Primer, kit and method for identifying magpie species

Abstract

The invention discloses a primer, a kit and a method for identifying the species of magpie. The primer comprises a primer SEQ ID NO.1 and a primer SEQ ID NO.2 of a targeted COI region; the primers of the target D-loop region are shown in SEQ ID NO.3 and SEQ ID NO.4. The invention also discloses a kit for detecting the target D The invention also provides a corresponding kit and a method for identifying the magpie species by using the primer. The specific PCR primer for the magpie, provided by the invention, targets a specific gene sequence of a species, and can stably realize accurate detection of DNA of the magpie in a complex environment medium. The method has the advantages of being easy and convenient to operate, high in sensitivity, rapid in detection and the like, is suitable for scenes such as species identification and environmental DNA monitoring, provides a reliable technical means for ecological research, species protection and dynamic monitoring of the ecological environment of the magpie, and has wide application prospects and practical value.

Description

Technical Field

[0001] The invention belongs to the technical field of species identification, and particularly relates to a primer, a kit and a method for species identification of gray magpie. Background Art

[0002] Birds are vital components of ecosystems, playing an irreplaceable role in maintaining biodiversity, regulating population structure, and maintaining ecological balance. By preying on insects and dispersing plant seeds, they participate in the material and energy cycles of ecosystems and are often used as indicators of ecological health. Accurate identification of bird species is fundamental to understanding their ecological functions and developing conservation measures, and is also a prerequisite for conducting ecological monitoring.

[0003] Gray Magpie ( Cyanopica cyanus ) belongs to the genus Pica (Pica spp.) of the family Corvidae in the order Passeriformes. Cyanopica The Gray Magpie is primarily found in eastern China. It prefers semi-open environments such as woodlands, parks, and orchards. It is highly adaptable and a carnivorous, omnivorous bird. It primarily feeds on insects (Hemiptera, Coleoptera, Lepidoptera, etc.) and their larvae, along with some fruits and seeds. It occasionally preys on small vertebrates or steals the young of other birds. According to statistics, an adult Gray Magpie can eliminate approximately 15,000 pine caterpillars annually, effectively protecting approximately 667 square meters of forest from pest infestation, making it a significant biocontrol species. Given its significant contribution to pest control and forest protection, the Gray Magpie is often considered an important indicator species for evaluating the quality of garden ecosystems.

[0004] Despite its prominent role in ecosystems, the identification and dynamic monitoring of the gray magpie still face technical bottlenecks. Currently, the mainstream gray magpie identification method, namely traditional morphological identification, relies too much on sample integrity and operator experience. It is difficult to handle morphologically similar species or incomplete samples. Traditional methods are cumbersome, inefficient, and prone to environmental disturbances, making them unable to meet the needs of current ecological research and species monitoring. While modern molecular methods have made some breakthroughs, they still have certain limitations. For example, metabarcoding and high-throughput sequencing can enable simultaneous detection of multiple species, but when dealing with birds with similar sequences, the phylogenetic resolution is insufficient, which can easily lead to cross-identification errors. Furthermore, this technology relies on database integrity and is relatively expensive, hindering the widespread use of routine ecological monitoring. Conventional PCR technology, despite its high sensitivity and ease of operation, lacks a specific primer design and validation system for gray magpies, resulting in a long-standing lack of application in gray magpie monitoring.

[0005] In view of this, it is very necessary to provide a rapid, sensitive and low-cost molecular identification method for Cinerea cirrhosae. Summary of the Invention

[0006] In order to solve at least one of the above problems, the present invention provides a primer, a kit and a method for species identification of Cinerea fasciatus.

[0007] In order to achieve the above object, the present invention adopts the following technical means: The first aspect of the present invention provides a primer for species identification of Cinerea grisea, comprising a primer targeting the COI region: the forward primer sequence is shown in SEQ ID NO.1, and the reverse primer sequence is shown in SEQ ID NO.2.

[0008] The second aspect of the present invention provides a primer for species identification of Cinerea grisea, comprising a primer targeting the D-loop region: the forward primer sequence is shown in SEQ ID NO.3, and the reverse primer sequence is shown in SEQ ID NO.4.

[0009] The third aspect of the present invention provides a kit for species identification of Cinerea grisea, comprising the primers described in the first and second aspects.

[0010] A fourth aspect of the present invention provides the use of the primers described in the first and second aspects for species identification of Cinerea cirrhosae. During the application result determination process, the target species is considered detected only when both pairs of specific primers detect a positive result. Double primer verification is performed to avoid false positives that may occur with a single primer.

[0011] A fifth aspect of the present invention provides a method for identifying species of gray magpie, comprising the following steps: S1. Extract genomic DNA of the sample to be tested; S2. Using the genomic DNA of the sample to be tested as a template, PCR amplification is performed using the primers described in claims 1 and 2; S3, the amplified products were detected by gel electrophoresis; If the gel electrophoresis diagrams of the two sets of amplified products show specific electrophoresis bands of 262 bp and 218 bp respectively, the sample to be tested is judged to be positive for the gray magpie DNA; otherwise, the sample to be tested is judged to be negative for the gray magpie DNA.

[0012] In some embodiments of the present invention, the sample to be tested can be a fresh feather sample with hair follicles, a feces sample, or an environmental sample such as soil, air, or water.

[0013] In some embodiments of the present invention, the PCR amplification system in step S2 is: 20-25 μL of MIX enzyme, 2 μL of forward primer, 2 μL of reverse primer, 25-30 ng of DNA template, and water to make up the system to 50 μL.

[0014] In some preferred embodiments of the present invention, the PCR amplification system in step S2 is: 25 μL of MIX enzyme, 2 μL of forward primer, 2 μL of reverse primer, 30 ng of DNA template, and water to make up the system to 50 μL.

[0015] In some embodiments of the present invention, in the PCR amplification system, the final concentration of the forward primer and the reverse primer is 0.4-0.5 μM.

[0016] In some embodiments of the present invention, when the primers described in claim 1 are used for amplification, the amplification conditions are: 95°C for 3 min; 95°C for 20 s for 34 cycles; 56°C for 30 s, 72°C for 30 s, 72°C for 5 min, and 12°C hold.

[0017] In some embodiments of the present invention, when the primers described in claim 2 are used for amplification, the amplification conditions are: 95°C for 10 min; 95°C for 30 s for 35 cycles; 55°C for 30 s, 72°C for 30 s, 72°C for 5 min, and hold at 12°C.

[0018] In some embodiments of the present invention, the method further comprises the step of recovering and purifying the PCR amplification product after agarose gel electrophoresis, and sequencing the sequencing result to compare the sequencing result with the known sequence.

[0019] Beneficial effects of the present invention Compared with the prior art, the present invention has the following beneficial effects: (1) High specificity. The gray magpie-specific PCR primers proposed in this invention can accurately identify and specifically amplify the target DNA fragment of gray magpie, avoiding nonspecific amplification of common related species (such as magpies, red-billed blue magpies, etc.) and non-target species in environmental samples, effectively ensuring the accuracy and reliability of qualitative identification.

[0020] (2) Strong adaptability. The primer targets species-specific gene sequences and can stably detect gray magpie-specific bands in bird feces samples containing a large amount of background DNA and environmental samples with extremely low target DNA abundance. It has good anti-interference ability against complex background DNA, significantly improving the detection efficiency under complex sample conditions.

[0021] (3) High efficiency. The rapid detection method based on conventional PCR and gel electrophoresis eliminates the expensive and time-consuming sequencing verification process, providing a simple and efficient detection method for ecological monitoring and environmental DNA research of gray magpies, greatly saving time and costs.

[0022] (4) Highly targeted application. This primer provides a dedicated technical tool for the qualitative detection of gray magpies, and is particularly suitable for practical application scenarios such as biodiversity surveys and wildlife protection, where the presence of gray magpies needs to be quickly and accurately confirmed.

[0023] (5) Convenient operation and easy promotion. The primer design is reasonable, the synthesis cost is low, the detection process is standardized, the operation is simple, and it is suitable for application under conventional laboratory conditions. It has good prospects for promotion and popularization. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The results of PCR amplification of DNA samples from gray magpie feathers using primers COI_Cy and D-loop_Cy in Example 2 of the present invention are shown; wherein lanes 1, 2, 3, 4, and 5 are gray magpie feathers from five different individuals; Figure 2 The results of PCR amplification of DNA from different bird feces samples using primers COI_Cy & D-loop_Cy in Example 3 of the present invention are shown, wherein lanes 1, 2, 3, 4, 5, 6, and 7 correspond to samples numbered HXQ-FB-1 to HXQ-FB-7, respectively; Figure 3 The results of PCR amplification of environmental (air) sample DNA using primers COI_Cy & D-loop_Cy & V12S-U in Example 4 of the present invention are shown, wherein lanes 1, 2, 3, 4, 5, 6, 7, and 8 correspond to samples numbered HXQ-KQ-1 to HXQ-KQ-8, respectively. DETAILED DESCRIPTION

[0025] The following examples are provided to illustrate preferred embodiments of the present invention. Those skilled in the art will appreciate that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to practice the present invention and, therefore, can be considered preferred embodiments of the present invention. However, those skilled in the art will appreciate from this disclosure that many modifications may be made to the specific embodiments disclosed herein while still achieving the same or similar results without departing from the spirit or scope of the present invention.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention belongs, and the disclosures herein and the materials they cite are hereby incorporated by reference. Those skilled in the art will recognize or be able to ascertain, through routine experimentation, many technical equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

[0027] The technical solution of the present application will be further described in detail below in conjunction with specific implementation methods.

[0028] Example 1 Primer design and evaluation 1. Sequence raw data acquisition and processing (1) Obtaining the mitochondrial genome of the target species and its closely related species.

[0029] Gray magpie ( Cyanopica cyanus ) and its congener and closely related species (such as the same family or order). Select the Nucleotide library in NCBI and enter the command in the search box: (( Cyanopica cyanus The collection included one mitochondrial genome of P. cinerea (NC_024821.1) and mitochondrial genomes of 12 closely related species. The specific AccessionIDs are shown in Table 1.

[0030] Table 1 Mitochondrial genomes of target species and their closely related species

[0031] (2) Multiple sequence alignment. MEGA was used to perform multiple sequence alignment on the obtained mitochondrial genome and generate an alignment file in FASTA format to provide a data basis for subsequent specific site analysis.

[0032] 2. Specific primer design and performance evaluation (1) Specific region screening This study identified regions with three or more base differences between the target species and closely related species as potential candidate regions for primer design. These differentiated regions provide clear targets for subsequent primer design.

[0033] (2) Primer design and screening Based on mitochondrial genome sequence alignment, highly variable regions (such as the D-loop) and conserved regions (such as interspecies variant sites in the COI and CytB genes) within the mitochondrial genome were prioritized as primer targets. By simultaneously designing two pairs of specific primers targeting different regions, a dual validation mechanism was implemented. This approach maintains high specificity and sensitivity while improving the accuracy and applicability of target species detection in complex samples. This approach is particularly suitable for field samples with complex backgrounds or severe DNA degradation. Primer design adhered to the following criteria: primer length was controlled between 18 and 24 bp, GC content was maintained at 45% to 55%, Tm was set at 58°C to 62°C, and amplification product length was controlled between 100 and 300 bp. Furthermore, attention was paid to avoiding continuous G / C base sequences at the 3' end of the primers to reduce the risk of nonspecific amplification. Primer dimers and hairpin structures, which could interfere with amplification specificity, were also avoided.

[0034] (3) Primer specificity assessment To ensure primer specificity, the designed primers were aligned with the full range of eukaryotic sequences in the NCBI Nucleotide Database (nt database) using the primer-BLAST tool. Validation parameters were set as follows: the nt database was selected, the species type was set to Eukaryota, the amplicon length was set based on the expected product length from the primer design, and all other parameters used the default values. Alignment analysis confirmed that the designed primers did not target homologous sequences from species other than the gray magpie, demonstrating good specificity. Finally, a pair of specific primers was selected for the COI and D-loop regions, respectively. Specific primer information is shown in Table 2, and these primers entered the subsequent experimental validation phase. During the result interpretation process, the target species was considered detected only if both pairs of specific primers detected a positive result. Double-primer validation was performed to prevent false positive reactions from single primers that could affect the final result.

[0035] Table 2 Primer sequence information

[0036] Example 2 Primer amplification feasibility verification test In order to evaluate the basic amplification ability of primers COI_Cy and D-loop_Cy for Cinerea spp., the following experiments were performed: 1. Sample Source and Processing This study collected feather samples from five different individuals of the gray magpie, strictly adhering to the standards of the "Technical Specifications for the Collection of Biological Genetic Resources (Trial Implementation)" (HJ628-2011). Given that DNA in feather samples is primarily found in hair follicles, fresh feathers with follicles present were preferred, with particular attention paid to feather tips free of hair to ensure the integrity and validity of the DNA in the samples. Genomic DNA was extracted using a DNA extraction kit (model: DP324-03) manufactured by Tiangen Biochemical Technology (Beijing) Co., Ltd. according to standardized procedures. The DNA obtained using this method will serve as a template for subsequent PCR amplification experiments, providing basic data support for further research.

[0037] 2. PCR test PCR reactions were set up for primers COI_Cy and D-loop_Cy, respectively. The PCR reaction system and amplification system of the COI_Cy primer are shown in Table 3; the PCR reaction system and amplification program of the D-loop_Cy primer are shown in Table 4.

[0038] Table 3 PCR reaction system and amplification procedure of COI_Cy primers

[0039] Table 4 PCR reaction system and amplification procedure of D-loop_Cy primers

[0040] After the PCR reaction is completed, the amplified products are analyzed by gel electrophoresis. The size of the products is determined by the position of the electrophoretic bands, and the presence of specific amplified bands is observed.

[0041] 3. Test results Gel electrophoresis results Figure 1 shown.

[0042] The results showed that primers COI_Cy and D-loop_Cy amplified the Cinerea grisea DNA samples, producing specific bands of the expected size: 262 bp and 218 bp, respectively (both band sizes were around 250 bp). This result demonstrates that the primer pair designed in this study can effectively amplify the target DNA fragment from Cinerea grisea samples and has good amplification feasibility.

[0043] Example 3 Verification of primer anti-interference and specificity in complex samples To evaluate the ability of the designed primers to effectively amplify the target species in a complex fecal matrix containing a large amount of background DNA and to resist interference from non-target substances, the following experiments were performed: 1. Sample Source and Processing In this study, fecal samples were collected from the gray magpie and six other closely related bird species with co-local distribution. Specific species information is shown in Table 5, covering species such as the magpie and the red-billed blue magpie.

[0044] Table 5 List of species in guano samples

[0045] Sample collection strictly adhered to the "Technical Specifications for the Collection of Biological Genetic Resources (Trial Implementation)" HJ628-2011, prioritizing bird droppings in granular form or attached to leaves. Immediately after collection, fecal samples were placed in test tubes containing a dedicated preservation solution to ensure sample stability and prepare for subsequent DNA extraction. For DNA extraction, a DNA extraction kit for soil samples (model: DZ301-03) manufactured by Ark Safety Biotechnology (Guangzhou) Co., Ltd. was used. Following standardized procedures, a mixed sample containing multiple components, including host bird genomic DNA, symbiotic microbial DNA, and DNA from undigested food residues, was successfully obtained. Using the extracted DNA as the reaction template, PCR amplification experiments were conducted using the COI_Cy and D-loop_Cy primers, which were experimentally validated for good amplification in Example 2, providing experimental basis for subsequent research.

[0046] 2. PCR test The same reaction system and amplification procedure as in Example 1 were used.

[0047] After the PCR reaction is completed, the amplified products are analyzed by gel electrophoresis. The presence of specific amplified bands is determined based on the position of the electrophoretic bands.

[0048] 3. Test results Gel electrophoresis results Figure 2 shown.

[0049] The results showed that in known gray magpie fecal samples (lane 1), primers COI_Cy and D-loop_Cy successfully amplified specific bands of the expected size: 262bp and 218bp specific electrophoresis bands (both band sizes were around 250bp). No target bands were observed in fecal samples from closely related and colocalized species. This result fully demonstrates that the primers designed in this study can effectively resist background DNA interference in complex fecal samples and accurately amplify the gray magpie target sequence. They have excellent anti-interference ability and species specificity, meeting the needs of complex sample detection.

[0050] Example 4 Validation of primer detection efficacy in environmental samples To verify the detection ability of the designed primers in real complex environmental samples, a cross-validation experiment was conducted by combining PCR and high-throughput sequencing technology. The specific contents are as follows: 1. Sample Source and Processing Eight air samples were collected from a park woodland frequented by gray magpies. The specific collection method was based on the "Technical Requirements and Testing Methods for Ambient Air Samplers" (HJ / T 375). Environmental DNA (eDNA) was extracted from these air samples using a DNA extraction kit (model: DP324-03) manufactured by Tiangen Biochemical Technology (Beijing) Co., Ltd. according to standardized procedures. The resulting samples contained target gray magpie DNA, as well as background DNA from symbiotic microorganisms, plants, and other organisms, forming a highly complex mixed system. The extracted samples were directly tested using the primers COI_Cy and D-loop_Cy, validated in Example 3, for subsequent PCR analysis.

[0051] 2. PCR test The reaction system and amplification procedure established in Example 1 were used to ensure consistency of experimental conditions.

[0052] After the PCR reaction is completed, the amplified products are analyzed by gel electrophoresis. The presence of specific amplified bands is determined based on the position of the electrophoretic bands.

[0053] 3. High-throughput sequencing cross-validation The universal primer pair V12S-U for vertebrates was used; V12S-UF: 5'-GTGCCAGCNRCCGCGGTYANAC-3'; V12S-UR: 5'-ATAGTRGGGTATCTAATCCYAGT-3'; product size: ~207 bp.

[0054] A second PCR amplification was performed on the same batch of environmental sample DNA. The products amplified using the three primer sets were purified and subsequently subjected to library construction, high-throughput sequencing, data quality control, and species annotation analysis. The sequencing results focused on the detection of the gray magpie, which was then compared with the PCR test results.

[0055] 4. Test results: Gel electrophoresis results Figure 3 shown.

[0056] The results showed that among the eight air samples, seven tested positive for Cinerea sibiricus DNA using both primers COI_Cy and D-loop_Cy. Lane 5 tested negative for Cinerea sibiricus. Using universal primer V12S-U, five samples tested positive for Cinerea sibiricus, while lanes 3, 5, and 8 tested negative.

[0057] The sequencing results are shown in Table 6.

[0058] Table 6 Environmental samples - sequencing results and PCR cross-validation

[0059] Note: “+” represents a positive test result, and “-” represents a negative test result.

[0060] The results showed that except for the inconsistent detection results of DNA samples corresponding to lanes 3 and 8 for primers COI_Cy, D-loop_Cy and V12S-U, the detection results of the other samples were completely consistent.

[0061] Cross-validation conclusion: Of the eight air samples, only one failed to detect Cinerea cirrhosae; the target species was detected in the remaining seven samples. PCR results showed that the detection results in lanes 3 and 8 were inconsistent among the three primer sets: primers COI_Cy and D-loop_Cy were both positive, while V12S-U was negative. Analysis combined with sequencing results confirmed that the primers designed in this paper have higher sensitivity and stronger specificity than V12S-U, enabling efficient and accurate identification of target species in low-abundance real-world environmental samples.

[0062] In summary, the proposed method for designing C. grisea-specific PCR primers targets species-specific gene sequences and can accurately and stably detect C. grisea DNA in complex environmental media. This method offers advantages such as ease of operation, high sensitivity, and rapid detection, making it suitable for species identification and environmental DNA monitoring. It provides a reliable technical tool for C. grisea ecological research, species conservation, and dynamic ecological monitoring, and has broad application prospects and practical value.

[0063] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the present application.

Claims

1. A primer for species identification of gray magpie, characterized in that: The primers targeting the COI region are as follows: the forward primer sequence is shown in SEQ ID NO.1, and the reverse primer sequence is shown in SEQ ID NO.

2.

2. A primer for species identification of gray magpie, characterized in that: The primers targeting the D-loop region are included: the forward primer sequence is shown in SEQ ID NO.3, and the reverse primer sequence is shown in SEQ ID NO.

4.

3. A kit for identifying species of gray magpie, characterized by: Comprising the primers according to claims 1 and 2.

4. Use of the primers described in claims 1 and 2 in species identification of Psoralea corylifolia.

5. A method for identifying species of gray magpie, characterized in that: The steps include: S1. Extract genomic DNA of the sample to be tested; S2. Using the genomic DNA of the sample to be tested as a template, PCR amplification is performed using the primers described in claims 1 and 2 respectively; S3, the amplified products were detected by gel electrophoresis; If the gel electrophoresis diagrams of the two sets of amplified products show specific electrophoresis bands of 262 bp and 218 bp respectively, the sample to be tested is judged to be positive for the gray magpie DNA; otherwise, the sample to be tested is judged to be negative for the gray magpie DNA.

6. The identification method according to claim 5, characterized in that: The PCR amplification system in step S2 is: 20-25 μL of MIX enzyme, 2 μL of forward primer, 2 μL of reverse primer, 25-30 ng of DNA template, and water to make up the system to 50 μL.

7. The identification method according to claim 6, wherein: In the PCR amplification system, the final concentrations of the forward primer and reverse primer were 0.4-0.5 μM.

8. The identification method according to claim 6, wherein: When the primers described in claim 1 are used, the amplification conditions are: 95°C for 3 minutes; 95°C for 20 seconds for 34 cycles; 56°C for 30 seconds, 72°C for 30 seconds, 72°C for 5 minutes, and hold at 12°C.

9. The identification method according to claim 6, wherein: When the primers described in claim 2 are used, the amplification conditions are: 95°C for 10 min; 95°C for 30 s for 35 cycles; 55°C for 30 s, 72°C for 30 s, 72°C for 5 min, and hold at 12°C.

10. The identification method according to any one of claims 5 to 9, characterized in that: The method also includes the steps of performing agarose gel electrophoresis on the PCR amplification product, recovering and purifying the product, and sequencing the product, and comparing the sequencing result with the known sequence.

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