Primer composition and kit for identifying testudinate species and application of primer composition and kit

Abstract

The invention discloses a primer composition and a kit for identifying testudinate species and application of the primer composition and the kit. The primer composition comprises primers (SEQ ID NO.1-6) for targeted amplification of specific marker genes 16S, COI and Cytb of testudinate mitochondrial species and an enrichment universal primer (SEQ ID NO.7). According to the invention, full lengths of three mitochondrial genes 16S, COI and Cytb are taken as targets, a specific primer group added with an enrichment tag and an enrichment primer are designed, and a targeted nanopore sequencing method capable of realizing testudinate species identification is established and optimized, and the method can simultaneously capture testudinate mitochondrial species specific marker gene information of a family species level, and can be used for identifying testudinate species. The problem that the existing DNA bar code is difficult to accurately identify the testudinate species due to the lack of database information is effectively solved; and each marker gene can obtain a complete coding sequence, so that genetic information is richer, and the method can be used for geographical traceability analysis of testudinate species.

Description

Technical Field

[0001] The present invention relates to the field of turtle species identification and the technical field of gene detection of their derived components, and in particular to a primer composition, a kit and applications thereof for turtle species identification. Background Art

[0002] As ancient creatures, turtles and tortoises (Tesudines) appeared more than 200 million years ago and have inestimable value in many fields such as scientific research, ecology, economy and culture. In terms of scientific research, their long evolutionary history provides precious samples for studying biological evolution; from an ecological perspective, turtles and tortoises play a key role in maintaining ecological balance; in terms of economy, turtle breeding and related product industries are quite large; in cultural terms, turtles and tortoises carry special meanings in many cultures. However, turtles and tortoises in my country are facing a grim situation. The "China Red Data Book of Endangered Animals" shows that among the more than 40 existing turtles and tortoises in China, 24 are endangered and 8 are critically endangered. It is urgent to establish efficient and accurate turtle species classification and identification technology, which is of great significance to protecting endangered species, maintaining biodiversity and regulating market order.

[0003] Traditional identification of turtles and tortoises is mainly based on morphological and anatomical characteristics, and is highly dependent on comparison with model specimens, requiring extremely high professionalism. DNA barcoding technology is the mainstream molecular identification technology in the field of species identification. This technology uses relatively short DNA fragments that are sufficiently variable, easy to amplify, and complete the identification of species. Its characteristics have both species specificity and inter-species diversity, and are not affected by the developmental stage, gender, and integrity of the specimen. In terms of specific operations, this technology is simple, convenient, easy to standardize, and has the advantages of strong versatility and high accuracy. However, traditional DNA barcoding technology has a single site, a short amplified fragment, and sometimes cannot accurately distinguish closely related species. In particular, there are many species of wild animals in the order Testudinata, and there are still gaps in the field of molecular research. Genetic information of many species of turtles and tortoises is missing, and it is difficult to accurately classify and identify based on a single DNA barcode. Rapid and accurate identification and classification of species in the order Testudinata based on existing genetic information is an important issue that needs to be solved urgently.

[0004] Nanopore sequencing is a third-generation sequencing technology based on single-molecule sequencing. Its core principle is to use a nanoscale protein pore (nanopore) as a biosensor, and determine the base sequence by detecting the current change caused by nucleic acid molecules passing through the nanopore, thereby achieving sequencing. Nanopore single-molecule sequencing has advantages such as being portable, real-time sequencing, and having extremely long read lengths. It can generate WGS data of a biological genome within 12 hours or even shorter, providing a new technology for molecular detection that is fast, accurate, high-throughput, traceable, with a high degree of automation, and the sequencing cost and cycle are more "affordable" compared to second-generation sequencing, and the data analysis software is also more "user-friendly". In recent years, it has been widely used in fields such as rapid clinical diagnosis, epidemiological investigation and monitoring, and pathogen genomics research. Currently, there are no research reports on using nanopore sequencing technology for the identification of turtle and tortoise species. Filling this gap in technology application and exploring the feasibility and effectiveness of nanopore sequencing technology in the identification of turtle and tortoise species is expected to open up a new path for the protection and identification of turtle and tortoise species. Summary of the Invention

[0005] The main object of the present invention is to propose a primer composition, kit and its application for the identification of turtle and tortoise species, aiming to solve the problem that the COI gene information of some turtle and tortoise species is incomplete, and the coverage and accuracy of conventional single barcode technology are difficult to meet the requirements of classification and identification.

[0006] To achieve the above object, the present invention first designs a set of primer compositions for the taxonomic identification of turtle and tortoise species. The primer compositions include primers (SEQ ID NO.1-6) targeting the amplification of mitochondrial species-specific marker genes 16S, COI, and Cytb of turtle and tortoise species and an enrichment universal primer (SEQ ID NO.7).

[0007] Preferably, a universal targeting enrichment tag is added to the 5' end of the primers. By amplifying and enriching specific fragments with the SEQ ID NO.7 universal primer, the efficiency of specifically capturing marker genes by targeted PCR can be significantly improved.

[0008] The primer composition includes a 16S full gene amplification primer pair with an enrichment tag at the 5' end composed of single-stranded DNA molecules shown in SEQ ID NO.1-SEQ ID NO.2; a COI full gene amplification primer pair with an enrichment tag at the 5' end composed of single-stranded DNA molecules shown in SEQ ID NO.3-SEQ ID NO.4; a Cytb full gene amplification primer pair with an enrichment tag at the 5' end composed of single-stranded DNA molecules shown in SEQ ID NO.5-SEQ ID NO.6; and a single-stranded DNA primer shown in SEQ ID NO.7 for the synchronous and efficient enrichment of specifically amplified fragments in the amplification system.

[0009] The three species-specific marker genes of the mitochondrial of Testudines amplified by the primer composition contain more classification sites than conventional DNA barcodes, and can more accurately identify Testudines species. This primer composition has both universality and specificity, and can efficiently capture and amplify the complete coding region genes of 16S, COI and Cytb of the mitochondrial of Testudines.

[0010] Preferably, the GC content of the primer is 40% - 60%, there is no secondary structure and repetition within the primer, there is no complementary sequence between and within the primers, the melting temperature of the primer is 55 - 65 °C, and the difference in melting temperature between the primers is less than 3 °C. The universality of Testudines species is taken into account.

[0011] The present invention also provides a kit or reagent for targeting and enriching species-specific marker genes of Testudines, and the active ingredient of the kit or reagent is the primer composition as described above.

[0012] Preferably, the kit or reagent further includes a targeted PCR amplification reagent.

[0013] Preferably, the targeted PCR amplification reagent includes a PCR reaction solution, a negative control, a positive control, and nuclease-free water. The PCR reaction solution includes a PCR buffer, MgCl 2 , dNTP, and Taq DNA polymerase.

[0014] Preferably, the positive control contains Testudines mitochondrial genes and inserts a LacZ tag sequence, and the negative control is the pBluescript II SK+ vector plasmid.

[0015] Preferably, the positive control is a plasmid DNA mixture containing three mitochondrial target genes of Testudines: the artificially synthesized mitochondrial gene 16S of Testudo graeca (NC_007692.1), with the LacZ sequence added in the middle as a gene tag, having a length of 1631 bp. The plasmid is cloned between the Not I and Kpn I sites of the pBluescript II SK+ vector as the positive control for the mitochondrial gene 16S of Testudines. The artificially synthesized mitochondrial gene COI of Testudo graeca (NC_007692.1), with the LacZ sequence added in the middle as a gene tag, having a length of 1573 bp. The plasmid is cloned between the Not I and Kpn I sites of the pBluescript II SK+ vector as the positive control for the mitochondrial gene COI of Testudines. The artificially synthesized mitochondrial gene Cytb of Testudo graeca (NC_007692.1), with the LacZ sequence added in the middle as a gene tag, having a length of 1169 bp. The plasmid is cloned between the Not I and Kpn I sites of the pBluescript II SK+ vector as the positive control for the mitochondrial gene Cytb of Testudines. The concentration of each plasmid is 10 5 copies / μL. The positive control in the kit can be used either by individually packaging each plasmid or by mixing the three plasmids in equal amounts. The concentration of the vector plasmid is also 10 5 copies / μL.

[0016] The present invention also provides a method for identifying Testudines species based on targeted nanopore sequencing of mitochondrial species-specific marker genes of Testudines, comprising the following steps (see the flowchart in Figure 1 ):

[0017] S1. Extract genomic DNA from the sample to be tested;

[0018] S2. Using the genomic DNA as a template, perform a PCR amplification reaction with the above-mentioned species-specific marker gene primers and enrichment universal primers to obtain a PCR mixture of the mitochondrial species-specific marker genes 16S, COI, and Cytb of Testudines. After purifying the product, obtain a sequencing library after ligating an identification tag (Barcode) and an adapter;

[0019] S3. Perform nanopore sequencing on the sequencing library;

[0020] S4. Assemble the data obtained from nanopore sequencing, conduct preliminary identification of the molecular identification library, and perform phylogenetic analysis: First, compare the 16S whole gene sequencing results with the molecular identification library to initially determine its taxonomic rank in the order Testudines. Then, select representative 16S whole gene sequences of different genera within the same family for phylogenetic analysis to classify the unknown Testudines species into the family and genus ranks; in the second step, conduct phylogenetic analysis on the COI or Cytb whole gene sequencing results together with the representative sequences of different species or subspecies within the same genus of the order Testudines. If any gene can classify the Testudines species into the species / subspecies rank, the result can be determined, thereby achieving accurate classification and identification of Testudines species.

[0021] Based on the targeted sequencing results and the gene information in the Testudines database, establish a two-level phylogenetic classification and identification method: First, blast the 16S whole gene sequencing results to obtain the taxonomic information of its related families and genera. Then, select representative 16S whole genes of different genera within the same family for phylogenetic analysis to classify the unknown Testudines species into the family and genus ranks; in the second step, blast the COI or Cytb whole gene sequencing results and select the corresponding sequences of different species or subspecies within the same genus that have been published for phylogenetic analysis respectively. If any gene can classify the Testudines species into the species / subspecies rank, the determination can be made, thereby achieving accurate classification and identification of Testudines species.

[0022] Since the COI gene information of some Testudines species is incomplete, the coverage and accuracy of conventional single barcode technology are difficult to meet the requirements of classification and identification. The present invention conducts species classification and identification based on the targeted sequencing of three mitochondrial species-specific marker genes of the order Testudines, obtaining richer species genetic information than conventional DNA barcode technology. It can not only effectively improve the accuracy and scope of application of Testudines species identification, but also effectively supplement the gene information of Testudines species and enrich the molecular database through third-generation sequencing.

[0023] Preferably, the test sample includes live animals and their tissues of Testudines species, Testudines products, or foods containing Testudines ingredients.

[0024] The present invention also proposes the application of the primer composition, the kit or reagent, and the method as described above in the identification of Testudines species.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0026] (1) Wide species spectrum identification and more accurate classification: The present invention uses three complete mitochondrial coding genes, 16S, COI, and Cytb, as targets. Based on the design of universal enrichment primers and the addition of enrichment tag-containing Chelonioidea-specific primer sets, a targeted nanopore sequencing method for Chelonioidea species identification is established and optimized. This method can simultaneously capture mitochondrial species-specific marker gene information at the family, genus, and species levels of Chelonioidea, effectively solving the problem that existing DNA barcodes are difficult to accurately classify and identify due to the lack of information in the Chelonioidea species database. Moreover, a complete coding gene sequence can be obtained for each marker gene, with richer genetic information. In addition to its existing functions, it can also be used for geographical traceability analysis of Chelonioidea species.

[0027] (2) Fast: The present invention uses third-generation nanopore sequencing technology, which can directly perform nanopore sequencing on the PCR products enriched with high efficiency. The species identification results of Chelonioidea can be obtained within a short time, and it can be directly classified to the species or subspecies rank.

[0028] (3) Sensitive: This method has high sensitivity. Detection of serially diluted genomic DNA of Kinosternon leucostomum shows that the sequencing sensitivity can reach 0.13 ng / μL.

[0029] (4) Specific: This method has strong specificity, can accurately distinguish Chelonioidea, avian, and fish species, and can classify Chelonioidea species to the species / subspecies rank.

[0030] (5) Wider range of applicable samples: It can identify samples from a single species source and can also identify Chelonioidea species in mixtures. At the same time, it provides a practical technical means for supplementing and improving the Chelonioidea species gene database. Description of the Drawings

[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

[0032] Figure 1 It is the flow chart of the targeted nanopore sequencing of mitochondrial species-specific marker genes of Chelonioidea of the present invention;

[0033] Figure 2 It is the sequence multiple alignment diagram of 16S of mitochondrial species-specific marker genes of Chelonioidea of the present invention;

[0034] Figure 3 It is the sequence multiple alignment diagram of COI of mitochondrial species-specific marker genes of Chelonioidea of the present invention;

[0035] Figure 4 This is the sequence multiple alignment diagram of the mitochondrial species-specific marker gene Cytb of Testudines in the present invention;

[0036] Figure 5 This is the PCR electrophoresis diagram of targeting and amplifying Kinosternon leucostomum using the primer composition in the present invention;

[0037] Figure 6 This is the phylogenetic tree diagram of 16S sequences of closely related genera in the family Kinosternidae of Testudines in the present invention;

[0038] Figure 7 This is the phylogenetic tree diagram of COI sequences of closely related species in the genus Kinosternon of the family Kinosternidae of Testudines in the present invention;

[0039] Figure 8 This is the phylogenetic tree diagram of Cytb sequences of closely related species in the genus Kinosternon of the family Kinosternidae of Testudines in the present invention;

[0040] Figure 9 This is the sensitivity diagram of the mitochondrial gene targeting nanopore sequencing method of Testudines in the present invention;

[0041] Figure 10 This is the PCR electrophoresis diagram of targeting and amplifying Testudo graeca using the primer composition in the present invention;

[0042] Figure 11 This is the phylogenetic tree diagram of 16S genes of closely related genera in the family Testudinidae of Testudines in the present invention;

[0043] Figure 12 This is the phylogenetic tree diagram of COI genes of closely related species in the genus Testudo of the family Testudinidae of Testudines in the present invention;

[0044] Figure 13 This is the phylogenetic tree diagram of Cytb genes of closely related species in the genus Testudo of the family Testudinidae of Testudines in the present invention.

[0045] The realization, functional characteristics and advantages of the object of the present invention will be further described with reference to the embodiments and the accompanying drawings. Detailed implementation manners

[0046] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be described clearly and completely below. For those not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase. In addition, the technical solutions between various embodiments can be combined with each other, but it must be based on the fact that those of ordinary skill in the art can implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0047] The technical solutions of the present invention will be further described in detail below in conjunction with specific embodiments and the accompanying drawings. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0048] In the present invention, the sample to be tested is a live animal of the Testudines species and its tissues, turtle shells, foods, and products. To improve the efficiency of nucleic acid extraction, especially when the sample to be tested is a food or product that may contain components of the Testudines species, pretreatment is required before DNA extraction.

[0049] In the present invention, the reaction system of the PCR amplification reaction is shown in Table 1 below.

[0050] Table 1 Reaction system of the PCR amplification reaction

[0051]

[0052]

[0053] The primers of 16S, COI, and Cytb are mixed in equal amounts to prepare primer pool A. Prepare the PCR system: add 10 μL of Q5 High-Fidelity 2X Master Mix with a concentration of 2X, 100 nM primer pool A, and 500 nM universal primer, 98 °C, 30 s, 1 cycle. Preferably, the reaction conditions for the targeted enrichment PCR are: 98 °C, 30 s, 62 °C, 30 s, 72 °C, 2 min, 30 cycles; 72 °C, 2 min, 1 cycle; 4 °C hold. The PCR products are purified with 0.8x magnetic beads and reserved for use.

[0054] Example 1 Primer composition for targeted enrichment of mitochondrial species-specific marker genes of Testudines

[0055] Select the mitochondrial genes 16S, COI, and Cytb of Testudines as the target regions. Based on multiple sequence alignments, design primers for the mitochondrial species-specific marker genes of Testudines (see the multiple sequence alignment and primer design regions in Figures 2 - 4 ). When designing primers, it is necessary to consider including the complete species-specific target genes and also take into account the generality of Testudines species. At the same time, to improve the PCR amplification efficiency, especially for trace genomic templates, add a universal enrichment tag to the 5' end of each primer, and use the universal primer and the primers of the mitochondrial species-specific marker genes of Testudines together as a primer pool. The primer length is about 20 bases, the GC content is 40% - 60%, there is no secondary structure and repeatability within the primer, there is no complementary sequence between and within the primers, the melting temperature (Tm value) of the primer is around 60, and the difference in melting temperature between primers is less than 3°C.

[0056] The nucleotide sequences of the primer pool are as follows. The italicized part is the enrichment tag primer sequence:

[0057] 1) 5'-ACTCCACGCTCAACCGATCTGTCGTAACAWGGTAAGYVTAC YGG-3' (SEQ ID NO.1)

[0058] 2) 5'-ACTCCACGCTCAACCGATCTTGTTAAGGAGAGRATTTGAATC TCTG-3' (SEQ ID NO.2)

[0059] 3) 5'-ACTCCACGCTCAACCGATCTRAGCTGCTTCTYTGAATTTGCA ATTC-3' (SEQ ID NO.3)

[0060] 4) 5'-ACTCCACGCTCAACCGATCT CATARWGGTTATGRTRTTGGC TTG-3' (SEQ ID NO.4)

[0061] 5) 5'-ACTCCACGCTCAACCGATCTTTTACTCGGACTCTAACCAAG-3' (SEQ ID NO.5)

[0062] 6) 5'-ACTCCACGCTCAACCGATCTGCGCTAGGAAGGATTTTAAC-3' (SEQ ID NO.6)

[0063] 7) 5'-ACTCCACGCTCAACCGATCT-3' (SEQ ID NO.7)

[0064] Among them, the primer pair for targeted amplification of the mitochondrial gene 16S of Testudines is composed of single-stranded DNA molecules shown in SEQ ID NO.1 and SEQ ID NO.2, which are respectively the common sense primer and antisense primer for the detection of the mitochondrial gene 16S of Testudines, and the 5'-end of the primer is provided with an enrichment tag sequence;

[0065] The primer pair for targeted amplification of the mitochondrial gene COI of Testudines is composed of single-stranded DNA molecules shown in SEQ ID NO.3 and SEQ ID NO.4, which are respectively the common sense primer and antisense primer for the detection of the mitochondrial gene COI of Testudines, and the 5'-end of the primer is provided with an enrichment tag sequence;

[0066] The primer pair for targeted amplification of the mitochondrial gene Cytb of Testudines is composed of single-stranded DNA molecules shown in SEQ ID NO.5 and SEQ ID NO.6, which are respectively the common sense primer and antisense primer for the detection of the mitochondrial Cytb gene of Testudines, and the 5'-end of the primer is provided with an enrichment tag sequence;

[0067] The universal primer for targeted enrichment of the mitochondrial 16S, COI, and Cytb genes of Testudines is the single-stranded DNA shown in SEQ ID NO.7, which is consistent with the enrichment tag sequence.

[0068] Example 2 Method for Targeted Enrichment of Species-Specific Marker Genes in the Mitochondria of Testudines

[0069] The method for targeted enrichment of mitochondrial genes of Testudines using multiplex PCR technology is as follows: Extract genomic DNA from the sample to be tested. Using the DNA as a template, perform PCR amplification with the primer pool in Example 1 to obtain various PCR products, and after purification, use them for nanopore sequencing. The specific steps are as follows:

[0070] 1. Pretreatment and DNA Extraction of the Sample to be Tested

[0071] For Testudines samples, the pretreatment and DNA extraction are carried out according to the following method:

[0072] For animal tissues, blood, swabs, etc. of Testudines, the total DNA can be directly extracted using the QIAamp DNA Mini Kit (Qiagen genomic DNA extraction kit, 51304) according to the instructions. Other equivalent genomic DNA extraction kits can also be used.

[0073] Turtle and tortoise animal-derived foods or products, turtle shells, etc.: Take 100 mg of the sample and add 200 μL of buffer ATL (from the DNA extraction kit), shake until completely suspended. Add 50 μL of Proteinase K solution, mix well, digest at 56 °C for 4 - 8 h, then take the supernatant and use the QIAamp DNA Mini Kit (Qiagen genomic DNA extraction kit, 51304) to extract total DNA according to the instructions. Other equivalent genomic DNA extraction kits can also be used.

[0074] When establishing the method of the present invention, oral swabs of living animals of Kinosternon leucostomum were collected as samples, named G28, and genomic DNA of G28 was extracted.

[0075] 2. Using the DNA of the test sample in step 1 as a template for targeted PCR amplification to obtain various PCR products

[0076] Using the genomic DNA of G28 obtained in step 1 as a template, using the pBluescript II SK+ vector plasmid as a negative control and a plasmid DNA mixture containing the corresponding target amplification genes (16S, COI, Cytb) as a positive control, respectively, perform PCR amplification using the primer pool in Example 1 to obtain various PCR products.

[0077] The negative control is the pBluescript II SK+ vector plasmid.

[0078] The positive control is a plasmid DNA mixture containing three mitochondrial genes 16S, COI, and Cytb of Testudines respectively.

[0079] Among them, for the positive control for detecting the mitochondrial 16S gene of Testudines: directly synthesize the 16S gene with a length of 1631 bp shown in SEQ ID NO.8 and containing the LacZ tag, and clone it between the Not I and Pst I sites of the pBluescript II SK+ vector. After sequencing confirmation, it is named pBSK-Tesudines-16S, which is the mother liquor of the positive control product for the mitochondrial 16S gene of Testudines;

[0080] The positive control plasmid for the mitochondrial gene 16S of Testudines is 1631 bp, and the sequence is as follows (SEQ ID NO.8):

[0081]

[0082] Among them, the positive control for detecting the mitochondrial COI gene of Testudines: directly synthesize the COI gene with a length of 1573 bp containing the LacZ tag shown in SEQ ID NO.9, and clone it between the Not I and Pst I sites of the pBluescript II SK+ vector. After sequencing confirmation, it is named pBSK-Tesudines-COI, which is the mother liquor of the positive control product for the mitochondrial COI gene of Testudines;

[0083] The positive control plasmid of the mitochondrial gene COI of Testudines is 1573 bp, and the sequence is as follows (SEQ ID NO.9):

[0084]

[0085] Among them, the positive control for detecting the mitochondrial Cytb gene of Testudines: directly synthesize the Cytb gene with a length of 1144 bp containing the LacZ tag shown in SEQ ID NO. 10, and clone it between the Not I and Pst I sites of the pBluescript II SK+ vector. After sequencing confirmation, it is named pBSK-Tesudines-Cytb, which is the mother liquor of the positive control product for the mitochondrial Cytb gene of Testudines.

[0086] The positive control plasmid of the mitochondrial gene Cytb of Testudines is 1169 bp, and the sequence is as follows (SEQ ID NO. 10):

[0087]

[0088] Targeted amplification PCR reaction system:

[0089] Table 2 Formulation of the reaction system

[0090] Component Volume or Concentration Q5 High-Fidelity 2X Master Mix 12.5 μl Specific Primer (Primer Pool A) 100 nM Universal Primer 500 nM DNA Template 1 μl DEPC Water 11 μl Total 25 μl

[0091] Among them, Q5 High-Fidelity 2X Master Mix was purchased from NEB; primers were synthesized by Shanghai Sangon Biotech Co., Ltd.; primer pool A was prepared by equally mixing three pairs of specific primers.

[0092] DEPC water: After twice distilling pure water, add DEPC to a final concentration of 0.1%, stir at 37°C for 12 h, and autoclave at 1.034×10 5 Pa for 15 minutes.

[0093] The reaction conditions for targeted multiplex PCR are: 98°C, 30 s, 1 cycle; 98°C, 30 s, 62°C, 30 s, 72°C, 2 min, 30 cycles; 72°C, 2 min, 1 cycle; 4°C hold.

[0094] The electrophoresis diagram of the PCR product is shown in Figure 5 . The PCR product was purified with 0.8x magnetic beads for later use.

[0095] Example 3 Establishment of a method for targeted nanopore sequencing of mitochondrial species-specific marker genes in Testudines

[0096] Operate according to the instructions of the nanopore sequencing related kit (Ligation Sequencing Kit SQK-LSK114; PromethlON sequencing chip R10, FLO-PRO114M, NANOPORE), specifically including the following steps:

[0097] 1. Two-round PCR reaction

[0098] Take the product of the first-round PCR reaction and perform barcode ligation PCR reaction according to the following table. Different barcode sequence primers are selected for different samples:

[0099] Table 3 Reagents for barcode ligation PCR reaction

[0100] Reagent Volume (μL) PCR Mix 10 Barcode 10 Previous PCR Product 5 Total 25

[0101] Table 4 Amplification program

[0102]

[0103] 2. Purification of PCR product:

[0104] ① Take a new 1.5 ml EP tube and evenly mix the PCR products connected with barcodes in equal volume.

[0105] ② Resuspend the AMPure XP magnetic beads by vortexing.

[0106] ③ Add 140 μL of AMPure XP magnetic beads to 200 μL of the mixed PCR products, mix by flicking the EP tube, and incubate at room temperature for 5 minutes.

[0107] ④ Place the EP tube on the magnetic stand until the eluate is clear and colorless, then remove the supernatant.

[0108] ⑤ Place the EP tube on the magnetic stand, wash the magnetic beads with 200 μL of freshly prepared 80% ethanol, take out the ethanol and discard it.

[0109] ⑥ Repeat the previous step.

[0110] ⑦ Centrifuge briefly and place the EP tube back on the magnetic stand to remove all residual ethanol. Dry for about 40 seconds, but do not dry to the extent that the particles crack.

[0111] ⑧ Remove the EP tube from the magnetic stand, resuspend the magnetic beads in 52 μL of EP, and incubate at room temperature for 2 minutes.

[0112] ⑨ Place the EP tube on the magnetic stand until the eluate is transparent and colorless.

[0113] ⑩ Transfer all the supernatant to a new 1.5 ml EB tube.

[0114] Take 1 μL of the purified product and detect the concentration using the Qubit dsDNA HS Assay Kit.

[0115] 3. Library construction

[0116] Table 5 Configuration system:

[0117] Component Dosage per Portion (μL) Second-round PCR Purified Product 35 Ligation Adapter (LA) 5 T4 DNA Ligase (Rapid) 10 2× Rapid Ligation Buffer 50 Total 100

[0118] Ligation and purification:

[0119] ① Place the PCR reaction tube on the PCR instrument; Program: 24 °C for 10 min.

[0120] ② Transfer the PCR products to a new 1.5 ml EP tube, add 80 μL of AMPure XP magnetic beads, mix by flicking the EP tube, and incubate at room temperature for 5 minutes.

[0121] ③ Place the EP tube on the magnetic stand until the eluate is clear and colorless, then remove the supernatant.

[0122] ④ Place the EP tube on the magnetic stand, wash the magnetic beads with 200 μL of SFB, remove the SFB and discard it.

[0123] ⑤ Repeat the previous step.

[0124] ⑥ Centrifuge briefly and place the EP tube back on the magnetic stand to remove all residual ethanol.

[0125] ⑦ Add 20 μL of EB elution buffer, remove the EP tube from the magnetic stand, and incubate at room temperature for 5 minutes.

[0126] ⑧ Place the EP tube on the magnetic stand until the elution buffer is clear and colorless.

[0127] ⑨ Transfer all the supernatant to a new 1.5 ml EB tube.

[0128] ⑩ Take 1 μL of the purified product and measure the concentration using the Qubit dsDNA HS Assay Kit.

[0129] 4. Sequencing and Chip Cleaning

[0130] ① Melt the Sequencing Buffer (SB), Library Beads (LIB), Flow Cell Tether (FCT), and Flow Cell Flush (FCF) at room temperature, vortex and mix well, centrifuge briefly, and keep on ice for later use.

[0131] ② Add 30 μL of FCT to one tube of FCF, vortex and mix well to prepare the priming mixture.

[0132] ③ Open the lid of the chip cleaning port and remove the air bubbles according to the following steps:

[0133] A. Set the 1000 μL pipette to 200 μL.

[0134] B. Gently insert the pipette tip into the cleaning port and hold it perpendicular to the plane of the chip.

[0135] C. Rotate the pipette wheel until the scale shows 220 - 230 μL, or until a small volume of buffer enters the pipette tip.

[0136] ④ Add 800 μL of the priming mixture to the chip cleaning port, avoiding the generation of air bubbles during the addition, and let it stand for 5 min.

[0137] ⑤ Prepare the loading library during the standing process:

[0138] Table 6 Loading Library

[0139] Ingredient Volume (μL) Sequencing Buffer (SB) 75 Library Beads (LIB) 51 DNA Library (100 ng) 24 Total 150

[0140] ⑥ Add the remaining 200 μL of the initiation mixture to the chip cleaning port, taking care to avoid introducing air bubbles during addition.

[0141] ⑦ Slowly add 75 μL of the loading mixture to the chip through the chip injection port.

[0142] ⑧ Close the chip injection port and the chip cleaning port, and load the chip into the GridION for sequencing.

[0143] 5. Sequencing data analysis

[0144] Download the mitochondrial gene sequences such as 16S, COI, and Cytb of Testudines species from the NCBI database, and construct a molecular identification library of Testudines mitochondrial genes after integration. Filter the generated sequence data to remove sequencing adapters and barcodes, and only retain the full-length sequence fragments with lengths between 1000 and 2000 bp to ensure the integrity and applicability of the data; perform similarity clustering analysis on the filtered sequences to reduce data redundancy. Compare the representative sequences after clustering with the constructed molecular identification library of Testudines, and classify and identify unknown Testudines species through the evolutionary analysis of the 16S, COI, and Cytb genes of Testudines species.

[0145] Due to the incomplete DNA barcode data of Testudines species, it is impossible to fixedly use a certain DNA barcode for classification, and it can only be determined according to the comparison results between the sequencing results and the gene information published in the NCBI database. When performing multiple alignments of the published mitochondrial genes of Testudines, it is found that the 16S gene is relatively more conserved, and the COI or Cytb genes have greater variation between species. Therefore, the determination of the targeted nanopore sequencing results of the present invention is divided into two steps: first, the 16S full gene sequencing results are preliminarily compared with the molecular identification library to obtain its taxonomic rank in Testudines, and then representative sequence information of different genera in the same family is selected for evolutionary analysis to classify the unknown Testudines species to the family and genus ranks; in the second step, the COI or Cytb full genes are respectively selected, and representative sequences of different species or subspecies in the same genus of Testudines are respectively used for evolutionary analysis. If any gene can classify the Testudines species to the species / subspecies rank, the result can be determined, thereby realizing the accurate classification and identification of Testudines species.

[0146] According to the above determination criteria, analyze and determine the nanopore sequencing results of the samples. The 16S gene sequence of G28 measured was preliminarily compared and identified with the sequences in the molecular identification library of Testudines, and the results showed that it had the highest similarity with the species of the family Kinosternidae; therefore, the 16S gene sequence of G28 was subjected to evolutionary analysis with the corresponding sequences of members of each genus in the same family, and the results are shown in Figure 6, it can be seen that G28 is in the same clade as the species of the genus Kinosternon, so G28 is classified as a species of the genus Kinosternon. Then, the COI gene sequence and Cytb gene sequence of G28 were respectively subjected to phylogenetic analysis with the published sequences of the species under the genus Kinosternon. The results are shown in Figure 7 and Figure 8 . It can be seen that both genes are in the same phylogenetic clade as the corresponding sequences of Kinosternon leucostomum. Therefore, it is comprehensively determined that G28 is from Kinosternon leucostomum.

[0147] Based on this, the targeted nanopore sequencing results of the G28 sample determined that the DNA sample was indeed Kinosternon leucostomum, which was consistent with the known taxonomic background of the sample, indicating that a targeted nanopore sequencing method for mitochondrial species-specific marker genes of Testudines was successfully established.

[0148] Example 4 Sensitivity and Specificity of the Targeted Nanopore Sequencing Method for Mitochondrial Genes of Testudines

[0149] 1. Sensitivity Test

[0150] Taking Kinosternon leucostomum as the test sample, the sensitivity test of the targeted nanopore sequencing method for Testudines species was carried out. Oral swabs of Kinosternon leucostomum were taken, and genomic DNA was extracted using the QIAamp DNA Mini Kit (Qiagen genomic DNA extraction kit, 51304). After the taken oral swabs were fully rinsed with 200 μL of 0.01 mol / L PBS at pH 7.4, 200 μL of buffer ATL was added, and the mixture was shaken well. Then, the supernatant was taken and total DNA was extracted using the QIAamp DNA Mini Kit (Qiagen genomic DNA extraction kit, 51304) according to the instructions. After DNA extraction, the nucleic acid concentration was measured to be 10.52 ng / μL using a Qbit fluorometer. The extracted genomic DNA of Kinosternon leucostomum was diluted 3-fold with nuclease-free water, and the obtained DNA template concentrations were 10.52 ng / μL, 3.51 ng / μL, 1.17 ng / μL, 0.39 ng / μL, 0.13 ng / μL, and 0.043 ng / μL in turn. Using the primer pool in Example 1, targeted PCR amplification was carried out with the above 6 groups of DNA with different concentrations as templates, and pBluescript II SK+ vector plasmid DNA was used as a negative control, and a mixture of plasmid DNAs of mitochondrial genes (16S, COI, Cytb) of Testudines was used as a positive control. After the 6 groups of targeted PCR products were purified, adapters were added respectively for nanopore sequencing. The results are shown in Figure 9, it can be seen that the PCR products with DNA template concentrations of 10.52 ng / μL, 3.51 ng / μL, 1.17 ng / μL, 0.39 ng / μL, 0.13 ng / μL, and 0.043 ng / μL all obtained the target sequences. Among them, the PCR product with a concentration of 0.13 ng / μL still obtained the sequencing results of 478 tortoise mitochondrial gene sequences, indicating that the sensitivity of the established targeted nanopore sequencing method for Testudines in the present invention is: 0.13 ng / μL DNA.

[0151] 2. Specificity test

[0152] Taking samples of Testudines species (white-lipped mud turtle, S1), Aves species (parrot, S2), and Actinopterygii species (bony fish, S3) with known species classification backgrounds as specificity verification samples, targeted nanopore sequencing was carried out. Appropriate amounts of S1 oral swabs, S2 parrot feathers, and S3 bony fish tissues were taken respectively, and the genomic DNA of the three samples was extracted using the QIAamp DNAMini Kit (Qiagen genomic DNA extraction kit, 51304). Then, using the primer pool in Example 1, the genomic DNA of the above three different species was used as a template for targeted PCR amplification, and the pBluescript II SK+ empty vector plasmid DNA was used as a negative control, and a plasmid DNA mixture containing gene-tagged Greek tortoise mitochondrial genes (16S, COI, Cytb) was used as a positive control. After purifying the 3 groups of targeted PCR products, different adapters were added for nanopore sequencing. After comparing the sequencing results with the molecular identification library sequences for preliminary species identification, only the sequencing identification result of S1 was the Testudines species white-lipped mud turtle, and the sequencing identification results of S2 and S3 samples were both non-Testudines species. The results are shown in Table 7, indicating that the established targeted nanopore sequencing method for Testudines in the present invention has good specificity.

[0153] Table 7 Specificity test of the targeted nanopore sequencing method for Testudines

[0154]

[0155] Example 5 Example of targeted nanopore sequencing of a live tortoise intercepted at the port

[0156] The method described in Example 2 was used to perform targeted nanopore sequencing on a live tortoise intercepted at the port, and the identification result was Testudo graeca nabeulensis.

[0157] 1. Extract tortoise genomic DNA

[0158] Since the intercepted animals were live, oral swab samples were taken and named 20240401-GUI. Genomic DNA was extracted using the QIAamp DNA Mini Kit (Qiagen genomic DNA extraction kit, 51304). After thoroughly rinsing the taken oral swabs with 200 μL of 0.01 mol / L PBS with pH 7.4, 200 μL of buffer ATL was added, and it was thoroughly shaken and mixed. Then, the supernatant was taken and total DNA was extracted using the QIAamp DNA Mini Kit (Qiagen genomic DNA extraction kit, 51304) according to the instructions.

[0159] 2. Perform targeted PCR amplification using the tortoise DNA extracted in step 1 as a template

[0160] Using the primer pool in Example 1, PCR was performed with tortoise genomic DNA as a template, and pBluescript IISK+ vector plasmid DNA was used as a negative control, and a plasmid DNA mixture of Testudines mitochondrial genes (16S, COI, Cytb) was used as a positive control. The PCR electrophoresis results are shown in Figure 10 , and the results showed that Testudines mitochondrial gene DNA was amplified in the tortoise DNA.

[0161] 3. Nanopore sequencing

[0162] Operate according to the nanopore sequencing kit instructions, purify the PCR product, add adapters, construct a library, and sequence.

[0163] 4. Result analysis

[0164] After 2 hours of nanopore sequencing, data analysis was carried out. The generated sequence data was filtered and cluster analyzed. Then, the 16S gene sequence of the measured tortoise 20240401-GUI was preliminarily compared and identified with the Testudines molecular identification library sequence. The results showed that it had the highest similarity with species in the family Testudinidae. Therefore, the 16S gene sequence of 20240401-GUI was subjected to phylogenetic analysis with the corresponding sequences of members of each genus in the same family. The results are shown in Figure 11 , and it can be seen that 20240401-GUI and species in the genus Testudo are in the same branch. Thus, 20240401-GUI was classified into the genus Testudo. Then, the COI gene sequence and Cytb gene sequence of 20240401-GUI were respectively subjected to phylogenetic analysis with the published sequences of species under the genus Testudo. The results are shown in Figure 12 and Figure 13。According to the evolutionary analysis of the COI gene sequence, 20240401-GUI and the corresponding sequence of Testudo graeca are in the same evolutionary branch; the evolutionary analysis of the Cytb gene shows that 20240401-GUI and the corresponding sequence of Testudo graeca nabeulensis are in the same evolutionary branch. Therefore, through comprehensive judgment, the species identification result of this live tortoise intercepted at the port is Testudo graeca nabeulensis.

[0165] The above are only the preferred embodiments of the present invention, and do not limit the patent scope of the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the patent protection scope of the present invention.

Claims

1. A primer composition for classification and identification of species of the order Testudinata, characterized in that: The primer composition comprises primers (SEQ ID NO.1-6) for targeted amplification of mitochondrial species-specific marker genes 16S, COI, and Cytb of the order Testudinata and a universal enrichment primer (SEQ ID NO.7).

2. The specific marker gene primers (SEQ ID NO. 1-6) according to claim 1, characterized in that: A universal target enrichment tag was added to the 5' end of each primer.

3. A kit or reagent for targeted enrichment of species-specific marker genes of the order Testudinata, characterized in that: The active ingredient of the kit or reagent is the primer composition according to any one of claims 1 to 2.

4. The kit or reagent according to claim 3, characterized in that The kit or reagent also includes a targeted PCR amplification reagent.

5. The kit or reagent according to claim 4, characterized in that The targeted PCR amplification reagent includes a PCR reaction solution, a negative control, a positive control, and nuclease-free water. The PCR reaction solution includes a PCR buffer, MgCl2, dNTP, and TaqDNA polymerase.

6. The kit according to claim 5, characterized in that The positive control comprises a turtle mitochondrial gene and is inserted with a LacZ tag sequence, and the negative control is a pBluescript II SK+ vector plasmid.

7. A method for identifying species of Testudinata based on targeted nanopore sequencing of mitochondrial species-specific marker genes of Testudinata, characterized in that: The steps include: S1, extracting genomic DNA from the sample to be tested; S2. Using the genomic DNA as a template, performing PCR amplification reaction using the species-specific marker gene primers and enrichment universal primers as claimed in claim 1, obtaining a PCR mixed product of the mitochondrial species-specific marker genes 16S, COI, and Cytb of the order Testudinata, and obtaining a sequencing library after purifying the product and connecting the identification tag (Barcode) and the adapter; S3, performing nanopore sequencing on the sequencing library; S4. Perform sequence assembly, preliminary identification with the molecular marker library, and phylogenetic evolution analysis on the data obtained from nanopore sequencing: First, the 16S whole-genome sequencing results are preliminarily compared with the molecular marker library to determine their taxonomic level in the order Testudinata, and then representative 16S whole-genome sequences from the same family but different genera are selected for evolutionary analysis to classify unknown turtle species into the family and genus level. In the second step, the COI or Cytb whole-genome sequencing results are separately analyzed together with representative sequences from different species or subspecies of the same genus in the order Testudinata for evolutionary analysis. If any gene can classify turtle species into the species / subspecies level, a judgment can be made, thereby achieving accurate classification and identification of species in the order Testudinata.

8. The method according to claim 7, characterized in that The samples to be tested include living animals of the order Testudinata and their tissues, turtle products or foods containing turtle ingredients.

9. Use of the primer combination according to claim 1, the kit or reagent according to claim 3, and the method according to claim 7 in the identification of species of the order Testudinata.

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