Primer, kit and method for identifying species of sparrow

Abstract

The invention discloses a primer, a kit and a method for identifying species of a sparrow. The primer comprises a primer SEQ ID NO.1 and a primer SEQ ID NO.2 of a targeted COI region, a primer SEQ ID NO.1 and a primer SEQ ID NO.2 of a targeted COI region, 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 species of the sparrow by using the primer. According to the specific PCR primer for the sparrow, a specific gene sequence of a targeted species is adopted, and accurate detection of DNA of the sparrow can be stably realized 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 sparrow, and has wide application prospects and practical values.

Description

Technical Field

[0001] The invention belongs to the technical field of species identification, and in particular relates to a primer, a kit and a method for species identification of tree sparrows. Background Art

[0002] Currently, species identification of tree sparrows still relies primarily on morphological identification, using phenotypic characteristics such as beak shape, body length, and plumage color. However, this method has significant drawbacks. Traditional morphological identification is limited by sample integrity, operator experience, and difficulty identifying juveniles and remains. This can easily lead to false positives, especially for birds with highly similar morphological structures to tree sparrows (such as the tree sparrow). This labor-intensive, inefficient, and environmentally damaging process makes it difficult to meet the demands of modern, efficient, and accurate identification.

[0003] Among the existing molecular detection technologies, from the molecular identification level, eDNA biological monitoring is mainly divided into two methods: multi-species monitoring based on macrobarcoding technology and single-species monitoring based on PCR technology.

[0004] Currently reported molecular identification of tree sparrows mostly relies on the former, such as the COI and CytB universal primers commonly used in bird species monitoring. The main principle of this technology is to design universal primers for the homologous DNA barcode regions of multiple species, and perform high-throughput sequencing on the obtained amplified products to achieve multi-species identification. However, this method also has obvious limitations. 1. Limited phylogenetic resolution: Existing macrobarcoding technology is often difficult to accurately distinguish when dealing with species with high sequence similarity, resulting in easy cross-reactions between tree sparrows and other similar species, requiring reliance on sequencing for secondary confirmation; 2. Species preference: Due to the species preference of macrobarcoding primers, species with lower abundance may be "masked", resulting in failure to detect the target species. 3. High cost and complexity: Macrobarcoding technology relies on high-throughput sequencing and requires huge database support, which increases the time and economic cost of detection. At the same time, the sequencing results are limited by the integrity of the database. Therefore, this type of technology is more suitable for biodiversity assessment. The latter approach involves designing specific primers based on the DNA sequence of the target species, then using PCR and multiplex PCR to detect the presence or absence of the target species for qualitative or quantitative analysis. Compared to macrobarcoding, it offers a simpler, cheaper, more sensitive, and more efficient method for qualitative identification. Currently, standards and a validation system for the design of specific primers for tree sparrows have not yet been established, creating a technical gap in the molecular identification of tree sparrows. Summary of the Invention

[0005] In order to solve at least one of the above problems, the present invention provides a primer, a kit and a method for identifying tree sparrow species.

[0006] 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 identifying tree sparrow species, 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.

[0007] The second aspect of the present invention provides a primer for identifying tree sparrow species, including 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.

[0008] The third aspect of the present invention provides a kit for identifying tree sparrow species, comprising the primers described in the first and second aspects.

[0009] The fourth aspect of the present invention provides the use of the primers described in the first and second aspects in identifying tree sparrow species.

[0010] A fifth aspect of the present invention provides a method for identifying tree sparrow species, 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 the first aspect and the second aspect respectively; S3, the amplified products were detected by gel electrophoresis; If the two groups of amplified products show specific electrophoretic bands of 200 bp and 259 bp respectively in the gel electrophoresis diagram, the tree sparrow DNA in the sample to be tested is determined to be positive; otherwise, the tree sparrow DNA in the sample to be tested is determined to be negative.

[0011] 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.

[0012] 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.

[0013] In a preferred embodiment 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.

[0014] 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.

[0015] In some embodiments of the present invention, when the primers of claim 1 are used, the amplification conditions are: 95°C for 3 min; 95°C for 20 s for 36 cycles; 56°C for 30 s, 72°C for 30 s, 72°C for 5 min, and 12°C hold.

[0016] In some embodiments of the present invention, when the primers of claim 2 are used, the amplification conditions are: 95°C for 5 min; 95°C for 20 s for 36 cycles; 55°C for 45 s, 72°C for 1 min, 72°C for 5 min, and 12°C hold.

[0017] 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.

[0018] Beneficial effects of the present invention Compared with the prior art, the present invention has the following beneficial effects: (1) High specificity and high resolution. This invention innovatively designs two pairs of tree sparrow-specific primers targeting the COI region and the D-loop region, respectively, which can significantly enhance species discrimination while ensuring amplification stability. It has been verified that the primers specifically amplify only the target gene fragment of the tree sparrow and have no effective amplification of the DNA of common related birds such as the house sparrow. This avoids the cross-reactions that may occur with existing primers, thereby improving the accurate identification of tree sparrows.

[0019] (2) Strong anti-interference ability. In bird droppings samples containing complex background DNA, the primers of the present invention can effectively resist interference from non-target species, avoid false positive results, and improve the accuracy of detection.

[0020] (3) Excellent environmental adaptability. In environmental samples with low DNA abundance, the primers of the present invention can effectively amplify the target tree sparrow DNA and have high sensitivity, enabling accurate and sensitive detection of the target species in complex environments.

[0021] (4) Improved eDNA monitoring efficiency. This invention provides a simple and sensitive detection method that can provide strong technical support for ecological monitoring, species protection, and environmental DNA monitoring of tree sparrows, thereby reducing monitoring costs and improving monitoring efficiency.

[0022] (5) Economical and easy to promote. The primers are reasonably designed, the synthesis cost is low, and the operation process is standardized, making them easy to apply and promote in routine laboratories. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1The results of PCR amplification of tree sparrow feather DNA samples using primers COI_Pm & D-loop_Pm in Example 2 of the present invention are shown; wherein lanes 1, 2, 3, 4, and 5 are feathers of five different tree sparrow individuals; Figure 2 The results of PCR amplification of DNA from different bird fecal samples using primers COI_Pm & D-loop_Pm in Example 3 of the present invention are shown. Lanes 1, 2, 3, 4, 5, 6, and 7 correspond to samples numbered SMQ-FB-1 to SMQ-FB-7, respectively. Figure 3 The results of PCR amplification of environmental (air) sample DNA using primers COI_Pm&D-loop_Pm&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 SMQ-KQ-1 to SMQ-KQ-8, respectively. DETAILED DESCRIPTION

[0024] 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.

[0025] 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.

[0026] The technical solution of the present application will be further described in detail below in conjunction with specific implementation methods.

[0027] Example 1 Primer design and evaluation 1. Sequence raw data acquisition and processing (1) Obtain the mitochondrial genome of the target species and its closely related species. Collect the tree sparrow ( Passer montanus ) 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: (( Passer montanusThe collection included one tree sparrow mitochondrial genome (NC_024821.1) and mitochondrial genomes of 10 closely related species. The specific AccessionIDs are shown in Table 1.

[0028] Table 1 Mitochondrial genomes of target species and their closely related species

[0029] (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.

[0030] 2. Specific primer design and performance evaluation (1) Finding specific regions Regions with at least three base differences between the target species and closely related species are considered potential candidate regions for primer design. These regions can provide clear targets for subsequent primer design.

[0031] (2) Specific primer screening Based on the comparison results of the mitochondrial genome, in order to enhance the stability of the detection and species discrimination ability, specific primers were designed in the interspecies variation regions in the conserved regions (such as COI and CytB) and the hypervariable regions (D-loop regions) with a large number of specific sites. The primer design principles are as follows: Key parameters: Primer length: 18-24bp; GC content: 40%-60%; Tm range: 55℃-65℃; product length range: 100-300bp.

[0032] During primer design, try to avoid primer dimers and hairpin structures that may lead to nonspecific amplification.

[0033] (3) Primer specificity assessment To evaluate the specificity of the primers designed in the present invention, the two pairs of primers designed in this paper were aligned with the nt library of NCBI using the primer-BLAST tool, and the aligned species covered all eukaryotic organisms.

[0034] The specific comparison parameters are set as follows: Parameter settings: Database: Nucleic acid library (nt); Species type: Eukaryote (Eukaryota); Amplicon length: It is set according to the designed primer amplification length; Other parameters: default.

[0035] The designed primers were compared and analyzed according to the above parameters to determine that the designed primers did not target homologous sequences of species other than tree sparrows, and the designed primers were considered to have good specificity.

[0036] Finally, a pair of specific primers were screened in the COI region and the D-loop region respectively to enter the subsequent experimental verification stage. The result judgment criteria were: only when the results of both pairs of primers were positive, the target species was considered to be detected. The specific information of the primers is shown in Table 2.

[0037] Table 2 Primer sequence information

[0038] Example 2 Amplification feasibility test To evaluate the basic amplification ability of primers COI_Pm and D-loop_Pm for tree sparrow, the following experiments were performed: 1. Sample Source and Processing Feather samples (n = 5) were collected from multiple individuals of tree sparrows. Specific collection guidelines were based on the "Technical Specifications for the Collection of Biological Genetic Resources (Trial Implementation)" (HJ628-2011). Because DNA in feather samples is primarily concentrated in hair follicles, feathers were collected from fresh, hair-tipped locations (including follicles) and free of hair. DNA was extracted from the samples using a DNA extraction kit (model: DP324-03) manufactured by Tiangen Biochemical Technology (Beijing) Co., Ltd. PCR amplification of the extracted DNA was performed using primers COI_Pm and D-loop_Pm.

[0039] 2. PCR test PCR reactions were set up using primers COI_Pm and COI_Pm, respectively. The PCR reaction system and amplification system of COI_Pm primers are shown in Table 3; the PCR reaction system and amplification program of COI_Pm primers are shown in Table 4.

[0040] Table 3 PCR reaction system and amplification procedure of COI_Pm primers

[0041] Table 4 PCR reaction system and amplification procedure of COI_Pm primers

[0042] After PCR amplification, gel electrophoresis was used to detect the size of the PCR product, and the presence of specific bands was determined based on the electrophoresis results.

[0043] 3. Test results Gel electrophoresis results Figure 1 shown.

[0044] The results showed that primers COI_Pm and D-loop_Pm successfully amplified the target product from five tree sparrow samples, generating specific electrophoresis bands of 200 bp and 259 bp, respectively (both bands were around 250 bp). This demonstrates that the primer pair designed in this paper can effectively amplify the target fragment from tree sparrow samples and has basic amplification feasibility.

[0045] Example 3 Anti-interference test and specificity verification 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 Fecal samples were collected from tree sparrows and their closely related and sympatric species (a total of 6 species), including house sparrows, mountain sparrows, etc. Specific species information is shown in Table 5 .

[0046] Table 5 List of species in guano samples

[0047] Sample collection principles follow the "Technical Specifications for Collection of Biological Genetic Resources (Trial Implementation)" (HJ628-2011). Samples are typically collected in the form of pelleted guano or from leaves. After collection, the samples are placed in tubes containing fecal preservation solution for subsequent DNA extraction. DNA extraction was performed using a DNA extraction kit (Model: DZ301-03) manufactured by Ark Safety Biotechnology (Guangzhou) Co., Ltd., aiming to obtain a complex mixture containing tree sparrow DNA and background DNA from a large amount of symbiotic microorganisms and undigested food residues. The extracted DNA samples were then subjected to PCR reactions using the primers COI_Pm and D-loop_Pm, validated in Example 2.

[0048] 2. PCR test The same reaction system and amplification procedure as in Example 1 were used.

[0049] After amplification, gel electrophoresis was used to detect the size of the PCR product, and the electrophoresis results were used to determine whether specific bands appeared in samples of tree sparrows and their closely related and co-localized species.

[0050] 3 Test results Gel electrophoresis results Figure 2 shown.

[0051] Gel electrophoresis results showed that primers COI_Pm and D-loop_Pm successfully amplified specific target bands in a known tree sparrow feces sample (lane 1): 200bp and 259bp specific electrophoresis bands (both amplified bands were around 250bp). No expected bands were observed in feces samples from closely related and sympatric bird species other than the tree sparrow. This demonstrates that the designed primers can effectively resist interference from background DNA in complex fecal matrices, successfully amplifying the target sequence with remarkable specificity, enabling the identification of the target species from other bird species, including closely related or sympatric species.

[0052] Example 4 Environmental sample test To evaluate the actual detection performance of the designed primers in real environmental samples (containing extremely complex microbial communities, trace DNA from multiple biological sources, and target DNA abundance is generally low), and to cross-validate using high-throughput sequencing, the following experiments were conducted: 1. Sample Source and Processing Eight air samples were collected from a wooded area in a park where tree sparrows are active. Specific collection principles were based on the "Technical Requirements and Testing Methods for Ambient Air Samplers" (HJ / T 375). eDNA was extracted from the air samples using a DNA extraction kit (model: DP324-03) manufactured by Tiangen Biochemical Technology (Beijing) Co., Ltd. The DNA extraction process for environmental samples followed standardized procedures to ensure a complex mixture containing the target tree sparrow DNA and other background DNA (such as DNA from symbiotic microorganisms, plants, or other species). The extracted DNA samples were then used for subsequent PCR experiments using the primers COI_Pm and D-loop_Pm, which were validated in Example 3.

[0053] 2. PCR test The same reaction system and amplification procedure as in Example 1 were used.

[0054] After amplification, gel electrophoresis was used to detect the size of the PCR product, and the electrophoresis results were used to determine whether specific bands appeared in samples of tree sparrows and their closely related and co-localized species.

[0055] 3 High-throughput sequencing cross-validation Use the universal vertebrate primer pair V12S-U: V12S-UF: 5'-GTGCCAGCNRCCGCGGTYANAC-3'; V12S-UR: 5′-ATAGTRGGGTATCTAATCCYAGT-3′; product size: ~207 bp).

[0056] PCR amplification was performed on the same batch of environmental DNA samples. Finally, the PCR products obtained by amplifying the two pairs of designed specific primers with the V12S-U primer were all subjected to subsequent major operations such as library construction, sequencing, quality control, and species annotation of the target fragments using a common technical solution. The sequencing results were analyzed, and attention was paid to the detection of tree sparrow sequences.

[0057] 4. Test results: The gel electrophoresis results after PCR amplification were as follows Figure 3 shown.

[0058] The results showed that among the 8 air samples, 7 samples (only lane 7 was negative) were detected as tree sparrow positive in the amplification results of primer COI_Pm, and 6 samples (only lanes 2 and 7 were negative) were detected as tree sparrow positive in the amplification results of primer D-loop_Pm; the results of the universal primer V12S-U were 5 samples (lanes 2, 3, and 7 were negative) were detected as tree sparrow positive.

[0059] The detection results of target species by high-throughput sequencing are shown in Table 6.

[0060] Table 6 Environmental samples - sequencing results and PCR cross-validation

[0061] Note: “+” represents a positive test result, and “-” represents a negative test result.

[0062] The results showed that except for the DNA sample corresponding to lane 3, which showed different amplification results between primers COI_Pm, D-loop_Pm and V12S-U, the tree sparrow detection results of other samples were completely consistent.

[0063] Cross-validation conclusion: Combining the results of PCR and high-throughput sequencing, the target species was detected in the remaining six of the eight air samples, except for the DNA samples corresponding to lanes 2 and 7, where no tree sparrow was detected. However, the PCR detection results in lanes 2 and 3 were inconsistent. Analysis combined with the sequencing results revealed that the discrepancies in the amplification results in lane 2 using primers COI_Pm, D-loop_Pm, and V12S-U may be due to the fact that the individual being tested and the target species were highly similar to the tree sparrow in a certain segment, but exhibited certain genetic differences in other segments. This resulted in a positive PCR result using primers COI_Pm, while negative PCR results using primers D-loop_Pm and V12S-U were negative. This demonstrates that dual-primer validation can, to a certain extent, reduce the impact of false positive reactions on result determination. Furthermore, the positive detection results for primers COI_Pm and D-loop_Pm in lane 3, while negative for V12S-U, indicate that the primers designed in this study have greater specificity and sensitivity than the universal primer V12S-U. These results confirm that the designed primers can sensitively and specifically detect tree sparrow DNA in complex, low-abundance, real-world environmental DNA samples. Furthermore, the combination of dual-primer detection further enhances the accuracy of species identification.

[0064] In summary, the present invention provides a method for designing PCR primer pairs for the specific detection of tree sparrows. These primer pairs target gene sequences unique to tree sparrows and can stably and specifically detect tree sparrow DNA in complex environmental samples. This method offers significant advantages, including simplicity, sensitivity, and efficiency, and is widely applicable to tree sparrow species identification and environmental DNA monitoring. Furthermore, the present invention can provide reliable technical support for tree sparrow ecological research, species conservation, and ecological environment monitoring, demonstrating strong application prospects and practical value.

[0065] 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 identifying tree sparrow species, 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 identifying tree sparrow species, 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 tree sparrow species, characterized in that: Comprising the primers according to claims 1 and 2.

4. Use of the primers according to claims 1 and 2 in identifying tree sparrow species.

5. A method for identifying tree sparrow species, 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 two groups of amplified products show specific electrophoretic bands of 200 bp and 259 bp respectively in the gel electrophoresis diagram, the tree sparrow DNA in the sample to be tested is determined to be positive; otherwise, the tree sparrow DNA in the sample to be tested is determined to be negative.

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 36 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 5 minutes; 95°C for 20 seconds for 36 cycles; 55°C for 45 seconds, 72°C for 1 minute, 72°C for 5 minutes, 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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