Specific real-time fluorescent PCR (Polymerase Chain Reaction) amplification primer, probe and detection method for identifying lutjanus purpureus
By designing specific real-time fluorescent PCR amplification primers and probes, and combining them with optimized PCR reaction procedures, the problem of accuracy in identifying purple snapper with closely related species has been solved. This enables rapid and sensitive identification and quantitative detection of purple snapper, which is suitable for distinguishing fish from multiple families and genera and for fishery resource management.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- HAINAN UNIV
- Filing Date
- 2026-03-31
- Publication Date
- 2026-05-15
AI Technical Summary
Existing technologies make it difficult to quickly and accurately identify the purple snapper and its closely related species. Traditional morphological methods are affected by the individual development stage and processing methods, and existing molecular detection methods have cross-reactivity and false negative results.
We designed specific real-time fluorescent PCR amplification primers and probes, and used single nucleotide polymorphism sites in the mitochondrial DNA of the red snapper and closely related species to screen for highly species-specific gene fragments for identification of the red snapper, combined with optimized PCR reaction procedures and fluorescent detection methods.
It enables rapid and accurate identification and quantitative detection of red snapper, distinguishing it from fish of multiple families and genera. It has high detection sensitivity, is easy to operate, and is applicable to both environmental DNA and biological DNA, ensuring the standardization of the fisheries market and the sustainable use of resources.
Smart Images
Figure CN122038601A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of molecular detection and species identification technology, specifically relating to a specific real-time fluorescent PCR amplification primer, probe, and detection method for identifying the purple snapper. Background Technology
[0002] Purple snapper ( Lutjanus argentimaculatus The red snapper (Siniperca purpureus) is an important marine economic fish belonging to the family Sinipercaidae in the order Perciformes, and is widely favored by consumers in the market. However, it is easily confused with some morphologically similar closely related species during processing or sales. Traditional morphological identification methods are affected by factors such as individual development stage and processing methods, resulting in limited accuracy and efficiency, making it difficult to meet the needs of rapid and accurate identification. Therefore, establishing a molecular identification method for the red snapper with high specificity, high sensitivity, and simple operation is of great significance for the sustainable development of fisheries. Real-time fluorescence PCR technology, with its advantages of high specificity, high sensitivity, and rapid detection, has been widely used in the field of species identification, providing an effective means to solve the above problems.
[0003] In existing research, the method of designing specific probe primers using fish mitochondrial DNA for species detection is widely used. Commonly selected gene fragments include cytochrome c oxidase subunit I (CoⅠ), D-loop, (pigmentocyte b) cytb, 16S rRNA, and 12S rRNA. However, in the design of specific probe primers for *Sinocyclocheilus purpuratus*, these common segments have limitations, exhibiting high conservation among closely related species. This makes it difficult to design specific probe primers that can accurately distinguish *Sinocyclocheilus purpuratus* from other closely related species, leading to potential cross-reactivity or false negative results in practical applications of existing detection methods, affecting the accuracy and reliability of identification. Summary of the Invention
[0004] In view of the shortcomings of existing technologies, this invention provides specific real-time fluorescent PCR amplification primers, probes, and detection methods for identifying *Syngonium purpureum*. The aim is to overcome the deficiencies in specificity and sensitivity in existing technologies, and to provide a molecular detection scheme capable of rapidly and accurately identifying and quantitatively detecting *Syngonium purpureum*.
[0005] The technical solution of this invention mainly includes the following: To overcome the technical bottleneck of effectively distinguishing between *Rhododendron rubiginense* and its closely related species using conventional mitochondrial genes, this study compared the mitochondrial DNA base sequences of *Rhododendron rubiginense* and its closely related species to screen for highly species-specific gene fragments as target regions. Within these specific regions, gene fragments were compared with those of other key closely related species (such as *Rhododendron leucogranum*). Lutjanus russellii Spotted snapper Lutjanus stellatusThere are multiple stable, species-specific single nucleotide polymorphism (SNP) sites among them (e.g., [elements not specified]). These SNP sites provide the molecular basis for designing highly specific probe primers and are key to achieving specific identification.
[0006] On the one hand, based on existing research results, this invention provides specific real-time fluorescent PCR amplification primers for identifying *Cyprinus purpureus*. The primers are one of primer pair A, primer pair B, or primer pair C. Primer pair A includes upstream primer ZHDDF1 and downstream primer ZHDDR1; primer pair B includes upstream primer ZHDDF2 and downstream primer ZHDDR2; primer pair C includes upstream primer ZHDDF3 and downstream primer ZHDDR3. The nucleotide sequence of upstream primer ZHDDF1 is shown in SEQ ID NO.1; the nucleotide sequence of downstream primer ZHDDR1 is shown in SEQ ID NO.2; the nucleotide sequence of upstream primer ZHDDF2 is shown in SEQ ID NO.4; the nucleotide sequence of downstream primer ZHDDR2 is shown in SEQ ID NO.5; the nucleotide sequence of upstream primer ZHDDF3 is shown in SEQ ID NO.7; and the nucleotide sequence of downstream primer ZHDDR3 is shown in SEQ ID NO.8.
[0007] A specific real-time fluorescent PCR amplification primer and probe composition for identifying purple snapper includes primer and probe group X consisting of primer pair A and probe ZHDDP1, primer and probe group Y consisting of primer pair B and probe ZHDDP2, or primer and probe group Z consisting of primer pair C and probe ZHDDP3. The nucleotide sequence of the probe ZHDDP1 is shown in SEQ ID NO.3. The nucleotide sequence of the probe ZHDDP2 is shown in SEQ ID NO.6. The nucleotide sequence of the probe ZHDDP3 is shown in SEQ ID NO.9.
[0008] Furthermore, based on the basic principle of real-time fluorescence PCR amplification, the probe is labeled with a fluorescent group at its 5' end and a fluorescence quencher group at its 3' end.
[0009] Furthermore, the present invention relates to a reagent for detecting purple snapper, the reagent comprising the specific real-time fluorescent PCR amplification primers or the specific real-time fluorescent PCR amplification primer-probe composition, and further comprising a real-time fluorescent PCR Master Mix.
[0010] In a second aspect, the present invention provides the use of the specific real-time fluorescent PCR amplification primers, the specific real-time fluorescent PCR amplification primer-probe composition, or the reagents described herein in any one of the following (1) to (2): (1) Distinguishing the purple snapper from other fish; (2) Preparation of products for identification of purple snapper.
[0011] Furthermore, the other fish species include snapper (family Lepidoptera). Lutjanidae Bigeye seabream family Priacanthidae Cardinalfish family Apogonidae Brilliant seabream family Acropomatidae Sushi Serranidae Carangidae Carangidae Cichlid family Cichlidae Eels Anguillidae One or more of them.
[0012] Furthermore, the other fish include one or more of the following: golden flame snapper (Flammulina gargarizans) of the family Snapperidae. Lutjanus fulviflamma Short-tailed bigeye snapper (family Bigeye snapperidae) Priacanthus macracanthus Cardinalfish family, four-lined cardinalfish Apogon quadrifasciatus , fine-striped cardinalfish Jaydia lineata Cardinalfish (Midline) Apogon kallopterus Japanese luminous seabream (family Phytoideidae) Acropoma japonicum South China Sea grouper (Sebastes) Epinephelus stictus Long-bodied round trevally of the trevally family Decapterus macrosoma High-body amber Seriola dumerili Cichlid family, Tilapia ziggii Coptodon zillii Eel family, spotted eel Anguilla marmorata .
[0013] Furthermore, the present invention provides a real-time fluorescent PCR detection method for red snapper, comprising the following steps: extracting DNA from the sample to be tested, performing real-time fluorescent PCR amplification using the primer and probe combination, and if the amplification product shows a fluorescence growth curve or a 132bp band appears after electrophoresis, it indicates that the sample to be tested contains red snapper DNA.
[0014] Furthermore, in the aforementioned real-time fluorescent PCR detection method for red snapper, the DNA of the sample to be tested includes DNA released into the environment by the organism through skin shedding, excretion, secretion, decomposition, etc., or DNA directly extracted from the organism.
[0015] Furthermore, the PCR reaction procedure was as follows: pre-incubation at 95°C for 30 seconds; then two-step amplification at 95°C for 10 seconds and 60°C for 30 seconds, repeated 40 times; finally, cooling at 95°C for 10 seconds and 60°C for 60 seconds.
[0016] Furthermore, each 20 μL PCR amplification system contains: 10 μL of 2×TaqMan Fast Real-Time Fluorescent PCR Master Mix, 0.8 μL each of forward and reverse primers, 0.4 μL of ZHDDP1 probe, 1.0 μL of DNA template, and ultrapure water to a final volume of 20 μL.
[0017] Furthermore, the upstream and downstream primers and probes are each pre-prepared into 10 μmol / L solutions before being added to the PCR amplification system.
[0018] The beneficial effects of this invention are: This invention screens highly species-specific gene fragments in the mitochondrial DNA of the red snapper as target regions. Specific primers and probes designed based on these target regions can accurately identify the red snapper.
[0019] The real-time fluorescence PCR detection method constructed in this invention has high specificity and can effectively distinguish the purple snapper from fish of multiple families and genera, including snapper and bigeye snapper. This invention can detect trace amounts (minimum detection concentration 30.5 × 10⁻⁶). -8 (ng / μL) of purple snapper DNA is suitable for detecting DNA extracted from organisms or environmental DNA.
[0020] This method is simple to operate. With an optimized PCR reaction procedure and system, amplification can be completed in a short time. The results can be determined based on the fluorescence growth curve or the electrophoretic band of a specific length. It provides a reliable technical means for the rapid identification and quantitative detection of purple snapper, which helps to ensure the standardization of the fishery market and the sustainable use of fishery resources. Attached Figure Description
[0021] Figure 1 : Schematic diagram of target sequence, primers, and probe detection.
[0022] Figure 2 Electrophoresis results of PCR reactions with different primers.
[0023] Figure 3 qPCR amplification curves of different samples. A1~A3: Purple snapper samples; CM: 11 other fish samples; N: blank control.
[0024] Figure 4 Detection limits of primers and probes. A1-A10: Fluorescence amplification curves of 10-fold gradient plasmid standards (A1: 305 ng / μL; A2: 30.5 ng / μL; A3: 30.5 × 10⁻⁶). -1 ng / μL; A4: 30.5×10 -2 ng / μL; A5: 30.5×10 -3ng / μL; A6: 30.5×10 -4 ng / μL; A7: 30.5×10 -5 ng / μL; A8: 30.5×10 -6 ng / μL; A9: 30.5×10 -7 ng / μL; A10: 30.5×10 -8 (ng / μL); A11: Blank control.
[0025] Figure 5 Standard curve of plasmid for the ND4L gene of the purple snapper.
[0026] Figure 6 Gel electrophoresis images of the two primer pairs (F1' R1' and F2' R2') initially designed. Figure 7 Gradient PCR gel electrophoresis imaging.
[0027] Figure 8 : Optimal annealing temperature curve for gradient qPCR. Detailed Implementation
[0028] To facilitate a clearer understanding of the technical content of this invention by those skilled in the art, the invention will be further described below in conjunction with specific embodiments.
[0029] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.
[0030] Example 1: Real-time fluorescent PCR detection method for environmental DNA of the purple snapper, comprising the following steps: 1. Design of specific primers and probes The following primers and probes were designed based on the mitochondrial DNA sequence of the purple snapper: Table 1 Primers and probes
[0031] Note: Primers and probes were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The detection principle of the above primers and probes is as follows: Figure 1As shown. Probe ZHDDP1 is an oligonucleotide probe with a fluorescent group attached to the 5' end and a quencher attached to the 3' end. When the probe is intact, the fluorescent signal emitted by the reporter group is absorbed by the quencher group. During PCR amplification, primers ZHDDF1 / ZHDDR1 undergo a thermal cycle of high-temperature denaturation and low-temperature annealing extension with the mitochondrial DNA of the red snapper, following the rules of polymerase chain reaction. Simultaneously, the added probe ZHDDP1 is complementary to the red snapper mitochondrial DNA. During the low-temperature annealing extension stage, the 5'-3' exonuclease activity of Taq enzyme in the reaction system degrades probe ZHDDP1, separating the reporter fluorescent group and the quencher fluorescent group. Thus, the fluorescence monitoring system can receive the fluorescent signal. That is, for each DNA strand amplified, one fluorescent molecule is formed, achieving complete synchronization between the accumulation of the fluorescence signal and the formation of the PCR product.
[0032] 2. Extraction of DNA from the muscle tissue of the purple snapper The dorsal fin muscle of the purple snapper was placed in a mortar and ground into powder with liquid nitrogen. DNA was extracted from the muscle tissue using the EZNA® TissueDNA Kit (Omega). The purple snapper used in the test was taken from the South China Sea.
[0033] 3. Real-time fluorescence PCR detection Real-time PCR amplification system (20 μL): 10 μL 2×TaqMan Fast Real-time PCR Master Mix (Sangon Biotech (Shanghai) Co., Ltd.), 0.8 μL each of forward and reverse primers ZHDDF1 / ZHDDR1 (10 μmol / L), 0.4 μL ZHDDP1 probe (10 μmol / L), 1.0 μL DNA template, and ultrapure water to a final volume of 20 μL. Real-time PCR amplification was performed using a Roche LightCycler microscope. ® 96. Perform the reaction on a real-time fluorescence PCR instrument. Turn on the instrument, set the PCR reaction conditions, and use the two-step amplification program: pre-incubation at 95°C for 30 seconds; then two-step amplification: 95°C for 10 seconds, 60°C for 30 seconds, cycle 40 times; finally, cool at 95°C for 10 seconds, 60°C for 60 seconds. Click "Run" to perform the real-time fluorescence PCR reaction. The reaction will be completed in about 1 hour. Save the file and open LightCycler. ® The analysis was performed using 96 software.
[0034] Real-time fluorescence PCR amplification showed that the specific primers ZHDDF1 / ZHDDR1 and probe ZHDDP1 for *Sinocyclocheilus purpureus* exhibited a fluorescence growth curve in the tested *Sinocyclocheilus purpureus* samples, indicating a strong increase in fluorescence signal and positive amplification (CT value < 35). The sample amplification curves are shown in the figure. Figure 3The tested ZHDD samples showed positive amplification, while the blank control showed no fluorescence signal and was negative. This indicates that primers ZHDDF1 / ZHDDR1 and probe ZHDDP1 can detect *Sinocyclocheilus purpureus*.
[0035] Example 2: Specificity verification of the purple snapper primers ZHDDF1 / ZHDDR1 and probe ZHDDP1, including the following steps: 1. Extraction of DNA from muscle tissue Muscle tissue DNA from purple snapper and other fish (see Table 2 for sample details) was collected in a mortar, ground into powder with liquid nitrogen, and then analyzed using EZNA. ® The Tissue DNA Kit (Omega) extracts DNA from muscle tissue samples.
[0036] Table 2 Sample Details
[0037] 2. Preliminary screening of primer pairs Three pairs of probe primers were designed in the initial stage of the experiment (sequences are detailed in Table 3). To preliminarily verify the specificity of the designed primers, the three pairs of probe primers ZHDDF1 / ZHDDR1, ZHDDF2 / ZHDDR2, and ZHDDF3 / ZHDDR3 were used to perform real-time quantitative PCR reactions with extracted muscle DNA from *Sinocyclocheilus purpureus*. The reaction products were then subjected to 1% agarose gel electrophoresis (marker 2000bp), and the electrophoresis results were analyzed using a MIULAB GIS-630 gel imaging analyzer. The results showed that the target band of the first pair of primers, ZHDDF1 / ZHDDR1, was clear and bright. Figure 2 Therefore, primers ZHDDF1 / ZHDDR1 were selected for subsequent experiments.
[0038] Table 3 Primers and probes
[0039] Note: Primers and probes were synthesized by Sangon Biotech (Shanghai) Co., Ltd. 3. Specificity verification of primers ZHDDF1 / ZHDDR1 and probe ZHDDP1 for purple snapper. Following the methods and steps described above, real-time fluorescence PCR amplification was performed. Primers ZHDDF1 / ZHDDR1 and probe ZHDDP1 showed a fluorescence growth curve in the tested purple snapper samples, indicating a strong increase in fluorescence signal and positive amplification. The sample amplification curve was referenced... Figure 3 The other 11 test samples (Table 2) and the blank control (N) showed no fluorescence signal, indicating a negative result. The sample amplification curves are referenced from... Figure 3This indicates that the real-time fluorescence PCR detected *Cyprinus purpurea*, demonstrating that primers ZHDDF1 / ZHDDR1 and probe ZHDDP1 can specifically detect *Cyprinus purpurea*.
[0040] Example 3: Specific probe primer sensitivity detection and standard curve Sangon Biotech (Shanghai) Co., Ltd. was commissioned to synthesize a plasmid standard for the target gene of *Syngonium purpureus* at a concentration of 305 ng / μL, which was then serially diluted 10-fold (range: 100–100). -10 Real-time fluorescence PCR was then performed, with ddH2O used as a negative control to test for contamination. The lowest detectable dilution concentration for this method is 30.5 × 10⁻⁶. -8 The concentration of ng / μL indicates that the designed primers and probes have high sensitivity. A standard curve was fitted by fitting the logarithm of the diluted plasmid standard concentration to the Ct value. Figure 4 The equation of the curve is: y = -3.244x + 39.28 (R² = 0.999) Comparative Example (1) The inventors initially designed two pairs of primers and probes based on a conserved region of the mitochondrial DNA of the red snapper (Table 4) and performed PCR amplification. However, in testing the primer specificity, it was found that not only the target species, the red snapper, amplified the band, but some non-target fish also showed similar results. It is speculated that the primer specificity is not high. Figure 6 Then, based on the target gene sequence, the three pairs of primers shown in Table 3 were redesigned. The electrophoresis results of the PCR products showed that the first pair of primers had good specificity for the purple snapper. Figure 2 Ultimately, ZHDDF1 and ZHDDR1 were selected for subsequent experiments.
[0041] Table 4 Primers and Probes
[0042] Note: Primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. Example 4: Determination of Annealing Temperature Optimizing qPCR reaction conditions is not a routine, predictable process. Inappropriate annealing temperatures can lead to detection failures, such as low primer sensitivity or poor specificity. Initially, the inventors used conventional agarose gel electrophoresis as the criterion, setting 11 temperature gradients within the range of 55°C to 65°C for testing. The results showed that the brightness was concentrated between 57°C and 61°C, and that problems such as tailing and unclear bands existed between 55°C and 58°C. Figure 7 Endpoint qualitative analysis alone cannot determine the optimal annealing temperature for real-time fluorescent PCR because gel electrophoresis cannot reflect subtle differences in amplification efficiency.
[0043] Subsequently, amplification was performed using a real-time fluorescence PCR instrument at the same temperature gradient, and fluorescence signals were acquired. Only at 60°C did the amplification curve peak earliest and the Ct value lowest ( Figure 8 When the fluorescence signal intensity reaches its highest point during the plateau phase, the curve is smooth and typical, indicating that the amplification efficiency is the highest, the specificity is the best, and the detection sensitivity is optimal at this time.
[0044] The above description is only a part of the specific embodiments of the technical solution of the present invention. These embodiments are only illustrative examples and are intended to help those skilled in the art understand the core concept of the present invention, and are not exhaustive enumerations of all embodiments of the present invention. The scope of protection of the present invention should be determined by the claims and their equivalents. Any adaptive modifications, equivalent substitutions of technical features, further optimizations and improvements implemented based on the basic principles and concepts disclosed in the present invention shall fall within the scope of protection of the present invention.
Claims
1. A specific real-time fluorescent PCR amplification primer for identifying *Cyprinus purpureus*, wherein the primer is one of primer pair A, primer pair B, or primer pair C, wherein primer pair A comprises upstream primer ZHDDF1 and downstream primer ZHDDR1, primer pair B comprises upstream primer ZHDDF2 and downstream primer ZHDDR2, and primer pair C comprises upstream primer ZHDDF3 and downstream primer ZHDDR3, characterized in that, The nucleotide sequence of the upstream primer ZHDDF1 is shown in SEQ ID NO.1, the nucleotide sequence of the downstream primer ZHDDR1 is shown in SEQ ID NO.2, the nucleotide sequence of the upstream primer ZHDDF2 is shown in SEQ ID NO.4, the nucleotide sequence of the downstream primer ZHDDR2 is shown in SEQ ID NO.5, the nucleotide sequence of the upstream primer ZHDDF3 is shown in SEQ ID NO.7, and the nucleotide sequence of the downstream primer ZHDDR3 is shown in SEQ ID NO.
8.
2. A specific real-time fluorescent PCR amplification primer and probe composition for identifying the purple snapper, characterized in that, The primer-probe set X, consisting of primer pair A and probe ZHDDP1 as described in claim 1, primer-probe set Y, consisting of primer pair B and probe ZHDDP2 as described in claim 1, or primer-probe set Z, consisting of primer pair C and probe ZHDDP3 as described in claim 1. The nucleotide sequence of the probe ZHDDP1 is shown in SEQ ID NO.
3. The nucleotide sequence of the probe ZHDDP2 is shown in SEQ ID NO.
6. The nucleotide sequence of the probe ZHDDP3 is shown in SEQ ID NO.
9.
3. A reagent for detecting purple snapper, characterized in that, The reagents include the specific real-time fluorescent PCR amplification primers of claim 1 or the specific real-time fluorescent PCR amplification primer-probe composition of claim 2, and also include real-time fluorescent PCR Master Mix.
4. The use of the specific real-time fluorescent PCR amplification primers of claim 1, the specific real-time fluorescent PCR amplification primer-probe composition of claim 2, or the reagent of claim 3 in any one of the following (1) to (2), characterized in that, (1) Distinguishing the purple snapper from other fish; (2) Preparation of products for identification of purple snapper.
5. The application according to claim 4, characterized in that, The other fish species include snapper (family Lepidoptera). Lutjanidae Bigeye seabream family Priacanthidae Cardinalfish family Apogonidae Brilliant seabream family Acropomatidae Sushi Serranidae Carangidae Carangidae Cichlid family Cichlidae Eels Anguillidae One or more of them.
6. The application according to claim 4, characterized in that, The other fish species include one or more of the following: Golden Flame Snapper (Fishfish) Lutjanus fulviflamma Short-tailed bigeye snapper (family Bigeye snapperidae) Priacanthus macracanthus Cardinalfish family, four-lined cardinalfish Apogon quadrifasciatus , fine-striped cardinalfish Jaydia lineata Cardinalfish (Midline) Apogon kallopterus Japanese luminous seabream (family Phytoideidae) Acropoma japonicum South China Sea grouper (Sebastes) Epinephelus stictus Long-bodied round trevally of the trevally family Decapterus macrosoma High-body amber Seriola dumerili Cichlid family, Tilapia ziggii Coptodon zillii Eel family, spotted eel Anguilla marmorata .
7. A real-time fluorescent PCR detection method for purple snapper, characterized in that, Includes the following steps: Extract DNA from the sample to be tested, and perform real-time fluorescent PCR amplification using the specific real-time fluorescent PCR amplification primers described in claim 1, the specific real-time fluorescent PCR amplification primer-probe composition described in claim 2, or the reagent described in claim 3. If the amplification product shows a fluorescence growth curve or a 132bp band appears after electrophoresis, it indicates that the sample to be tested contains red snapper DNA.
8. The real-time fluorescent PCR detection method for purple snapper according to claim 7, characterized in that, The DNA sample to be tested includes DNA released into the environment by an organism or DNA extracted directly from an organism.
9. The real-time fluorescent PCR detection method for purple snapper according to claim 7, characterized in that, The PCR reaction procedure was as follows: pre-incubation at 95°C for 30 seconds; then two-step amplification at 95°C for 10 seconds and 60°C for 30 seconds, repeated 40 times; finally, cooling at 95°C for 10 seconds and 60°C for 60 seconds.
10. The real-time fluorescent PCR detection method for purple snapper according to claim 7, characterized in that, Each 20 μL PCR amplification system contains: 10 μL of 2×TaqMan Fast Real-Time Fluorescent PCR Master Mix, 0.8 μL each of forward and reverse primers, 0.4 μL of probe, 1.0 μL of DNA template, and ultrapure water to a final volume of 20 μL.