Specific primer and method for rapidly identifying species of trachidermus fasciatus with high sensitivity

Abstract

The invention belongs to the technical field of molecular identification, and relates to a high-sensitivity specific primer and method for rapidly identifying species of trachidermus fasciatus. A forward primer sequence and a reverse primer sequence of the specific primer are respectively shown as SEQ ID NO.1 and SEQ ID NO.2 in a sequence table. The identification method comprises the following steps: (1) respectively extracting genome DNAs (deoxyribonucleic acids) of adult tracheilus fasciatus, juvenile tracheilus fasciatus and sibling species of leptocarpus aldrianus and durachis fasciatus by adopting a phenol-chloroform method; (2) respectively diluting the DNA sample by 10 times, 100 times and 1000 times, and carrying out PCR (Polymerase Chain Reaction) amplification on the primer by using the diluted DNA sample as a template; and (3) carrying out gel electrophoresis detection and result judgment on the amplification product. The method disclosed by the invention simplifies a conventional trachidermus fasciatus germplasm identification process, is simple and convenient to operate, short in period and low in cost, can quickly and accurately complete molecular identification of trachidermus fasciatus, and is particularly suitable for screening a large batch of samples.

Description

Technical Field

[0001] This invention belongs to the field of molecular identification technology and relates to a highly sensitive and rapid specific primer and method for identifying the species of Songjiang perch. Background Technology

[0002] Songjiang perch ( Trachidermusfasciatus Heckel, 1840), belongs to the order Perciformes ( Perciformes ), Culteridae ( Cottidae ), Songjiang perch ( Trachidermus Efficient and accurate germplasm identification and resource monitoring of the Songjiang perch is a key technical issue in current conservation and management efforts. Currently, species identification of the Songjiang perch mainly relies on morphological characteristics. However, due to significant morphological differences at different developmental stages, coupled with its external morphological similarities to closely related species, conventional morphological methods have obvious limitations in practical identification, especially in accurately distinguishing the target species in early developmental stages or environmental samples. Furthermore, current monitoring of wild population distribution mainly relies on traditional fisheries survey methods, which are low in sensitivity and labor-intensive, making it difficult to meet the monitoring and assessment needs of endangered populations. Therefore, developing highly sensitive genetic markers and efficient molecular detection methods for identifying the Songjiang perch has significant scientific and practical application value.

[0003] In recent years, molecular marker technology has been widely used in species identification and resource monitoring. Conventional molecular methods, such as mitochondrial markers... COI While sequencing identification of genes or D-loop regions has high resolution, its operation process is complex, usually involving DNA extraction, target gene amplification, sequencing and alignment analysis, and the detection cycle is long (about 2-3 days), which is not conducive to high-throughput and rapid screening.

[0004] Existing research has shown that highly conserved and specific regions often exist in the genomes of species. By designing specific primers targeting these regions and combining them with PCR amplification and agarose gel electrophoresis, rapid species identification without sequencing can be achieved. However, current primer designs are mostly based on conventional fragments, limiting their ability to detect degraded samples or trace amounts of DNA, and there is still room for improvement in sensitivity and specificity. Therefore, developing highly sensitive species-specific primers for the Songjiang perch and constructing a simple and reliable germplasm identification method can not only significantly improve detection efficiency but also contribute to the precise protection of its germplasm resources and the scientific development of genetic breeding work, possessing significant theoretical and practical value. Summary of the Invention

[0005] This invention aims to address the problems of insufficient sensitivity, cumbersome procedures, and long detection times in existing methods for identifying Songjiang perch germplasm. It provides a highly sensitive species-specific primer for Songjiang perch based on short-fragment design and a rapid molecular identification method. This method optimizes the conventional germplasm identification process, enabling efficient and accurate identification of Songjiang perch germplasm resources in a shorter time.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a highly sensitive and rapid specific primer for identifying the species of the Songjiang perch, wherein the forward and reverse primers of the specific primer are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0007] This invention also provides a method for rapid identification of Songjiang perch species, comprising the following steps: (1) Genomic DNA was extracted from the sample using the phenol-chloroform method at a concentration of approximately 10–60 ng / μL; (2) The DNA was diluted 10 times, 100 times and 1000 times respectively, and PCR amplification was performed using the specific primers described above; (3) The PCR products were identified and analyzed by agarose gel electrophoresis.

[0008] When a clear and specific band appears at 92 bp in the amplified product, the sample is determined to contain perch; if the band does not appear, perch can be excluded, indicating that the sample is other fish (such as closely related species like scorpionfish, scorpionfish, etc.).

[0009] The PCR amplification reaction system in step (2) is as follows: total volume 25 μL, including 12.5 μL of 2×PCR Mix, 1 μL of diluted genomic DNA template, 0.5 μL each of forward and reverse primers (10 μM), and 10.5 μL of sterile double-distilled water.

[0010] The PCR amplification reaction conditions in step (2) are as follows: 95 °C pre-denaturation for 5 min; 95 °C denaturation for 30 s, 59 °C annealing for 30 s, 72 °C extension for 50 s, for 35 cycles; and finally 72 °C extension for 5 min.

[0011] After electrophoresis of the amplified product in step (3) on a 1.5% agarose gel (1×TAE buffer, voltage ≤ 5 V / cm) for 40 min, the electrophoresis image is recorded using a gel imaging system for determination. 2 μL of the PCR product is loaded for detection.

[0012] The present invention also provides the use of the specific primers in the preparation of products for rapid identification of Songjiang perch species, said products being reagents or kits.

[0013] The present invention also provides a reagent or kit for rapid identification of the species of Songjiang perch, comprising the aforementioned specific primers.

[0014] Compared with the prior art, the present invention has the following beneficial effects: (1) High sensitivity and high specificity: The species-specific primers for perch provided in this invention are for perch. Cytb The design of specific mutation sites in the gene can effectively distinguish the Songjiang perch from its closely related species, and can achieve stable amplification in DNA samples with extremely low concentrations (approximately 0.03-0.06 ng / μL), greatly improving the detection sensitivity; (2) Short fragment design improves adaptability: The primer amplification fragment designed in this invention is only 92 bp. The short fragment design can maintain a high amplification success rate in severely degraded or trace samples, and is suitable for detection of adult fish, fry, tissue residue samples and environmental water samples (eDNA), effectively breaking through the dependence of traditional methods on sample quality; (3) Short detection cycle and low cost: The identification method of the present invention is simple and efficient to operate. It only requires conventional PCR amplification and agarose gel electrophoresis to complete the identification. No DNA sequencing or nucleic acid probes are required. It only takes a few hours from sample processing to result interpretation. The detection cost is significantly reduced compared with traditional molecular identification methods. It is suitable for large-scale and rapid accurate identification of Songjiang perch species, which significantly improves the practical value and application prospects of this technology in ecological monitoring and resource management. (4) Wide range of applications: Existing technologies are mostly concentrated on laboratory or organizational sample testing, and have not yet covered the monitoring of Songjiang perch populations in natural waters. This invention is not only applicable to species identification under laboratory conditions, but can also be combined with environmental DNA technology to achieve highly sensitive monitoring of the distribution and population dynamics of Songjiang perch in natural waters, providing efficient and reliable technical support for fishery resource assessment and endangered species protection. Attached Figure Description

[0015] Figure 1 Mitochondria of the Songjiang perch in the example Cytb A schematic diagram showing the sequence comparison results between the gene-specific reverse primer design region and eight closely related species; Figure 2 The agarose gel electrophoresis image is shown to verify the amplification efficiency of the specific primers for the perch in this example. Detailed Implementation

[0016] The present invention will be further described below with reference to the accompanying drawings and specific application examples. It should be understood that these application examples are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0017] The identification method provided by this invention includes: (1) Two adult and two juvenile *Perequa spp.* were collected as test subjects, and two *Sculpinus esculentus* and two *Sculpinus multispinus* were collected as closely related control species. Genomic DNA was extracted from each sample using the phenol-chloroform method, and the DNA concentration was determined using Nanodrop. The DNA concentrations of adult *Perequa spp.* were 36.9 ng / μL and 31.8 ng / μL, respectively; the DNA concentrations of juvenile *Perequa esculentus* were 53.6 ng / μL and 55.8 ng / μL, respectively. The DNA concentrations of *Sculpinus esculentus* were 28.8 ng / μL and 31.6 ng / μL, respectively; the DNA concentrations of *Sculpinus multispinus* were 16.8 ng / μL and 14.0 ng / μL, respectively.

[0018] (2) The extracted DNA samples were diluted to 10, 100 and 1000 times respectively and used as PCR templates. PCR amplification was performed using the designed specific primers. (3) Agarose gel electrophoresis analysis of the amplification products: When using the DNA of the Songjiang perch as a template, a clear specific amplification band can be seen at 92 bp; while no amplification band is seen when using the DNA of the E. erinaceus and the E. multi-spined scorpion as templates.

[0019] The specific primers for the Songjiang perch provided in this invention possess both high sensitivity and high specificity, enabling stable amplification in extremely low-concentration DNA samples, overcoming the common amplification failure problem in trace or degraded samples. Their short 92 bp design effectively avoids the problem of large-fragment amplification failure caused by improper sample storage or excessive storage time, significantly improving the stability and applicability of the detection. This method is simple to operate, has a short detection cycle, and can quickly and accurately complete the species identification of the Songjiang perch. It is particularly suitable for large-scale sample screening and field monitoring applications, and has broad application prospects.

[0020] Example 1 This example provides a highly sensitive and rapid specific primer for identifying the species of the Songjiang perch. Mitochondrial genome sequences of the Songjiang perch and eight closely related species of sculpins were downloaded from the NCBI database and analyzed using BioEdit software. Cytb Multiple sequence alignment analysis was performed on the gene. Due to the close relationship between the Songjiang perch and its closely related species... Cytb Due to the high degree of sequence conservation, it is difficult to simultaneously identify regions with significant interspecific differences at both ends of the amplified fragment. Therefore, primer design focuses on the specific recognition function of the 3′ end, screening out a specific variant segment that can be used for species identification. This specific variant segment, within a 4 bp range, was selected where there are at least two base differences between the Songjiang perch and its closely related species. Cytb The segment serves as the 3' end target site for the PCR reverse primer (TF-R) (see...). Figure 1Based on this, and taking into account parameters such as annealing temperature, GC content, and amplified fragment length, forward primers TF-F were designed using Primer Premier 5 software, ultimately obtaining a pair of highly specific *Perch spp.* Cytb Gene amplification primers, primer sequences are as follows: Forward primer TF-F: 5'-CTAAACTCCGACGCAGATA-3' (SEQ NO.1); Reverse primer TF-R: 5'-GCTGTAAGGGCAATCAAC-3' (SEQ NO.2).

[0021] Example 2: Identification experiment of Songjiang perch using the specific primers of Example 1. (1) Muscle tissue samples (20 mg each) from 2 adult Songjiang perch, 2 juvenile Songjiang perch, 2 sculpins of the genus *Sculpins esculpinus*, and 2 sculpins of the genus *Sculpins multispinosa* were collected, and genomic DNA was extracted using the phenol-chloroform method. The original DNA concentrations of the adult and juvenile Songjiang perch were 36.9, 31.8, 53.6, and 55.8 ng / μL, respectively; the original DNA concentrations of the genus *Sculpins esculpinus* were 28.8 and 31.6 ng / μL, respectively; and the original DNA concentrations of the genus *Sculpins multispinosa* were 16.8 and 14.0 ng / μL, respectively. The DNA from Songjiang perch, sculpins esculpinus, and sculpins multispinosa were diluted 10-fold, 100-fold, and 1000-fold, respectively, and used as templates for PCR amplification.

[0022] (2) PCR amplification was performed using the specific primers for Songjiang perch designed in this invention: The PCR amplification system consisted of a total volume of 25 μL, including 12.5 μL of 2×PCR Mix, 1 μL of DNA template, 0.5 μL each of forward and reverse primers at a concentration of 10 μM / μL, and the volume was made up with sterile double-distilled water.

[0023] The PCR amplification program was as follows: 95 °C pre-denaturation for 5 min; 95 °C denaturation for 30 s, 59 °C annealing for 30 s, 72 °C extension for 50 s, 35 cycles; and a final extension at 72 °C for 5 min.

[0024] (3) Take 2 μL of the amplification product for 1.5% agarose gel electrophoresis (1×TAE buffer, voltage ≤5 V / cm, 40 min), and observe the amplification results using a UV gel imaging system (see Figure 2 ). Figure 2In the diagram: M represents the DL2000 DNA Marker; A1–A6 are amplified bands from DNA samples of two juvenile Songjiang perch at 10-fold (5.36 ng / μL, 5.58 ng / μL), 100-fold (0.54 ng / μL, 0.56 ng / μL), and 1000-fold (0.05 ng / μL, 0.06 ng / μL) dilutions, respectively; B1–B6 are amplified bands from DNA samples of two adult Songjiang perch at 10-fold (3.69 ng / μL, 3.18 ng / μL), 100-fold (0.37 ng / μL, 0.32 ng / μL), and 1000-fold (0.04 ng / μL, 0.03 ng / μL) dilutions, respectively; C1–C6 are amplified bands from DNA samples of two scorpionfish at 10-fold (2.88 ng / μL, 3.16 ng / μL), 100-fold (0.37 ng / μL, 0.32 ng / μL), and 1000-fold (0.04 ng / μL, 0.03 ng / μL) dilutions, respectively; Amplification bands of DNA from two scorpionfish at dilutions of 10-fold (0.29 ng / μL, 0.31 ng / μL) and 1000-fold (0.03 ng / μL, 0.03 ng / μL), respectively; D1–D6 are amplification bands of DNA from two scorpionfish at dilutions of 10-fold (1.68 ng / μL, 1.40 ng / μL), 100-fold (0.17 ng / μL, 0.14 ng / μL) and 1000-fold (0.02 ng / μL, 0.01 ng / μL), respectively.

[0025] The results showed that both adult and juvenile perch samples exhibited clear and specific bands at 92 bp, and stable amplification was achieved even at extremely low DNA concentrations after 1000-fold dilution. In contrast, no amplification signals were observed in samples of *Sepia esculenta* and *Sepia multispinosa* at any concentration gradient, further verifying that the designed primers have high species specificity for *Sepia esculenta* and do not cross-amplify with closely related species.

[0026] In summary, the species-specific primers for *Perequa spp.* described in this invention can achieve stable short-fragment specific amplification in both adult and juvenile *Perequa spp.* at extremely low DNA concentrations, while showing no amplification signal in closely related species such as *Sculpins esculpinus* and *Sculpins multispinus*. This method has significant advantages in terms of ease of operation, high sensitivity, and strong specificity, making it suitable for rapid large-scale identification of *Perequa spp.* germplasm resources and environmental DNA monitoring, providing an efficient and reliable molecular detection tool for the conservation, artificial breeding, and resource assessment of *Perequa spp.*

Claims

1. A highly sensitive and rapid specific primer for identifying the species of *Perequa spp.*, characterized in that: The forward and reverse primer sequences of the specific primers are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

2. A method for rapid identification of the species of Songjiang perch, characterized in that, Includes the following steps: (1) Genomic DNA was extracted from the sample to be tested using the phenol-chloroform method, and the concentration of the original DNA solution was 10–60 ng / μL; (2) The extracted DNA samples were diluted 10, 100 and 1,000 times respectively, and used as DNA templates for PCR amplification using the specific primers described in claim 1; (3) Gel electrophoresis detection and result determination of PCR amplification products: If a clear amplification band appears at 92 bp, the sample is determined to be a Songjiang perch; If no bands appear, the sample is not a species of Songjiang perch.

3. The method according to claim 2, characterized in that: The total volume of the PCR amplification reaction system described in step (2) is 25 μL, including 12.5 μL of 2×PCR Mix, 1 μL of diluted DNA template, 0.5 μL each of forward and reverse primers, and 10.5 μL of sterile double-distilled water.

4. The method according to claim 2, characterized in that: The PCR amplification reaction conditions in step (2) are as follows: pre-denaturation at 95 °C for 5 min, followed by 35 cycles, each cycle including denaturation at 95 °C for 30 s, annealing at 59 °C for 30 s, extension at 72 °C for 50 s; and finally extension at 72 °C for 5 min.

5. The method according to claim 2, characterized in that: In step (3), the amplification products were detected by 1.5% agarose gel electrophoresis. The electrophoresis buffer was 1×TAE, the voltage was controlled at ≤5 V / cm, the electrophoresis time was 40 min, 2 μL of PCR amplification products were taken for detection, and the image was recorded using a gel imaging system for result analysis and judgment.

6. The use of the specific primers according to claim 1 in the preparation of products for rapid identification of *Perch spp.* species, characterized in that: The product is a reagent or kit.

7. A reagent or kit for rapid identification of *Perch spp.* species, characterized in that: It contains the specific primers as described in claim 1.

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