 35 or no Ct value were considered negative. Only the *Catfish brevicus* sample showed a significant fluorescence signal and was considered positive. No effective amplification was detected in other closely related species distributed in the same region and the blank control. This confirms that the primer-probe combination designed in this invention has extremely high species specificity and can accurately distinguish *Catfish brevicus* from easily confused closely related species without cross-reaction.
[0076] Example 3: Field monitoring application of environmental DNA of *Catfish simonii* in the Hongshui River Basin
[0077] The main stream of the Hongshui River and its major tributaries were selected as the survey area. A total of 17 typical sampling points were set up in the survey area, numbered S01-S17.
[0078] Traditional resource surveys and eDNA water sample collection were carried out simultaneously at 17 locations. With the assistance of experienced local fishermen, trial fishing was conducted using specialized fishing gear for catfish, such as drift nets and fish traps. Each location operated continuously for 24 hours, recording whether catfish were caught. Before hauling in the nets, 1L of mixed surface and mid-layer water samples were collected at the same sampling point. After on-site filtration and pretreatment, total DNA was extracted as a test template.
[0079] The kit described in this invention was used to quantitatively detect environmental DNA samples from 17 sites. A standard curve was constructed using plasmid standards containing the Cytb gene fragment of the catfish. The volume was converted based on the filtered water volume, elution volume, and sample loading volume, and finally converted to the number of DNA copies per unit volume of water.
[0080] A positive result is defined as a Ct value ≤ 35 with a typical amplification curve, while a negative result is defined as no Ct value or Ct > 35. This kit detected significant positive signals at all sampling points where traditional surveys successfully captured samples of *Catfish simonii*, showing a high degree of consistency with the results of traditional surveys. The kit also detected positive signals at some locations where samples were not captured by traditional surveys, enabling the detection of hidden distribution points missed by traditional methods. Simultaneously, it can accurately exclude negative results from non-distribution points, confirming the excellent performance of this kit in field surveys and its suitability for widespread application in monitoring rare fish resources in complex water systems.
[0081] The method of use and working principle of this invention are as follows:
[0082] Instructions for use: First, collect water or sediment samples from the water body to be tested for pretreatment. Vacuum filter the water sample through a filter membrane to enrich environmental DNA. Grind the sediment sample with liquid nitrogen and extract the total DNA from the pretreated sample as a detection template. Then, use the primer and probe combination in the kit, along with qPCR amplification premix and other supporting components, to construct a real-time fluorescent PCR amplification system. Perform real-time fluorescent PCR amplification according to the set reaction program. During the amplification process, set up positive and negative controls simultaneously to control the effectiveness of the experiment. Finally, determine the results based on the amplification curve and Ct value criteria to achieve qualitative detection of *Catfish spp.* in the water.
[0083] Working Principle: The kit uses the Cytb gene of the mitochondria of the catfish (Cytb) as a specific detection target. The core working principle involves real-time fluorescent PCR amplification using a primer-probe combination designed for this target gene. The upstream and downstream primers specifically bind to the target gene sequence in the sample DNA and initiate the DNA amplification reaction. The TaqMan-MGB probe simultaneously binds to the specific region of the target gene. During PCR amplification, the exonuclease activity of Taq polymerase degrades the bound probe, separating the fluorescent reporter group at the 5' end from the quencher group at the 3' end and releasing a fluorescent signal. The accumulation of the fluorescent signal is completely synchronized with the formation of the PCR product. The fluorescent signal is collected in real-time using a quantitative PCR instrument. By combining the amplification curve and Ct value, the presence of catfish environmental DNA in the sample can be determined. Simultaneously, the minor groove binding on the probe enhances the specificity of sequence recognition. The UDG enzyme in the kit effectively reduces false positives caused by aerosol contamination, further ensuring the accuracy of the detection results.
[0084] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Those skilled in the art can readily implement the present invention based on the description and drawings above. However, any modifications, alterations, and variations made by those skilled in the art without departing from the scope of the present invention using the disclosed technical content are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, and variations made to the above embodiments based on the essential technology of the present invention are still within the protection scope of the present invention.
Claims
1. A DNA detection kit for the environmental environment of the long-rumped catfish, characterized in that, The invention includes a primer and probe composition for real-time fluorescence PCR detection based on the Cytb gene of the mitochondria of the catfish (Cyprinus longissimus). The composition includes an upstream primer, a downstream primer, and a TaqMan-MGB probe. The nucleotide sequence of the upstream primer is shown in SEQ ID No. 1, the nucleotide sequence of the downstream primer is shown in SEQ ID No. 2, and the nucleotide sequence of the TaqMan-MGB probe is shown in SEQ ID No.
3. The 5' end of the TaqMan-MGB probe is modified with a fluorescent reporter group, and the 3' end is coupled with a minor groove binder and a non-fluorescent quencher group.
2. The environmental DNA detection kit for *Catfish bream* according to claim 1, characterized in that, The working concentrations of the upstream primer, downstream primer, and TaqMan-MGB probe are all 10 μmol / L.
3. The environmental DNA detection kit for *Catfish bream* according to claim 1, characterized in that, The kit also includes qPCR amplification premix, DNA extraction reagent, positive control and negative control, wherein the qPCR amplification premix is 2×ProbeqPCRSuperMix containing UDG enzyme.
4. The environmental DNA detection kit for *Catfish bream* according to claim 3, characterized in that, The positive control is plasmid DNA containing the amplified fragment between SEQ ID No. 1 and SEQ ID No. 2 of the Cytb gene of the long-rumped catfish, or the genomic DNA of the long-rumped catfish. The negative control is nuclease-free ultrapure water.
5. The environmental DNA detection kit for *Catfish bream* according to claim 1, characterized in that, The method of use includes the following: the primer and probe composition is used to amplify the total DNA of the sample to be tested using real-time fluorescent PCR to complete the qualitative detection of the long-rumped catfish.
6. The environmental DNA detection kit for *Catfish bream* according to claim 5, characterized in that, The test samples are water samples or bottom sediment samples from the water area to be tested. The water samples are vacuum filtered through a filter membrane to enrich environmental DNA, and the bottom sediment samples are ground with liquid nitrogen. The total DNA extracted from the treated samples is used as a template for real-time fluorescent PCR amplification.
7. The environmental DNA detection kit for *Catfish bream* according to claim 5, characterized in that, The total volume of the real-time fluorescence PCR amplification system is 20 μL, including 10 μL of 2×ProbeqPCRSuperMix, 0.4 μL of 10 μmol / L upstream primer, 0.4 μL of 10 μmol / L downstream primer, 0.4 μL of 10 μmol / L TaqMan-MGB probe, 0.4 μL of 50×PassiveReferenceDye, 1 μL of DNA template, and the remaining volume is made up with nuclease-free ultrapure water.
8. The environmental DNA detection kit for *Catfish spp.* according to claim 5, wherein the real-time fluorescence PCR reaction program is as follows: pre-denaturation at 94℃ for 5 min, followed by 94℃ for 5 s and 60℃ for 30 s, for a total of 40 cycles. After amplification, samples with a Ct value ≤ 35 and a typical S-shaped amplification curve are judged as positive, and samples with a Ct value > 35 or no amplification curve are judged as negative.