Animal mitochondrial bar code multiple PCR amplification and nanopore sequencing species identification kit

Abstract

The invention discloses a species identification kit for animal mitochondrial bar code multiplex PCR amplification and nanopore sequencing, and relates to the technical field of species identification. The species identification kit comprises optimized primer pairs aiming at four hyper-variable genes (Cytb, COI, 16S rRNA and 12S rRNA) of mitochondria, by introducing R, Y, D and other degenerate bases at key sites, the binding capacity of the primers and template DNA of different species is remarkably improved, the problems that an existing universal primer is prone to mismatch, low in efficiency and even failed in amplification during cross-species amplification are solved, and the species identification kit has the advantages of being high in specificity, high in specificity and high in specificity. The method can be used for species identification of a mixed DNA sample containing at least two different animal species sources. The species identification primer combination is applied to preparation of an animal species identification kit, and the detection coverage range of the kit is expanded. According to the animal species identification method provided by the invention, a multiple PCR amplification system and a nanopore length reading sequencing technology are combined, congeneric sibling species with highly similar sequences can be distinguished, and the method has good accuracy.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of species identification, and particularly relates to a species identification kit for animal mitochondrial barcode multiplex PCR amplification and nanopore sequencing. BACKGROUND

[0002] Animal species identification plays a crucial role in ecological monitoring, food safety and forensic science. Traditional identification methods mainly include morphology, behavior, ecological and distribution information, and acoustic identification. Morphological identification, as the most classic method, relies on external morphology and anatomical features, but its identification accuracy is highly dependent on the classification experience of professional personnel, and its identification ability for similar species, juveniles or damaged samples is limited. Behavior identification distinguishes species by their unique behavior patterns (such as bird songs and bat echolocation), but it is still limited by observation conditions and behavioral plasticity. Ecological and distribution information can be used to assist identification by using the geographical distribution and habitat preference of species, which has a clear application scenario but low resolution, and is mainly used for preliminary screening.

[0003] With the rapid development of molecular biology technology, DNA identification methods have become increasingly popular and have significantly improved the accuracy and applicability of species identification. Such methods amplify and sequence specific DNA fragments (such as mitochondrial COI gene) and compare them with reference databases, which can achieve higher identification resolution than traditional methods, especially for similar species, cryptic species or trace / degraded samples. However, current molecular identification still faces several key technical bottlenecks: including the insufficient variation rate of commonly used barcode genes (such as COI, Cytb, 12S rRNA, 16S rRNA) in some groups, leading to high species misjudgment rate; the inconsistent amplification efficiency of universal primers among different species, with limited coverage; and the insufficient resolution of single-gene barcodes for closely related species due to limited information.

[0004] Iterative development of DNA sequencing technology has continuously promoted species identification. First-generation Sanger sequencing has high precision and long read length, but its low throughput and high cost make it difficult to meet the needs of large-scale screening. Second-generation high-throughput sequencing technology (such as Illumina) has greatly improved sequencing throughput and reduced cost, but due to short read length (usually ≤300 bp), it is difficult to accurately analyze complex genomic regions or implement long fragment amplicon analysis, limiting its application in closely related species differentiation. Third-generation sequencing technology (such as nanopore sequencing) has the advantages of long read length and portability, supporting real-time, on-site rapid detection. With the improvement of sequencing accuracy and optimization of signal analysis algorithms, nanopore sequencing is increasingly used in various fields.

[0005] In summary, although the existing animal species identification technologies have their own advantages, they still have the problems of poor universality, insufficient coverage of polymorphic regions, difficulty in distinguishing closely related species, and difficulty in balancing high precision and high throughput. Therefore, there is an urgent need in the field for a new identification method that integrates multi-gene information, efficiently compatible primers, simple operation and controllable cost. SUMMARY

[0006] The technical problem to be solved by the present application is based on at least one of the defects involved in the background art, and a species identification method and matching products based on animal mitochondrial barcode multiplex PCR amplification and nanopore sequencing are proposed, which realizes faster, more accurate and more extensive animal species identification by optimizing primer design, establishing an efficient multiplex amplification system, and combining long-read sequencing technology.

[0007] To solve the above problems, the present application proposes the following technical solutions: In a first aspect, the present application provides a species identification primer combination for animal mitochondrial barcode multiplex PCR amplification and nanopore sequencing, which comprises at least one of the following primer pairs: a primer pair specific for amplifying Cytb gene, the nucleotide sequence of which is shown in SEQ ID NO. 1-2; a primer pair specific for amplifying COI gene, the nucleotide sequence of which is shown in SEQ ID NO. 3-4; a primer pair specific for amplifying 16S rRNA gene, the nucleotide sequence of which is shown in SEQ ID NO. 5-6; a primer pair specific for amplifying 12S rRNA gene, the nucleotide sequence of which is shown in SEQ ID NO. 7-8; wherein, In sequence SEQ ID NO. 2, base R represents degenerate base A / G, and base D represents A / G / T; In sequence SEQ ID NO. 3, base Y represents degenerate base C / T; In sequence SEQ ID NO. 4, base R represents degenerate base A / G, and base Y represents degenerate base C / T.

[0008] In a second aspect, the present application provides the use of the species identification primer combination in the preparation of a kit for animal species identification.

[0009] In a third aspect, the present application provides a species identification kit for animal mitochondrial barcode multiplex PCR amplification and nanopore sequencing, comprising the species identification primer combination of the first aspect, and the kit is used for species identification of a mixed DNA sample containing at least two different animal species.

[0010] Further, the species identification kit further comprises sequencing reagents for long-read sequencing of the amplification products of the species identification primer combination.

[0011] Furthermore, the long-read sequencing is nanopore sequencing.

[0012] Fourthly, the present invention provides a method for animal species identification, comprising the following steps: Genomic DNA is extracted from the target sample and amplified by singleton PCR or multiplex PCR using the species identification primer combination or the kit described above. The amplification products were subjected to gel electrophoresis, and the size of the target fragment in the amplification products was verified based on the electrophoretic bands. Nanopore sequencing is performed on the amplified products, and the sequencing data is subjected to quality control and comparative analysis to achieve species identification.

[0013] Furthermore, the amplified products were subjected to nanopore long-read sequencing.

[0014] Furthermore, in the method, the reaction system for singleton PCR amplification includes: 25 μL 2×Phanta MaxMaster Mix, 2 μL primer pair with an upstream primer to downstream primer volume ratio of 1:1, 3-8 ng DNA template, and RNase-free water to make up the total reaction system volume to 50 μL.

[0015] Furthermore, in the method, the reaction system for multiplex PCR amplification includes: 25 μL 2×Phanta MaxMaster Mix, 10 μL primer mixture, 3-8 ng DNA template, and RNase-Free Water to make up the total volume of the reaction system to 50 μL.

[0016] In the multiplex PCR amplification reaction system, the volume ratio of each primer pair in the primer mixture is COI:Cytb:16S rRNA:12S rRNA = 2.5:1.5:0.6:0.4. The volume ratio of the upstream primer to the downstream primer in the primer pair is 1:1.

[0017] Furthermore, in the method, the reaction program for singleton PCR amplification or multiplex PCR amplification is as follows: pre-denaturation at 95℃ for 3 min; denaturation at 95℃ for 15 s, annealing at 52℃ for 30 s, extension at 72℃ for 30 s, for a total of 25-30 cycles; final extension at 72℃ for 5 min; and incubation at 4℃.

[0018] In the method described, the size of the target fragment detected by gel electrophoresis must be between 400-700 bp.

[0019] Fifthly, the present invention also provides the application of the species identification primer combination, the species identification kit, or the method described herein in animal species identification.

[0020] Compared with the prior art, the technical effects achieved by the present invention include: This invention provides a species identification kit for animal mitochondrial barcoding multiplex PCR amplification and nanopore sequencing. It 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 problems of mismatch, low efficiency, and even amplification failure that often occur with existing universal primers during cross-species amplification. This carefully designed degeneracy ensures that the primers are widely adaptable to highly diverse animal groups such as insects, fish, birds, and mammals, and can be used to prepare animal species identification kits, expanding the detection coverage of the kits.

[0021] This invention provides a method for animal species identification using primer combinations or kits, which can utilize single primer pairs for singleton PCR amplification or multiple primer pairs for multiplex PCR amplification. Furthermore, by integrating primers for these four markers into an optimized multiplex PCR reaction system, a detection method for simultaneously amplifying four target gene fragments in a single tube and single reaction is achieved. This approach not only significantly increases detection throughput and saves valuable sample DNA and reagent costs, but more importantly, the joint detection of multiple genes constitutes an inherent redundant verification system. When a specific marker in a species has insufficient resolution due to evolutionary conservation or the presence of pseudogenes (e.g., COI is not significantly different in some closely related species), the remaining markers can provide complementary genetic information, thus mutually verifying each other and jointly ensuring the accuracy and reliability of the identification results, effectively minimizing the risk of misjudgment that may be caused by a single gene marker.

[0022] Furthermore, the identification method of this invention combines multiplex PCR amplification with nanopore long-read sequencing technology. Nanopore technology has no stringent limitations on amplicon length; the primer pairs of this invention can obtain amplified fragments of 400-700 bp, with each read covering the complete target gene region, thus obtaining richer phylogenetic information, which is crucial for distinguishing closely related species with highly similar sequences. Simultaneously, this approach is compatible with high-throughput technology, allowing for parallel sequencing of a large number of samples in a single run, solving the problems of low throughput and high cost associated with traditional Sanger sequencing. The advantages of nanopore long reads also avoid potential errors in assembling complex sequences during second-generation short-read sequencing, making it particularly suitable for fragments with length polymorphism, thereby achieving a higher success rate in confirmatory identification of degraded samples. Attached Figure Description

[0023] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 Gel electrophoresis images of multiplex PCR amplification results for sheep DNA samples of different concentrations.

[0025] Figure 2 This is a flowchart of methods for identifying animal species. Detailed Implementation

[0026] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the embodiments described below are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0027] It should be understood that the terminology used in this specification of embodiments of the invention is for the purpose of describing particular embodiments only and is not intended to limit the embodiments of the invention. As used in this specification of embodiments of the invention and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms unless the context clearly indicates otherwise.

[0028] This invention provides a primer set for species identification using animal mitochondrial barcode multiplex PCR amplification and nanopore sequencing. The primer set comprises at least one of the following primer pairs: a specific primer pair for amplifying the Cytb gene, with nucleotide sequences as shown in SEQ ID NO. 1-2; a specific primer pair for amplifying the COI gene, with nucleotide sequences as shown in SEQ ID NO. 3-4; a specific primer pair for amplifying the 16S rRNA gene, with nucleotide sequences as shown in SEQ ID NO. 5-6; and a specific primer pair for amplifying the 12S rRNA gene, with nucleotide sequences as shown in SEQ ID NO. 7-8.

[0029] In sequence SEQ ID NO.2, the base R represents the degenerate bases A / G, and the base D represents A / G / T; In sequence SEQ ID NO.3, the base Y represents the degenerate base C / T; In sequence SEQ ID NO.4, base R represents degenerate bases A / G, and base Y represents degenerate bases C / T.

[0030] In this embodiment of the invention, the optimized primer length is 18-28 bp, and the GC content is 40%-60%. The specific primer pairs are shown in Table 1.

[0031] Table 1 Primer pairs for four hypervariable genes This invention optimizes universal primers for four hypervariable genes in animal mitochondrial DNA (mtDNA), introducing degenerate bases (R / Y / D, etc.) to increase species adaptability. The amplified fragments vary in size, with longer fragments containing more genetic information, thus improving the accuracy of species identification. Specifically: COI (Cytochrome c Oxidase Subunit I) – Universal Barcode, Species-Level Resolution; Cytb (cytochrome b) – commonly used in vertebrates, providing species / subspecies resolution; 16S rRNA & 12S rRNA – highly conserved, suitable for high-order classification and degradation samples.

[0032] This invention provides an application of the above-described species identification primer combination in the preparation of a kit for animal species identification.

[0033] In one embodiment, the present invention provides a species identification kit for animal mitochondrial barcode multiplex PCR amplification and nanopore sequencing, comprising the aforementioned species identification primer combination.

[0034] In a specific embodiment, the kit provided by the present invention comprises an amplification enzyme mixture, primers (primer mixture), and a positive control. The amplification enzyme mixture comprises hot-start Taq DNA polymerase (0.05 U / μL), dNTPs (0.2 mM), MgCl2 (2.5 mM), and buffer (pH 8.3); the positive control is genomic DNA of a known species.

[0035] See Figure 2 This is a flowchart of an animal species identification method provided by an embodiment of the present invention. The method for animal species identification provided by the present invention includes the following steps: S1. Extract genomic DNA from the target sample and perform singleton or multiplex PCR amplification using the species identification primer combination or the kit described above.

[0036] Total DNA was extracted from the target sample as a template for subsequent amplification of genomic DNA (gDNA), with an input amount of 3-8 ng, preferably 5 ng. This invention uses Vazyme's 2×Phanta Max Master Mix as the amplification reagent. Specific amplification reaction systems are shown in Tables 2 and 3, and amplification reaction conditions are shown in Table 4.

[0037] Table 2 Reaction system for singleton PCR amplification Table 3. Reaction system for multiplex PCR amplification In this specific embodiment, the primer mixture (mix) was prepared by mixing primer pairs in a volume ratio of COI:Cytb:16S rRNA:12S rRNA = 2.5:1.5:0.6:0.4. The volume ratio of the upstream primer to the downstream primer in each primer pair was 1:1.

[0038] Table 4. Reaction procedures for singleton or multiplex PCR amplification S2. Take the amplification product for gel electrophoresis detection, and verify the size of the target fragment in the amplification product based on the electrophoretic bands.

[0039] In practice, the amplification product fragments based on the four sets of primers of this invention are all of different sizes. The applicability of the primers used can be verified by several parallel amplifications and repeated amplifications, and the size of the amplified fragments can be detected as corresponding to each target within the range of 400-700bp, which is beneficial to the quality control of the experiment.

[0040] S3. Perform nanopore sequencing on the amplified products, perform quality control and comparative analysis on the sequencing data, and achieve species identification.

[0041] In practice, the obtained multiplex PCR amplification products are used to construct DNA sequencing libraries for nanopore sequencing. After sequencing, bioinformatics methods are used to perform quality control and comparative analysis of the sequencing data. By comparing and analyzing genetic markers from different species, the goal of accurately identifying the species of samples using multiple genetic markers is achieved.

[0042] The present invention utilizes molecular biology identification technology to effectively distinguish morphologically similar animal species. It can identify fragments (such as hides and meat products), feces, and even environmental DNA samples (DNA fragments in water or soil), and is effective for undetermined species and cryptic species.

[0043] Example 1 This embodiment is based on the method for animal species identification provided by the present invention, and uses multiplex PCR amplification technology to identify a single species sample: Experimental Methods: The sensitivity of mtDNA multi-segment multiplex amplification (mtDNA) to single samples was evaluated using serially diluted sheep DNA. DNA extracted from mutton samples was serially diluted 1-12 times (specific concentrations are shown in Table 5). Using the original DNA solution and its diluted samples as templates, multiplex PCR amplification was performed according to the reaction system in Table 3 and the reaction conditions in Table 4. The amplification products were detected by gel electrophoresis. Figure 1 (and nanopore sequencing and data analysis).

[0044] Table 5 Sample Dilution Gradients Figure 1 As can be seen, the four target mitochondrial genes (COI, Cytb, 12S rRNA, 16S rRNA) can be synchronously and clearly amplified at concentrations of 1-0.125 ng / ul.

[0045] When the sample input amount is 125pg, according to the existing amplification system, there are still 4 weak bands. This shows that the present invention has extremely low requirements for the sample input amount and can still amplify the target fragment at the pg level.

[0046] The amplification product of the 125 pg DNA template was used to construct a sequencing library using the Oxford Nanopore Rapid Library Conversion Kit. Sequencing was then performed on a GridION sequencer (QiCarbon 384 chip), and the results are shown in Table 6. Analysis using our self-developed bioinformatics software successfully identified the species as sheep (Ovis aries).

[0047] Table 6. Nanopore sequencing analysis results The results in Table 6 indicate that the amplification products generated using 125 pg of DNA as the starting template in this embodiment are sufficient to support reliable species identification.

[0048] As can be seen, all genes exhibit extremely high sequence similarity (all above 99.4%). Relying solely on conserved 12S or 16S rRNA genes would introduce uncertainty in identification results (failing to distinguish between Ovis aries and Ovisorientalis with 100% accuracy). However, this invention overcomes the problem of insufficient resolution of single conserved genes by simultaneously analyzing four genes and utilizing the decisive evidence provided by the high-resolution COX1 and CYTB genes. Through multi-gene joint analysis, comprehensively utilizing information from conserved and variable genes, the invention overcomes the limitation of insufficient resolution of single DNA barcodes in specific taxa, achieving species-level accuracy in identification. The results are highly reliable, fully demonstrating the accuracy and effectiveness of the method in multi-gene joint identification.

[0049] Example 2 This embodiment is based on the method for animal species identification provided by the present invention, and uses multiplex PCR amplification technology to identify mixed species samples: Experimental Methods: The sensitivity of the mtDNA multiplex amplification program to mixed samples was evaluated using beef (Bos indicus) and pork (Sus scrofa) samples. Beef and pork DNA were separately mixed to prepare mixed samples with different mass ratios (the composition ratio of the mixed samples with a total input of 1 ng is shown in Table 7). Multiplex PCR amplification was performed using the reaction system in Table 3 and the reaction conditions in Table 4. The amplification products were detected by gel electrophoresis and nanopore sequencing, and the data were analyzed. The results are shown in Table 8.

[0050] Table 7 Composition Ratio of Mixed Samples Table 8 Sequencing data for each mixed sample As can be seen, in all test gradients (10%-50%), the species ratios measured by the species identification kit and method provided by this invention are highly consistent with the theoretical formulation ratios, with a maximum deviation of only 2%. This proves that this invention can not only qualitatively detect species components in mixed samples, but also perform highly accurate quantitative analysis. Even in sample S1 where the proportion of bovine DNA is as low as 10%, the detection rate (9.5%) is still very accurate, demonstrating that this invention has excellent detection capabilities even for components with low concentrations.

[0051] The above results demonstrate that this invention can be effectively applied to the identification of mixed species samples, and can highly accurately reflect the true composition ratio of each species in the mixed sample, exhibiting excellent quantitative detection capabilities. This gives it great application potential in fields requiring precise knowledge of the composition of mixtures, such as food safety (e.g., identification of adulteration in meat products) and ecological research (e.g., dietary analysis).

[0052] In the above embodiments, the descriptions of each embodiment have different focuses. For parts that are not described in detail in a certain embodiment, please refer to the relevant descriptions in other embodiments.

[0053] The above description describes specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in the present invention, and these modifications or substitutions should all be covered within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A primer set for species identification by multiplex PCR amplification and nanopore sequencing of animal mitochondrial barcoding, characterized in that... The primer combination comprises at least one of the following primer pairs: a specific primer pair for amplifying the Cytb gene, with nucleotide sequences as shown in SEQ ID NO. 1-2; a specific primer pair for amplifying the COI gene, with nucleotide sequences as shown in SEQ ID NO. 3-4; a specific primer pair for amplifying the 16S rRNA gene, with nucleotide sequences as shown in SEQ ID NO. 5-6; and a specific primer pair for amplifying the 12S rRNA gene, with nucleotide sequences as shown in SEQ ID NO. 7-8; wherein, In sequence SEQ ID NO.2, the base R represents the degenerate bases A / G, and the base D represents A / G / T; In sequence SEQ ID NO.3, the base Y represents the degenerate base C / T; In sequence SEQ ID NO.4, base R represents degenerate bases A / G, and base Y represents degenerate bases C / T.

2. The use of the species identification primer combination as described in claim 1 in the preparation of a kit for animal species identification.

3. A species identification kit for animal mitochondrial barcoding multiplex PCR amplification and nanopore sequencing, characterized in that, The species identification kit comprises the species identification primer combination of claim 1 and is used for species identification of mixed DNA samples containing at least two different animal species.

4. The species identification kit as described in claim 3, characterized in that, The species identification kit also contains amplification products suitable for nanopore long-read sequencing of the primer combinations used for species identification.

5. A method for identifying animal species, characterized in that, Includes the following steps: Genomic DNA is extracted from the target sample and amplified by singleton PCR or multiplex PCR using the species identification primer combination described in claim 1 or the species identification kit described in claim 3 or 4. The amplification products were subjected to gel electrophoresis to verify the size of the target fragment in the amplification products. Nanopore sequencing was performed on the amplified products, and the sequencing data were subjected to quality control and comparative analysis to achieve species identification.

6. The method as described in claim 5, characterized in that, The reaction system for multiplex PCR amplification includes: 25 μL 2×Phanta Max Master Mix, 10 μL primer mixture, 3-8 ng DNA template, and RNase-free water to make up the total volume of the reaction system to 50 μL.

7. The method as described in claim 6, characterized in that, In the primer mixture, the volume ratio of each primer pair is COI:Cytb:16S rRNA:12S rRNA = 2.5:1.5:0.6:0.

4.

8. The method as described in claim 5, characterized in that, The reaction program for both singleton PCR amplification and multiplex PCR amplification is as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 52℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 25-30 cycles; 72℃ final extension for 5 min; and incubation at 4℃.

9. The method as described in claim 4, characterized in that, The size of the target fragment for gel electrophoresis detection must be between 400-700 bp.

10. The application of a species identification primer combination as described in claim 1, a species identification kit as described in claim 3 or 4, or the method described in any one of claims 5-9 in animal species identification.

Images