Primer combination for analyzing bird species information based on environmental biological samples, and design method and application thereof

Abstract

The application discloses a primer combination for analyzing bird species information based on an environmental biological sample and a design method and application thereof, and relates to the technical field of bird species diversity analysis. By using the design method, the primer combination for analyzing bird species information based on an environmental biological sample can be designed, the specificity of the primer can be enhanced, and the amplification of non-target groups can be reduced on the basis of ensuring wide coverage of bird species, so that more efficient and more accurate amplification and data analysis can be realized in bird species identification and ecological monitoring. The primer combination has wide coverage, high resolution and strong specificity, and improves the reliability of bird species information analysis.

Description

Technical Field

[0001] This application relates to the field of bird species diversity analysis technology, and in particular to a primer combination, its design method, and its application for analyzing bird species information based on environmental biological samples. Background Technology

[0002] Birds, as vital components of ecosystems, play a crucial role in maintaining ecological balance, species diversity, and ecological functions. Their diversity and wide distribution make them important subjects in ecology, environmental monitoring, and biodiversity research. Accurate identification and distribution surveys of bird species are fundamental in fields such as species conservation, bird migration studies, and ecological monitoring. With the impacts of environmental change and human activities, many bird species face threats such as habitat loss, habitat fragmentation, and even extinction; therefore, timely monitoring of bird species is of paramount importance.

[0003] Traditional methods for investigating and identifying bird species rely on direct observation, using morphological characteristics to distinguish specific species. However, this method is time-consuming, labor-intensive, and can potentially damage the environment. It is inefficient in detecting true biodiversity and cannot meet the needs of large-scale, efficient monitoring.

[0004] With the development of molecular biology techniques, species identification methods based on environmental DNA (eDNA) have gradually become an important means of bird species identification. It can overcome the limitations of traditional morphological methods and obtain information on species composition in the environment non-invasively by extracting DNA from environmental samples such as water, soil or air. It is widely used in species monitoring and ecological research and is currently an efficient and inexpensive means of species identification for assessing environmental biodiversity.

[0005] The COI gene (cytochrome oxidase I gene) is a commonly used DNA barcode, widely applied in species identification and taxonomic studies across various groups, including birds. However, despite its promising potential as an eDNA barcode in avian research, existing COI primers still have limitations, particularly in large-scale avian species identification and ecological monitoring studies, where their effectiveness is somewhat restricted. The reasons are as follows:

[0006] 1) Currently, there are no universal COI primers specifically designed for birds. Most existing COI primers are designed to be cross-species universal. While these universal primers can theoretically amplify COI gene sequences from multiple bird species, their overly broad coverage often results in the amplification of non-target groups, especially in invertebrates, during avian eDNA studies. This leads to significant interference from background noise in subsequent experimental results, increasing the complexity and inaccuracy of data analysis and impacting species diversity assessment. Furthermore, existing primers struggle to effectively distinguish between similar species, particularly in areas with high bird species diversity. They often fail to accurately differentiate subtle genetic differences between species, leading to errors in species identification and affecting the accuracy of ecological monitoring and species conservation research.

[0007] 2) The limited number of COI primer designs targeting bird species are also primarily focused on a few common bird species, especially in specific bird conservation or ecological studies. This limitation means that the primers cannot cover the widely distributed bird populations in nature, and therefore cannot meet the needs of large-scale bird species identification.

[0008] In summary, a major challenge facing avian eDNA research in large-scale bird species identification and monitoring is how to enhance primer specificity, reduce the amplification of non-target groups, improve data reliability, and further enhance the accuracy of bird species identification while ensuring broad coverage of bird populations. Summary of the Invention

[0009] To solve at least one of the above-mentioned technical problems, the technical solution adopted in this application is as follows.

[0010] The first aspect of this application provides a method for designing primer combinations based on environmental biological samples for analyzing bird species information, including the following steps:

[0011] S1, obtain the nucleic acid sequences of COI genes from multiple bird species, and remove redundancy to obtain representative sequences;

[0012] S2, perform multiple sequence alignment on the representative sequence obtained in step S1, and obtain the conserved region based on the alignment results;

[0013] S3, After selecting the conserved region for primer design, design primers based on the base frequencies at each site within that conserved region:

[0014] For a given site, if the frequency of a base is not less than 90%, then that base is used directly; if the frequency of all bases is less than 90%, then primer degenerates the bases using the following method:

[0015] If the frequencies of the two lowest-frequency bases are both less than 10%, but the sum of their frequencies is not less than 10%, then the site should be designed as a degenerate base N. Otherwise, degenerate primers should be designed based on all bases with frequencies not less than 10%.

[0016] In this application, those skilled in the art can obtain the nucleic acid sequences of the COI genes of the bird species from any publicly available or non-public database, including but not limited to NCBI, BOLD, and MODIRI. In one specific embodiment of this application, the inventors obtained nucleic acid sequences of the COI genes of 55,497 bird species, covering 39 orders, 203 families, 1,867 genera, and 6,177 species-level barcodes of the bird species.

[0017] In some embodiments of this application, in step S1, the redundancy removal involves classifying sequences with a sequence similarity exceeding 99% as belonging to the same category and retaining one as the representative sequence.

[0018] In some specific embodiments of this application, a sequence quality control step is included before redundancy removal. Specifically, the following types of sequences are removed:

[0019] i) Sequences with a length lower than the first threshold;

[0020] ii) Sequences with fuzzy information exceeding the second threshold.

[0021] In some preferred embodiments of this application, the first threshold is 550 bp.

[0022] In some preferred embodiments of this application, the fuzzy information refers to an "N" or "-" base. In some more preferred embodiments of this application, the second threshold is 50%.

[0023] In some embodiments of this application, those skilled in the art can use any multiple sequence alignment software or tool to perform multiple sequence alignment on the representative sequence. Such multiple sequence alignment software or tools include, but are not limited to, ClustalOmega, MAFFT, MUSCLE, T-Coffee, Kalign, PRANK, DIALIGN, ProbCons, POA, FSA, Geneious, BioEdit, MEGA, SeaView, and NCBI BLAST.

[0024] In this application, the conserved region refers to a region that is highly similar across different bird species. In some embodiments of this application, those skilled in the art can obtain the conserved region using any multiple sequence alignment result analysis software or tools.

[0025] In some embodiments of this application, the base frequency of the site refers to the proportion of each base at that site in the alignment results. For example, for a certain site, if A appears 1000 times, C appears 1000 times, T appears 1000 times, and G appears 1000 times, then the base frequency of each base is 25%.

[0026] In some specific embodiments of this application, degenerate bases are designed according to the table below.

[0027]

[0028] For example, for a target site, the frequencies of A, T, C, and G are 45%, 5%, 8%, and 42%, respectively. The frequencies of the two least frequent bases (T and C) are both less than 10%, but their sum is 13%, which is more than 10%. In this case, the site is designed as N. As another example, for a target site, the frequencies of A, T, C, and G are 45%, 5%, 3%, and 47%, respectively. The frequencies of the two least frequent bases (T and C) are both less than 10%, and their sum is 8%, which is less than 10%. In this case, based on frequency... For a site with more than 10% of bases (A and G), a degenerate base R is designed. For another site, if the frequencies of A, T, C, and G are 35%, 15%, 3%, and 47%, respectively, and one of the two least frequent bases has a frequency of not less than 10%, then a degenerate base H is designed for the site based on the bases with a frequency of more than 10% (A, T, and C). For yet another site, if the frequencies of A, T, C, and G are all 25%, that is, the frequencies of A, T, C, and G all exceed 10%, then a degenerate base N is designed for the site.

[0029] In some embodiments of this application, in order to balance primer universality and amplification efficiency, the first to third bases at the 3' and / or 5' ends are not designed as degenerate bases for the conserved regions used to design primers.

[0030] Furthermore, for any primer designed for the conserved region: if the number of degenerate bases exceeds 30% of the primer length, then delete the primer; if the number of degenerate bases in all primers designed for the conserved region exceeds 30% of the primer length, then select at least one primer and perform the following treatment: alternately replace the degenerate bases at the 3' and 5' ends with the most frequent major terms until the number of degenerate bases does not exceed 30% of the primer length.

[0031] In some embodiments of this application, a step of validating the designed primers is also included.

[0032] In some specific embodiments of this application, during verification, the primer pair consisting of SEQ ID No. 6 and SEQ ID No. 7 and / or the primer pair consisting of SEQ ID No. 8 and SEQ ID No. 9 are compared.

[0033] In some specific embodiments of this application, coverage, resolution, and / or specificity are used as performance metrics for evaluation during verification.

[0034] For coverage, a simulated amplification method was used. Specifically, sequence alignment tools were used, and the target sequence of the COI gene of a certain bird species was considered suitable for amplification if the following conditions were met:

[0035] (1) The matching length between the primer and the target sequence reaches 80% or more of the primer length;

[0036] (2) The total number of mismatched bases between the primers and the target sequence is no more than 3;

[0037] (3) The primer amplifies the target sequence fragment length of 100bp~300bp.

[0038] For resolution, multiple species were randomly selected from each order of birds within the amplifiable range. Amplification product sequences were extracted and redundancy removed. The resolution of each primer at the species / genus / family level was then calculated. Evaluating the primer amplification efficiency and the resolution of the target gene region ensures that the primers can effectively distinguish between different species.

[0039] For specificity, the amplified species composition, excluding birds, is statistically analyzed, categorized into vertebrates (excluding birds) and invertebrates. The binding of primers to non-target sequences is assessed to avoid non-specific amplification.

[0040] The second aspect of this application provides a primer combination for analyzing bird species information based on environmental biological samples, wherein the primer combination is designed using any of the design methods described in the first aspect of this application.

[0041] A third aspect of this application provides a primer combination for analyzing bird species information based on environmental biological samples, specifically including primers shown in SEQ ID No. 1 to SEQ ID No. 5, wherein SEQ ID No. 1 and SEQ ID No. 3 form primer pairs with SEQ ID No. 2, and SEQ ID No. 4 and SEQ ID No. 5 form primer pairs.

[0042] The fourth aspect of this application provides a kit for analyzing bird species information based on environmental biological samples, including the primer combination described in the third aspect of this application.

[0043] In some embodiments of this application, the kit further includes DNA extraction reagents, DNA purification reagents, PCR amplification reagents, and / or sequencing reagents.

[0044] The fifth aspect of this application provides a method for analyzing bird species information based on environmental biological samples, comprising the following steps:

[0045] Obtain DNA samples from environmental biological samples;

[0046] The DNA sample was amplified by PCR using the primer combination described in the second or third aspect of this application.

[0047] The amplified products were used to construct a library and sequence the sequences. The sequencing data were then analyzed to obtain information about the bird species.

[0048] In some embodiments of this application, the PCR amplification procedure is as follows:

[0049] 95℃ for 5 min; 95℃ for 30 sec, 50℃ for 30 sec, 72℃ for 30 sec, 36 cycles; 72℃ for 8 min; 4℃ Hold.

[0050] In this application, the environmental biological samples include, but are not limited to:

[0051] In this application, the bird species information includes, but is not limited to, bird diversity information.

[0052] Compared with the prior art, the invention title of this application has the following beneficial effects:

[0053] Using the design method of this application, primer combinations for analyzing bird species information based on environmental biological samples can be designed. While ensuring broad coverage of bird species, primer specificity can be enhanced and the amplification of non-target groups can be reduced, so as to achieve more efficient and accurate amplification and data analysis in bird species identification and ecological monitoring.

[0054] The primer combinations in this application have broad coverage (more than 5,000 species-level barcodes), high resolution, and strong specificity, which can significantly reduce the non-specific amplification phenomenon commonly found in environmental samples, especially the interference of invertebrate pollution on bird species identification, and improve the reliability of the data.

[0055] The primer combinations described in this application exhibit greater versatility in large-scale bird species identification, avoiding the experimental complexity caused by designing multiple primers and frequently changing them in such studies. By reducing non-specific amplification and optimizing experimental resource utilization, experimental costs are significantly reduced, improving economic efficiency. These primers can be directly applied to bird species identification of environmental samples and high-throughput analysis of multiple species, saving time and resources.

[0056] It should be understood that the description in this section is not intended to identify key or essential features of the embodiments of this application, nor is it intended to limit the scope of this application. Other features of this application will become readily apparent from the following description. Attached Figure Description

[0057] The above and other objects, features, and advantages of exemplary embodiments of this application will become readily apparent from the following detailed description taken in conjunction with the accompanying drawings. Several embodiments of this application are illustrated in the drawings by way of example and not limitation, in which:

[0058] Figure 1 A schematic diagram of the process upon which the design in Embodiment 1 of this application is based is shown;

[0059] Figure 2 The highly conserved regions and base frequency distributions for primer design obtained in Example 1 of this application are shown.

[0060] Figure 3 The location of the five primer pairs in Example 1 of this application on the COI gene is shown;

[0061] Figure 4 The amplification numbers of each primer combination for bird species, vertebrates (excluding birds), and invertebrates in Example 2 of this application are shown.

[0062] Figure 5 The results of bird species detection in a single-group DNA sample from Example 3 of this application are shown. Detailed Implementation

[0063] Unless otherwise stated, implied from the context, or as is customary in the art, all parts and percentages in this application are based on weight, and all testing and characterization methods used are concurrent with the filing date of this application. Where applicable, any patent, patent application, or disclosure relating to this application is incorporated herein by reference in its entirety, and its equivalent patent families are also incorporated herein by reference, in particular the definitions of relevant terms in the art disclosed in such documents. If any definition of a specific term disclosed in the prior art is inconsistent with any definition provided in this application, the definition provided in this application shall prevail.

[0064] The terms “comprising,” “including,” “having,” and their derivatives do not exclude the presence of any other components, steps, or processes, regardless of whether such other components, steps, or processes are disclosed in this application. To eliminate any doubt, unless expressly stated otherwise, all compositions using the terms “comprising,” “including,” or “having” in this application may contain any additional additives, excipients, or compounds. Conversely, except for those necessary for operational performance, the term “substantially constitutes…” excludes any other components, steps, or processes described below with respect to that term. The term “consisting of…” does not include any components, steps, or processes not specifically described or listed. Unless expressly stated otherwise, the term “or” refers to the individual members listed or any combination thereof.

[0065] To make the technical problems, technical solutions and beneficial effects solved by this application clearer, the following detailed description is provided in conjunction with embodiments.

[0066] The following examples are used to illustrate preferred embodiments of this application. Those skilled in the art will understand that the techniques disclosed in the examples represent technologies discovered by the inventors that can be used to implement this application, and therefore can be considered preferred embodiments of this application. However, those skilled in the art should understand from this specification that many modifications can be made to the specific embodiments disclosed herein, still yielding the same or similar results, without departing from the spirit or scope of this application.

[0067] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains, and all materials cited herein and referenced by them are incorporated herein by reference.

[0068] Those skilled in the art will recognize, or can learn through routine experimentation, many equivalents of the specific embodiments of the invention described herein. These equivalents will be included in the claims.

[0069] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0070] Example 1: Primer Design for Bird Species Diversity Analysis

[0071] This embodiment combines Figure 1 This application describes the primer design method used for bird species diversity analysis.

[0072] 1. Obtain the avian COI gene sequence

[0073] (1) Acquisition of avian COI gene nucleic acid sequence data

[0074] A total of 55,497 avian COI gene nucleic acid sequences were downloaded from multiple databases (including NCBI, BOLD, and MODIRI), covering 39 orders, 203 families, 1,867 genera, and 6,177 species-level barcodes. These data provided the inventors with a comprehensive sequence basis for primer design.

[0075] (2) Nucleic acid sequence preprocessing

[0076] The acquired sequences were quality controlled to remove the following types of sequences:

[0077] i) The length is lower than the set threshold (<550 bp);

[0078] ii) Sequences with fuzzy information exceeding a set threshold (where “N” or “-” occupies 50% of the total sequence length).

[0079] The cd-hit tool was used to remove redundancy from the sequences, and a sequence similarity threshold of 99% was set (i.e., sequences with more than 99% similarity were considered to be of the same class) to obtain representative sequences and improve data accuracy.

[0080] (3) Multiple sequence alignment

[0081] Multiple sequence alignment software (such as muscle and maft) is used to perform multiple sequence alignment on the preprocessed representative sequences to generate alignment files in FASTA format, providing a data foundation for subsequent analysis of conserved regions.

[0082] 2. Conserved sequence identification and primer design

[0083] (1) Finding conservative sequences

[0084] Import the alignment results (FASTA format alignment file) into Jalview software for conservative region analysis.

[0085] Universal primer design typically involves large-scale data alignment, which may contain unreliable sequences or misaligned regions. Therefore, it's necessary to remove low-quality sequences from the alignment results, such as sequences containing numerous missing bases ("-" frequency > 99% at a certain site in the alignment file) and low-quality sequences with obvious alignment misalignments. This processing ensures high-quality alignment results while improving the accuracy of conserved region identification. Ultimately, conserved regions in the avian COI gene are identified. The selected conserved regions are based on those located at the gene ends, while the middle region may contain highly variable fragments.

[0086] Furthermore, highly conserved regions of at least 20 base pairs were selected as candidate sites. Special attention was paid to the design of the 3' end, avoiding the selection of low-complexity regions or fragments with excessively high GC content (e.g., exceeding 60%) to reduce the risk of nonspecific amplification.

[0087] The highly conserved regions and base frequency distributions obtained for primer design are as follows: Figure 2 As shown, there are three regions, and the length of the amplified products is less than 300 bp, which is suitable for short-read sequencing platforms such as Illumina HiSeq.

[0088] (2) Degenerate base design

[0089] Based on the results of multiple sequence alignment and the base frequency distribution of the target sequence, the principles for introducing degenerate bases are set according to requirements, as follows:

[0090] i) Conditions for introducing degenerate bases

[0091] For a given target site, if the frequency of a base is not less than 90%, then that base is used directly; if the frequencies of all bases are less than 90% (i.e., at least two or more bases have frequencies of not less than 10%), then multiple bases coexist relatively evenly, and we should consider introducing degenerate bases.

[0092] If the frequencies of the two lowest-frequency bases are both less than 10%, but the sum of their frequencies is not less than 10%, then the site is designed as a degenerate base N. Otherwise, all bases with frequencies not less than 10% are selected, and the corresponding degenerate bases are generated according to the IUPAC rules, as shown in Table 1. For example, for a target site, the frequencies of A, T, C, and G are 45%, 5%, 8%, and 42%, respectively. The frequencies of the two lowest-frequency bases (T and C) are both less than 10%, but the sum of their frequencies is 13%, which is more than 10%, so the site is designed as N. As another example, for a target site, the frequencies of A, T, C, and G are 45%, 5%, 3%, and 47%, respectively. The frequencies of the two lowest-frequency bases (T and C) are both less than 10%, and the sum of their frequencies is 8%, which is less than 10%. In this case, according to the frequency... For a site with more than 10% of bases (A and G), a degenerate base R is designed. For another site, if the frequencies of A, T, C, and G are 35%, 15%, 3%, and 47%, respectively, and one of the two least frequent bases has a frequency of not less than 10%, then a degenerate base H is designed for the site based on the bases with a frequency of more than 10% (A, T, and C). For yet another site, if the frequencies of A, T, C, and G are all 25%, that is, the frequencies of A, T, C, and G all exceed 10%, then a degenerate base N is designed for the site.

[0093] Table 1 Degenerate base design rules

[0094]

[0095] ii) Restrictions on the number and type of degeneracy

[0096] The number of degenerate bases should be controlled within 30% of the primer length to balance primer universality and amplification efficiency. The introduction of degenerate bases should be concentrated in the middle of the sequence, rather than at the ends, to improve amplification stability and primer binding reliability. Avoid introducing degenerate bases at the primer ends (especially the 3' end) to reduce the risk of nonspecific amplification and primer dimer formation.

[0097] For any highly conserved region used for primer design, primers are designed according to the above design rules to obtain multiple candidate primer pairs. For any primer, if the number of degenerate bases exceeds 30% of the primer length, the primer is deleted. If, for this region, the number of degenerate bases in all candidate primers exceeds 30% of the primer length, at least one candidate primer is randomly selected for degenerate base replacement: first, the first degenerate base at the 3' end is replaced with the most frequent base; then, the first degenerate base at the 5' end is replaced with the most frequent base. If the number of degenerate bases still exceeds 30% of the primer length, the first degenerate base at the 3' end is replaced with the most frequent base again, and so on, until the number of degenerate bases does not exceed 30% of the primer length.

[0098] After the above design and screening, the primer sequences of three highly conserved regions were obtained as shown in Table 2.

[0099] Table 2 Primers for 3 highly conserved regions

[0100]

[0101] The inventors also synthesized two existing pairs of universal vertebrate primers: VCOI-U (-F / R) and Mod_RepCOI (-F / R) to verify the superiority of the primer combinations screened in this application.

[0102] VCOI-UF: CAYGCHTTTGTNATRATYTTYTT (SEQ ID No. 6)

[0103] VCOI-UR:GGRGGRTADACDGTYCANCCNGT (SEQ ID No. 7)

[0104] Mod_RepCOI_F:TNTTYTCMACYAACCACAAAGA (SEQ ID No. 8)

[0105] Mod_RepCOI_R:CARAAGCTYATGTTRTTYATDCG (SEQ ID No. 9)

[0106] The location of the three primer pairs and the two universal primer pairs mentioned above on the COI gene is shown in Table 2. Figure 3 As shown.

[0107] Example 2 Primer performance evaluation

[0108] Primer performance metrics include coverage, resolution, and specificity. Simulated amplification was performed using the BLAST tool for comparison, with the following settings:

[0109] Query Coverage: The matching length between the primer and the target sequence must reach ≥80% of their total length to ensure that the alignment region has a sufficient degree of matching;

[0110] Tolerance range for mismatched bases: The total number of mismatched bases between the primer and the target sequence is ≤3. If there are two consecutive mismatches at the 3' end, it is considered non-amplifiable even if other conditions are met;

[0111] Alignment fragment length limitations: The target amplified fragment length is primarily 100-300 bp, while allowing for some individual variation (within 600 bp) to meet the requirements of next-generation sequencing. Results exceeding this range are considered invalid.

[0112] Database selection: A bird COI gene database that has undergone quality control and redundancy removal was used as a comparison reference.

[0113] Furthermore, 100 species from each order of birds were randomly selected from the amplifiable bird species, and the amplification product sequences were extracted and redundancy removed. The resolution of each primer at the species / genus / family level was then calculated. Specifically:

[0114] Among the selected 100 species, the amplified sequences of these species were dereduplicated (100% similarity). Assuming that 90 sequences were obtained after dereduplicated sequences, the species-level resolution of the primer is 90%.

[0115] Next, the remaining 10 redundant sequences are analyzed. If there are identical sequences among species from different genera (for example, a sequence from a species in genus A is identical to a sequence from a species in genera B and C), it means the primers cannot distinguish them, and the genus-level resolution is calculated. Assuming these 100 species are distributed across 50 different genera, then based on the number of redundant sequences, the genus-level resolution is: 1 - 2 / 50 = 96%.

[0116] By analogy, continue calculating the resolution at the subject and item levels.

[0117] Assessing primer amplification efficiency and target gene region resolution ensures that primers can effectively distinguish between different species.

[0118] The coverage and resolution of each primer combination for bird species are shown in Tables 3 and 4, respectively. The amplification counts for bird species, vertebrates (excluding birds), and invertebrates are shown in Tables 5 and 6. Figure 4 As shown.

[0119] Table 3. Coverage of bird species for each primer combination

[0120]

[0121] Table 4. Resolution of each primer combination for bird species

[0122]

[0123] Table 5. Number of species amplified by each primer combination for different species.

[0124]

[0125] From Table 3 and Figure 4 As can be seen, in terms of coverage, the three primer pairs in this application all achieved a species-level coverage of over 80%, amplifying more than 5,000 species-level barcodes of birds, significantly outperforming VCOI-U and Mod_RepCOI.VertCOI_7216. Furthermore, the primer pairs in this application also demonstrated higher universality in terms of genus / family / order-level coverage.

[0126] As shown in Table 4, in terms of resolution, the three primer pairs in this application all have a resolution of around 90% at the species level, which is roughly equivalent to VCOI-U and Mod_RepCOI.VertCOI_7216.

[0127] From Table 5 and Figure 4As can be seen, in terms of specificity, the three primer pairs in this application are significantly more specific than VCOI-U, greatly limiting the amplification of non-target groups, including vertebrates (excluding birds) and invertebrates. Compared with Mod_RepCOI.VertCOI, the effect on limiting invertebrates is roughly equivalent. Mod_RepCOI.VertCOI is superior to the three primer pairs in this application in limiting vertebrates (excluding birds). However, since the primer pairs in this application are mainly used for bird species identification research and do not completely avoid the amplification of non-avian animals, considering that some specificity will inevitably be lost while maintaining bird universality, and for vertebrates (excluding birds), compared to invertebrates, birds have limited ecological contact with other vertebrates (such as mammals and reptiles). The probability of non-avian vertebrate DNA contamination in bird samples is relatively low, and its impact on bird identification results is small. Furthermore, even if a small amount of non-avian vertebrate sequence signals are amplified during amplification, subsequent bioinformatics analysis (such as alignment filtering) can easily eliminate the interference caused by these species.

[0128] Example 3: Artificially constructed DNA mixed samples to verify primer amplification performance

[0129] 1. Mixed DNA samples

[0130] The target DNA, derived from birds, includes Gallus gallus , Apus nipalensis , Passer domesticus Forty bird species were selected, covering the main lineages of birds and representing a wide range of species.

[0131] Background DNA was derived from vertebrates (excluding birds) and invertebrates. Fifteen vertebrate species (excluding birds) were randomly selected, covering amphibians, mammals, fish, reptiles, etc., including... Carassius auratus , Lithobates catesbeianus , Gekko chinensis etc.; 15 representative species of invertebrates were randomly selected, including some insects and mollusks, including Enpinanga assamensis , Polypedilum bingoparadoxum , Atractomorpha sinensis wait;

[0132] 2. Single-group DNA pooled samples

[0133] Bird DNA was mixed in equal quantities to a uniform concentration (10 ng / µL). Two template concentration settings were used for the PCR reaction:

[0134] High concentration: 30 ng / reaction;

[0135] Low concentration: 3 ng / reaction.

[0136] Single-group DNA pooled samples were used to test the detection efficiency and sensitivity of different primer combinations for target species.

[0137] 3. Multi-group DNA pooled samples

[0138] Construct a mixed sample of target DNA (avian DNA) and background DNA (non-avian vertebrate DNA + invertebrate DNA). There are three ratios of avian DNA: vertebrate DNA (excluding birds): invertebrate DNA:

[0139] High: 70% : 10% : 20%;

[0140] Medium: 50% : 20% : 30%;

[0141] Low: 30% : 30% : 40%.

[0142] The total DNA concentration of the mixed samples was set to 10 ng / µL to simulate DNA abundance under different environmental conditions. The PCR reaction template concentration was set to 30 ng / reaction.

[0143] Multi-group DNA pooled samples are used to detect the specificity of different primer combinations for target groups.

[0144] The detailed PCR reaction system and execution procedure used in the experiment are as follows:

[0145] Reaction system:

[0146] Total reaction volume: 50µL

[0147] Reaction components:

[0148] 1) Taq DNA polymerase (Novoprotein): 25µL

[0149] 2) Forward primer (10µM): 2µL

[0150] 3) Reverse primer (10µM): 2µL

[0151] 4) DNA template: Add according to the experimental design.

[0152] 5) Sterile water: Add to a final volume of 50µL

[0153] Amplification procedure:

[0154] 95℃ for 5 min; 95℃ for 30 sec, 50℃ for 30 sec, 72℃ for 30 sec, 36 cycles; 72℃ for 8 min; 4℃ Hold. 4. Sample Detection

[0155] The target fragment was amplified, library constructed, and sequenced using common techniques in the field, and the data were subjected to quality control and species annotation according to the general procedure.

[0156] 5. Results Analysis

[0157] The results of bird species detection in single-group DNA samples are shown in Table 6 and Figure 5 As shown.

[0158] Table 6. Results of bird species detection in single-group DNA samples

[0159]

[0160] Note: The table shows the detection status of each species at "high / low" concentrations, with "+" indicating a positive detection and "-" indicating a negative detection.

[0161] From Table 6 and Figure 5 It can be seen that primer combinations AvianCOI-U-1 and AvianCOI-U-2 showed better detection rates for bird species than primer combinations VCOI-U and Mod_RepCOI.VertCOI_7216. Even at low concentrations, the detection rates of primer combinations AvianCOI-U-1 and AvianCOI-U-2 reached nearly 90% (35 / 40 for both), indicating that both primer combinations have good detection efficacy and sensitivity. Primer combination AvianCOI-U-3 showed slightly lower detection efficacy than VCOI-U (33 / 40) at high concentrations (31 / 40), but the detection rate was the same at low concentrations (31 / 40). Mod_RepCOI.VertCOI_7216 only achieved about half of the total detection rate for birds (high concentration: 24 / 40, low concentration: 21 / 40).

[0162] The species detection results of DNA samples from multiple groups are shown in Tables 7 to 10.

[0163] Table 7 Statistical results of DNA samples from multiple groups

[0164]

[0165] Table 8 Species Detection Results of Bird DNA Samples

[0166]

[0167] Note: The table shows the detection results of each species at different mixing ratios (70: 10: 20; 50: 20: 30; 30: 30: 40). "+" indicates a positive result; "-" indicates a negative result.

[0168] Table 9. Species Detection Results of Vertebrate (excluding birds) DNA Samples

[0169]

[0170] Note: The table shows the detection results of each species at different mixing ratios (70: 10: 20; 50: 20: 30; 30: 30: 40). "+" indicates a positive result; "-" indicates a negative result.

[0171] Table 10 Species Detection Results of Invertebrate DNA Samples

[0172]

[0173] Note: The table shows the detection results of each species at different mixing ratios (70: 10: 20; 50: 20: 30; 30: 30: 40). "+" indicates a positive result; "-" indicates a negative result.

[0174] As shown in Tables 7-10, under different group mixing ratios, when the abundance of bird species is at a medium to high level, the reads of the target group obtained using each primer combination reached over 80%. Regarding limiting non-specific amplification of vertebrates other than birds, the order was: AvianCOI-U-3 > Mod_RepCOI.VertCOI_7216 > AvianCOI-U-1 > AvianCOI-U-2 >> VCOI-U. Regarding limiting non-specific amplification of invertebrates, the three primer combinations in this application outperformed VCOI-U and Mod_RepCOI.VertCOI_7216, limiting the amplification rate of this group to approximately 1%, effectively restricting the impact of non-specific amplification of invertebrates on the target species.

[0175] Example 4: Detection results of bird species information in environmental samples

[0176] To further verify the detection efficacy of the primer pair designed in Example 1 in a real environment, the inventors conducted multi-media environmental sample collection at the Shenzhen Bay Futian Mangrove Wetland as the ecological sampling area, including air and water samples, totaling 6 samples (4 air samples and 2 water samples). The specific sample collection method is as follows:

[0177] Water samples were collected in accordance with the following standards: Technical Specifications for Environmental Monitoring of Nearshore Marine Areas Part VI: Biological Monitoring of Nearshore Marine Areas (HJ442.6); Marine Monitoring Specifications Part VII: Ecological Investigation and Biological Monitoring of Nearshore Pollution (GB17378.7); Technical Specifications for Identification of Environmental DNA (eDNA) of Fish and Shellfish (DB11_T 2023-2022); and Technical Methods for Monitoring Environmental DNA of Freshwater Organisms (Draft for Comments) (DB 32 / T).

[0178] The water sampler and sampling bottles were disinfected with a 10% bleach solution before use, and the personnel collecting the samples changed disposable gloves after each sampling. 1L of water was collected from each sampling point. The collected water samples were stored under refrigeration and transported back to the laboratory. Within 24 hours, they were filtered using a vacuum pump onto a 0.45μm cellulose acetate membrane. The equipment was disinfected before and after each sample filtration to remove residual DNA and prevent cross-contamination. The filter membrane was placed in a freezer and immediately stored at -20°C.

[0179] Air sample collection shall be conducted in accordance with the "Technical Requirements and Testing Methods for Ambient Air Samplers" HJ / T 375.

[0180] A medium-flow particulate sampler was selected for aerosol enrichment. The sampling personnel wore disposable gloves and installed a 90mm × 0.45μm glass fiber filter membrane into the air sampler. The eDNA enrichment parameters were adjusted as follows: (1) Sampling method: self-aspiration, 120L / min (±2.0%); (2) Sampling time: 4h; (3) Inlet velocity: 0.3m / s. After sample collection, the aerosol filter membrane was removed and placed in a storage tube, stored at -20℃, and shipped.

[0181] The collected samples were extracted and amplified, library constructed, and sequenced according to a unified amplification system (see Example 3). The data were then subjected to quality control and species annotation according to a general procedure.

[0182] The final test results are shown in Table 11. AvianCOI-U-1, AvianCOI-U-2, and AvianCOI-U-3 detected 14, 10, and 12 bird species, respectively. VCOI-U and Mod_RepCOI.VertCOI_7216 detected 7 and 6 bird species, respectively. In addition, some non-avian species were also detected, mainly including fish and invertebrates (such as zooplankton and insects). AvianCOI-U-1, AvianCOI-U-2, and AvianCOI-U-3 detected 3, 3, and 2 of these species, respectively. VCOI-U and Mod_RepCOI.VertCOI_7216 detected 14 and 3 of these species, respectively. The AvianCOI-U primer set significantly reduced interference from non-target groups in real-world environments, exhibiting significantly higher specificity than VCOI-U. Furthermore, its ability to detect bird species was also superior to both VCOI-U and Mod_RepCOI.VertCOI_7216. This demonstrates that the VCOI-U primer set of this invention still exhibits significant advantages and maintains reliable bird detection capabilities even in complex and diverse environments.

[0183] Table 11 Species Detection Results of Environmental Samples

[0184]

[0185] In summary, based on in-depth mining and precise analysis of a large amount of data, this application has achieved effective identification and optimization of target primer sequences, and comprehensively evaluated primer performance. The designed primer combination shows significant advantages over existing primers in terms of both coverage and specificity of bird species, and is suitable for occasions such as environmental samples with complex species composition and where bird diversity assessment is required.

[0186] All references to this application are incorporated herein by reference as if each reference were individually incorporated herein by reference. Furthermore, it should be understood that after reading the foregoing teachings of this application, those skilled in the art can make various alterations or modifications to this application, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A primer combination for analyzing bird species information based on environmental biological samples, characterized in that, Includes primers shown in SEQ ID No. 1 to SEQ ID No. 5, wherein SEQ ID No. 1 and SEQ ID No. 3 form primer pairs with SEQ ID No. 2, and SEQ ID No. 4 and SEQ ID No. 5 form primer pairs.

2. A kit for analyzing bird species information based on environmental biological samples, characterized by, Includes the primer combination described in claim 1.

3. The kit of claim 2, wherein It also includes DNA extraction reagents, DNA purification reagents, PCR amplification reagents, and / or sequencing reagents.

4. A method of analyzing bird species information based on environmental biological samples, characterized by, Includes the following steps: Obtain DNA samples from environmental biological samples; The DNA sample was amplified by PCR using the primer combination described in claim 1. The amplified products were used to construct a library and sequence the sequences. The sequencing data were then analyzed to obtain information about the bird species.

5. The method of claim 4, wherein, The PCR amplification procedure is as follows: 95℃ for 5 min; 95℃ for 30 sec, 50℃ for 30 sec, 72℃ for 30 sec, 36 cycles; 72℃ for 8 min; 4℃ Hold.

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