A dual-gene-specific primer set for rapid identification of chiropteran species and a method for applying the same

Abstract

This invention discloses a dual-gene specific primer set for rapid identification of Chiroptera species and its application method. The primer set includes primer pair COI-F / COI-R for amplifying the mitochondrial COI gene and primer pair Cytb-F / Cytb-R for amplifying the Cytb gene, with nucleotide sequences shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4, respectively. Experiments have demonstrated that this primer set exhibits good interspecific specificity and intraspecific conservation, effectively distinguishing Chiroptera from other closely related mammals and accurately identifying cryptic species and juvenile samples that are difficult to distinguish morphologically. This invention employs dual-gene markers for mutual verification, significantly improving the accuracy and reliability of identification, providing a powerful molecular biology tool for rapid and accurate identification of Chiroptera species, and has significant application value in fields such as biodiversity surveys, disease tracing, and ecological conservation.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and in particular to a dual-gene specific primer set for rapid identification of Chiroptera species and its application method. Background Technology

[0002] Bats, or Chiroptera, are the second most diverse group of mammals, boasting exceptional species diversity and a global distribution (excluding polar regions). They play crucial roles in ecosystems, such as controlling agricultural and forestry pests and dispersing pollen and seeds, thus playing a vital role in maintaining ecological balance. Furthermore, as natural hosts for various zoonotic viruses, bats are of significant interest in public health and disease tracing.

[0003] However, accurate identification of Chiroptera species has always been a challenge. Traditional morphological identification methods mainly rely on external features, tooth structure, forearm length, and other indicators. These methods have significant limitations: First, many Chiroptera species are highly similar in external morphology (such as several species within the Rhinolophus family), making them difficult to distinguish by the naked eye and conventional measurements alone; second, morphological features are often lacking in juveniles, remains, feces, and food boluses frequently encountered in field surveys, making effective identification impossible; furthermore, recent studies in molecular systematics have revealed the existence of numerous cryptic species, further increasing the difficulty of traditional classification.

[0004] The development of molecular biology techniques has provided new solutions for species identification. Mitochondrial DNA, characterized by its rapid evolutionary rate, maternal inheritance, high intraspecific conservation, and significant interspecific variation, is frequently used as a molecular marker for species identification. Among these, the cytochrome C oxidase subunit I (COI) gene is widely recognized as the "DNA barcode" of the animal kingdom, performing exceptionally well in species identification and the discovery of cryptic species; the cytochrome b (Cytb) gene, with its moderate evolutionary rate, is widely used in phylogenetic reconstruction and population genetic structure analysis.

[0005] In DNA barcoding research for the identification of Chiroptera species, current techniques primarily employ universal primers for single-gene marker amplification. However, Chiroptera mitochondrial gene sequences exhibit significant variation at the family and genus level, leading to decreased stability of universal primer binding to the template and large fluctuations in amplification efficiency. Although some studies have attempted to improve amplification results by optimizing annealing temperature, highly specific primer systems designed for the mitochondrial genome characteristics of Chiroptera groups, and their systematic application in the collaborative identification of multiple gene loci, have not yet been adequately reported in the literature. Furthermore, relying solely on the size of electrophoretic bands carries the risk of misjudgment and cannot meet the needs of accurate identification.

[0006] The prior art, patent application number CN121320558A, discloses a species identification kit for animal mitochondrial barcoding multiplex PCR amplification and nanopore sequencing, belonging to the field of species identification technology. This species identification kit contains optimized primer pairs targeting four hypervariable mitochondrial genes (Cytb, COI, 16S rRNA, and 12S rRNA). By introducing degenerate bases such as R, Y, and D at key sites, the binding ability of the primers to template DNA from different species is significantly improved, overcoming the problem of mismatch, low efficiency, or even amplification failure when using existing universal primers for cross-species amplification. It can be used for species identification of mixed DNA samples containing at least two different animal species. While this invention combines a multiplex PCR amplification system with nanopore long-read sequencing technology, enabling the differentiation of closely related species with highly similar sequences, it cannot be used to accurately identify Chiroptera species. Summary of the Invention

[0007] To address the technical bottlenecks in existing bat-like species identification methods, such as unstable amplification efficiency, insufficient specificity, and limited developmental resolution of single-gene marker systems, this invention provides a dual-gene specific primer set and its application method for rapid identification of bat-like species. This primer set is designed based on the mitochondrial COI and Cytb dual-gene markers, enabling efficient and accurate identification of bat-like species. This invention utilizes a dual-gene marker primer set for bat-like species identification with high specificity and sensitivity, and establishes a standardized identification process combined with sequencing verification. This dual verification mechanism improves the accuracy of identification, which has significant practical implications for bat-like resource surveys, biodiversity conservation, and epidemiological tracing.

[0008] This invention discloses a dual-gene-specific primer set for rapid identification of Chiroptera species. The primer set is a primer set for amplifying the COI gene and the Cytb gene. The primer pair for amplifying the COI gene has the upstream primer sequence shown in SEQ ID NO.1 and the downstream primer sequence shown in SEQ ID NO.2. The primer pair for amplifying the Cytb gene has the upstream primer sequence shown in SEQ ID NO.3 and the downstream primer sequence shown in SEQ ID NO.4.

[0009] Preferably, the primer pair used to amplify the COI gene has an amplification fragment length of 653 bp, and the primer pair used to amplify the Cytb gene has an amplification fragment length of 835 bp.

[0010] Primer pairs used to amplify the COI gene: Upstream primer COI-F: 5'-GACCAAATCTATAATGTTATTGT-3' (SEQ ID NO.1); Downstream primer COI-R: 5'-AAGGGTTCTTTTTTCCCTGAATAGT-3' (SEQ ID NO.2); The amplified fragment size is 653 bp.

[0011] Primer pair used to amplify the Cytb gene: Upstream primer Cytb-F: 5'-TAGGAATCTGCCTAGCACTACAAAT-3' (SEQ ID NO.3); Downstream primer Cytb-R: 5'-ATGCTGCGTTGTTTAGAGGTGTG-3' (SEQ ID NO.4); The amplified fragment size is 835 bp.

[0012] The present invention also discloses a kit for rapid identification of Chiroptera species, the kit comprising the primer set disclosed above.

[0013] Preferably, the kit further includes at least one of water, multiplex PCR buffer, dNTPs, and DNA polymerase.

[0014] This invention also discloses the application of the above-mentioned dual-gene specific primer set and kit in the rapid identification of Chiroptera species.

[0015] This invention also discloses a method for designing specific primer sets for rapid identification of Chiroptera species, comprising the following steps: (1) Download the mitochondrial genome sequences of various families and genera of Chiroptera and common non-Chiroptera mammals from the GenBank database; (2) Extract the COI gene and Cytb gene sequences respectively, and use DNAMAN software to perform multiple alignments to screen out specific conserved regions that are highly conserved among Chiropteran species and significantly different from other mammals. (3) Based on the specific conserved region, primer pairs for the COI gene and primer pairs for the Cytb gene were designed using bioinformatics software, and the target primer set was obtained through specificity verification. Finally, primer pairs for amplifying the COI gene were obtained, with the upstream primer sequence shown in SEQ ID NO.1 and the downstream primer sequence shown in SEQ ID NO.2; and primer pairs for amplifying the Cytb gene, with the upstream primer sequence shown in SEQ ID NO.3 and the downstream primer sequence shown in SEQ ID NO.4.

[0016] This invention also discloses a method for identifying Chiroptera species, comprising the following steps: S1, Take the tissue sample to be tested, and separate and extract the DNA to be tested; S2, using the DNA to be tested as a template, PCR amplification is performed using the COI primer pair and Cytb primer pair described in claim 1, respectively, to obtain the amplification product; S3. The amplified products are detected and analyzed. If the COI gene amplifies a 653 bp band and the Cytb gene amplifies an 835 bp band, the amplified products are sequenced. The sequences obtained from sequencing are compared and analyzed with the database. The taxonomic position of the sample to be tested is determined based on sequence homology and the constructed phylogenetic tree.

[0017] Preferably, in step S2, the PCR amplification reaction system is 20 μL and includes: 2.0 µL of 10× PCR Buffer (containing Mg²⁺); 1.6 µL of 2.5 mM dNTP mixture; 0.2 µL of Taq DNA polymerase (5 U / µL); 1.0 µL each of 10 µM upstream primer and 10 µM downstream primer; 1.0 µL of DNA template; and ddH₂O to bring the total volume to 20 μL.

[0018] In any of the above schemes, the preferred method is that in step S2, the PCR amplification reaction program is as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1 min, for a total of 35 cycles; 72℃ final extension for 10 min.

[0019] In any of the above schemes, the preferred method is that in step S3, the classification status of the sample to be tested is determined based on sequence homology. The determination criterion is: if the homology between the sequencing sequence and the corresponding gene sequence of the Chiroptera species reaches more than 98%, the sample to be tested is determined to be a Chiroptera species; a phylogenetic tree of the sequencing sequence and the corresponding gene sequence is constructed to accurately determine the specific Chiroptera species. Beneficial effects

[0020] (1) The dual-gene specific primer set and its application method for rapid identification of Chiroptera species disclosed in this invention have double verification and high accuracy: This invention adopts the COI and Cytb dual-gene marker strategy. Both primer pairs can be amplified independently and corroborate each other, effectively avoiding misjudgment caused by single gene sequence variation or pseudogene interference, and significantly improving the reliability of the identification results.

[0021] (2) Rigorous design method: Specific conserved regions were screened by multi-species sequence comparison and verified by electronic simulation, which ensured the universality of primers in the Chiroptera group.

[0022] (3) Scientific Judgment Logic: It abandons the crude model of judging species solely by the size of electrophoretic bands and establishes a standard judgment process of "specific amplification + sequencing verification + construction of phylogenetic tree" to ensure that the identification results have molecular phylogenetic basis and provide a reliable tool for the accurate identification of Chiroptera species.

[0023] (4) Good practicality: The experimental procedure does not rely on expensive reagent kits, is easy to operate, and has low cost, making it suitable for promotion and application in grassroots laboratories and large-scale field surveys. Attached Figure Description

[0024] Figure 1 This is a schematic diagram showing the location of the COI gene and Cytb gene-specific primer pairs on the mitochondrial genome in Example 1 of the present invention; Figure 2 This is a schematic diagram of electrophoresis used to verify the species-specific amplification of the COI gene primer pair in Example 2 of the present invention. Figure 3 This is a schematic diagram of electrophoresis used to verify the species-specific amplification of the Cytb gene primer pair in Example 2 of the present invention. Figure 4 This is a phylogenetic tree of some species of Chiroptera constructed based on the COI gene sequence in Example 3 of the present invention; Figure 5 This is a phylogenetic tree of some species of Chiroptera constructed based on the Cytb gene sequence in Example 3 of the present invention; Figure 6 This is a schematic diagram of the process for rapid identification of Chiroptera species provided in an embodiment of the present invention. Detailed Implementation

[0025] To better understand the technology and advantages of the present invention, the present invention will be further described below through specific embodiments and in conjunction with the accompanying drawings. Example

[0026] A method for designing specific primer sets for rapid identification of Chiroptera species. Figure 6 A flowchart illustrating the rapid identification of Chiroptera species provided in this embodiment of the invention includes the following steps: Download mitochondrial genome sequences from various families and genera of Chiroptera and common non-Chiroptera mammals from the GenBank database; COI and Cytb gene sequences were extracted separately, and multiple alignments were performed using DNAMAN software to screen out specific conserved regions that are highly conserved among Chiropteran species and significantly different from other mammals. Based on the specific conserved regions, primer pairs for the COI gene and primer pairs for the Cytb gene were designed respectively, and the target primer set was obtained through specificity verification, which was used to amplify the COI gene primer pair.

[0027] In practice, primer design and synthesis are performed first. Sequence alignment and analysis: The complete mitochondrial genome sequences of major Chiroptera groups (including Horseshoe Bats, Bats, and Hoofed Bats) and common non-Chiroptera mammals (such as Rodents and Lagomorphs) were downloaded from the NCBI GenBank database. Multiple alignments of the COI and Cytb gene sequences were performed using DNAMAN bioinformatics software.

[0028] Primer screening: Regions highly conserved among Chiropteran species and exhibiting significant base differences from non-chiropteran mammals were identified as specifically conserved regions. Candidate primers were designed within these selected conserved regions using DNAMAN software. Primers were selected to amplify the longest possible gene sequences to obtain more species-specific information. Specificity was then validated using the NCBI Primer-BLAST online tool to ensure a high degree of match between the primer sequences and the Chiropteran genome.

[0029] Primer sequences: Two pairs of specific primers were finally obtained through screening. The primer positions are shown in the diagram below. Figure 1 As shown. Among them, Figure 1 A corresponds to the COI gene. Figure 1 B corresponds to the Cytb gene. Numbers without parentheses indicate the position within the corresponding gene, while numbers within parentheses indicate the position within the mitochondrial genome. The COI primer pair amplifies a fragment primarily located in the first half of the COI gene region, with a length of 653 bp; the Cytb primer pair amplifies a fragment covering most of the Cytb gene coding region, with a length of 835 bp. Specific primer information is shown in Table 1. For the primer pair used to amplify the COI gene, the upstream primer sequence is shown in SEQ ID NO.1, and the downstream primer sequence is shown in SEQ ID NO.2; for the primer pair used to amplify the Cytb gene, the upstream primer sequence is shown in SEQ ID NO.3, and the downstream primer sequence is shown in SEQ ID NO.4. The primers were synthesized by a biotechnology company.

[0030] Table 1 Primer sequence related information Example 2: Methods for identifying Chiroptera species (a) Sample collection and DNA extraction 1. Experimental materials: Fifteen individuals from three families of Chiroptera were collected and selected as experimental samples. Specific sample information is as follows: Horseshoe Bats (Rhinolophus pilosa): Rhinolophus piriformis, Rhinolophus maximus, Rhinolophus sinensis, Rhinolophus mesenteroides, Rhinolophus philippinensis; Bats: Common Winged Bat, Eyre's Tube-nosed Bat, Hassler's Tube-nosed Bat, Dark Brown Bat, Velvet Mountain Bat, South China Water Rat Ear Bat, Great Brown Bat; Hoofed bats: Great hoofed bat, Przewalski's hoofed bat, and Angle's small hoofed bat.

[0031] 2. DNA extraction: Take approximately 20 mg of muscle or liver tissue from the above samples and extract total DNA using the conventional phenol-chloroform method or a commercial tissue genomic DNA extraction kit. Determine the DNA concentration and purity (OD260 / OD280 ratio between 1.8 and 2.0) using a NanoDrop spectrophotometer, adjust the concentration to 10.0–100.0 ng / µL, and store at -20°C for later use.

[0032] (II) PCR amplification and electrophoresis detection 1. PCR reaction system: The genomic DNA of the above 15 samples was amplified by PCR using the COI primer pair and Cytb primer pair prepared in Example 1, respectively. The total volume of the reaction system was 20 μL, containing: The PCR reaction system is shown in Table 2: Table 2 PCR reaction system 2. The PCR reaction procedure is shown in Table 3: Pre-denaturation at 94℃ for 3 min; Denaturation at 94℃ for 30 s, annealing at 55℃ for 30 s, extension at 72℃ for 1 min, for a total of 35 cycles; Final extension at 72℃ for 10 min; Store at 4°C.

[0033] Table 3 PCR reaction procedure 3. Electrophoresis detection: The PCR amplification products were spotted onto a 1.0% agarose gel and electrophoresed at 120V for 30 min in 1.0 × TAE electrophoresis buffer. The results were observed and recorded using a gel imaging system.

[0034] The results are as follows Figure 2As shown, using COI primer pairs for amplification, all Chiroptera samples showed a bright, single, specific band at approximately 653 bp, with no extraneous bands. In the figure, lane 1 is the DL2000 DNA Marker; lanes 2-6 are for Horseshoe Bats, namely *Rhinolophus prygius*, *Rhinolophus maximus*, *Rhinolophus sinensis*, *Rhinolophus mesenteroides*, and *Rhinolophus philippinensis*; lanes 7-13 are for Bats, namely *Pterocarya spp.*, ... and *Pterocarya spp.*, respectively; lanes 14-16 are for Hoofed Bats, namely *Pterocarya spp.*, *Pterocarya spp.*, and *Pterocarya spp.*, respectively.

[0035] like Figure 3 As shown, amplification using Cytb primer pairs resulted in a bright, single, specific band at approximately 835 bp in all Chiropteran samples. The lane arrangement in the figure is consistent with... Figure 2 Consistent.

[0036] The results show that the dual-gene primer set designed in this invention has good amplification efficiency and specificity, and can be effectively applied to the identification of Chiroptera species.

[0037] Example 3: Sequencing and Sequence Analysis To further verify the accuracy of the amplification products, the PCR amplification products obtained in Example 2 were sent to a biotechnology company for bidirectional sequencing (Sanger sequencing).

[0038] The sequencing results were assembled and proofread using DNAMAN software. After removing primer sequences, the intermediate target gene fragment sequence was obtained. The obtained sequence was then subjected to BLAST homology analysis in the NCBI GenBank database.

[0039] The comparison results are shown in Table 4: Table 4. Homology analysis of COI and Cytb gene sequencing results. The COI and Cytb gene sequences of all samples showed high homology (similarity >98%) with the reference sequences of the corresponding species in the database, and could accurately distinguish morphologically similar species such as *Rhinolophus chinensis* and *Rhinolophus sinensis*. Sequencing results confirmed the specificity of PCR amplification, indicating that the primer set designed in this invention can accurately identify Chiroptera species and provides reliable molecular data for further species classification and phylogenetic analysis.

[0040] To more accurately confirm the taxonomic position of species, especially to distinguish morphologically similar species, the COI and Cytb gene sequences of the 15 samples obtained above were compared with the reference sequences of relevant species downloaded from GenBank. A phylogenetic tree was constructed using the Neighbor-Joining (NJ) method in MEGA11 software, and node support was tested using the Bootstrap method (1000 replicates). The results are as follows: Figure 4 and Figure 5 As shown in the figure, the samples to be tested are marked with ▲ (corresponding to sample numbers 01-15 in Table 4), and the reference sequences are labeled with GenBank accession numbers.

[0041] Homology analysis of sequencing results and phylogenetic tree construction jointly confirmed the specificity of PCR amplification, indicating that the primer set designed in this invention can accurately identify Chiroptera species and provide reliable molecular data for further species classification and phylogenetic analysis.

[0042] In summary, this invention discloses a dual-gene specific primer set for rapid identification of Chiroptera species and its application method. The primer set includes primer pair COI-F / COI-R for amplifying the mitochondrial COI gene and primer pair Cytb-F / Cytb-R for amplifying the Cytb gene, with nucleotide sequences shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4, respectively. Based on sequence alignment of the mitochondrial COI and Cytb genes in Chiroptera species, this invention designed two pairs of specific primers and achieved efficient and specific amplification of Chiroptera species by optimizing the PCR reaction system. Experiments show that this primer set has good interspecific specificity and intraspecific conservation, with amplified band sizes of 653 bp (COI) and 835 bp (Cytb), respectively. It can effectively distinguish Chiroptera from other closely related mammals and accurately identify cryptic species and juvenile samples that are difficult to distinguish morphologically. This invention employs dual gene markers for mutual verification, significantly improving the accuracy and reliability of identification. It provides a powerful molecular biology tool for the rapid and accurate identification of Chiroptera species and has significant application value in fields such as biodiversity surveys, disease tracing, and ecological protection.

[0043] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A dual-gene-specific primer set for rapid identification of Chiroptera species, characterized in that: The primer set is a primer set for amplifying the COI gene and the Cytb gene; The primer pair used to amplify the COI gene has the upstream primer sequence shown in SEQ ID NO.1 and the downstream primer sequence shown in SEQ ID NO.

2. The primer pair used to amplify the Cytb gene has the upstream primer sequence shown in SEQ ID NO.3 and the downstream primer sequence shown in SEQ ID NO.

4.

2. The dual-gene-specific primer set for rapid identification of Chiroptera species according to claim 1, characterized in that: The primer pair used to amplify the COI gene has an amplification fragment length of 653 bp, and the primer pair used to amplify the Cytb gene has an amplification fragment length of 835 bp.

3. A kit for rapid identification of Chiroptera species, characterized in that, The kit includes the primer set as described in claim 1.

4. The reagent kit according to claim 3, characterized in that, The kit also includes at least one of water, multiplex PCR buffer, dNTPs, and DNA polymerase.

5. The application of the dual-gene-specific primer set according to claim 1 and the kit according to claim 3 or claim 4 in the rapid identification of Chiroptera species.

6. A method for designing a specific primer set for rapid identification of Chiroptera species, characterized in that, Includes the following steps: (1) Download the mitochondrial genome sequences of various families and genera of Chiroptera and common non-Chiroptera mammals from the GenBank database; (2) Extract the COI gene and Cytb gene sequences respectively, and use DNAMAN software to perform multiple alignments to screen out specific conserved regions that are highly conserved among Chiropteran species and significantly different from other mammals. (3) Based on the specific conserved region, primer pairs for the COI gene and primer pairs for the Cytb gene were designed respectively, and the target primer set was obtained through specificity verification. The primer pairs used to amplify the COI gene are shown in SEQ ID NO.1 and the downstream primer sequence is shown in SEQ ID NO.

2. The primer pair used to amplify the Cytb gene has the upstream primer sequence shown in SEQ ID NO.3 and the downstream primer sequence shown in SEQ ID NO.

4.

7. A method for identifying species of the order Chiroptera, characterized in that, Includes the following steps: S1, Take the tissue sample to be tested, and separate and extract the DNA to be tested; S2, using the DNA to be tested as a template, PCR amplification is performed using the COI primer pair and Cytb primer pair described in claim 1, respectively, to obtain the amplification product; S3, the amplification products are detected and analyzed. If the COI gene amplifies a 653 bp band and the Cytb gene amplifies an 835 bp band, the amplification products are sequenced. The sequences obtained from sequencing are compared and analyzed with databases, and the taxonomic position of the sample is determined based on sequence homology and the constructed phylogenetic tree.

8. The identification method according to claim 7, characterized in that, In step S2, the PCR amplification reaction system is 20 μL and includes: 2.0 µL of 10× PCR Buffer (containing Mg²⁺); 1.6 µL of 2.5 mM dNTP mixture; 0.2 µL of Taq DNA polymerase (5 U / µL); 1.0 µL each of 10 µM upstream primer and 10 µM downstream primer; 1.0 µL of DNA template; and ddH₂O to bring the total volume to 20 μL.

9. The identification method according to claim 7, characterized in that, In step S2, the PCR amplification reaction program is as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1 min, for a total of 35 cycles; 72℃ final extension for 10 min.

10. The identification method according to claim 7, characterized in that, In step S3, the taxonomic position of the sample to be tested is determined based on sequence homology. The determination criteria are as follows: if the homology between the sequencing sequence and the corresponding gene sequence of the Chiroptera species reaches more than 98%, the sample to be tested is determined to be a Chiroptera species; a phylogenetic tree of the sequencing sequence and the corresponding gene sequence is constructed to accurately determine the specific Chiroptera species.

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