Universal primers for the complete sequence of mitochondrial genome of the family Betyphaidae and amplification method and application thereof

Abstract

The present invention discloses universal primers for the complete sequence of mitochondrial genomes of Silphidae insects and an amplification method and application thereof. By dividing the mitochondrial genome into 6 mutually overlapping fragments, performing common PCR amplification, Sanger sequencing and sequence splicing, the complete sequence information of the mitochondrial genomes of Silphidae insects can be quickly obtained. The invention has low requirements on operating instruments and analysis equipment, is easy to implement, requires a small number of primer pairs, and the complete sequence of the entire mitochondrial genome can be obtained from the total DNA of a single individual, thus avoiding interference from genomic DNA of multiple individuals. The invention has been verified in the amplification of the mitochondrial genomes of 5 species of Silphidae insects, has important significance for obtaining more complete mitochondrial sequences of Silphidae insects, further inferring the kinship between the genera of Silphidae and the phylogenetic relationship between the families of the superfamily Paederoidea, and also provides a reference for studying the evolution and phylogenetic relationship of the suborder Polyphaga.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to universal primers for the complete sequence of mitochondrial genomes of Euryptidae insects and an amplification method and application thereof. Background Art

[0002] Silphidae belongs to the superfamily Staphylinoidea of ​​the suborder Polyphaga of the order Coleoptera. The body length is 7 to 45 mm (usually 12 to 20 mm), including the largest species in the superfamily Staphylinoidea; the body color is mostly dark, black or dark brown, but the subfamily Nicrophorinae often has bright orange-red markings or bands on the elytra, which is extremely beautiful; most of the insects in the Silphidae family are scavengers, feeding on animal carcasses or other decaying matter; some are obligate or facultative predators, and their prey includes snails, earthworms and small insects. As a major type of necrophilous insect, Silphidae plays an important role in forensic research and application. my country has an extremely rich species of Silphidae insects, and is the origin and distribution center of Nicrophorinae. In recent years, most of the new genera and species published in the world are from southwest my country and neighboring countries and regions. There are currently 75 species of Euphorbiidae recorded in my country, accounting for 40% of the total number of known species in the world.

[0003] Mitochondrial genomes have the characteristics of relatively small molecular weight, maternal inheritance and high mutation rate, and are widely used in population genetics, molecular evolution, comparative and evolutionary genomics, etc. At present, there are only more than 10 complete mitochondrial sequences of the family of necrophilous insects published in Genbank. As a key group of necrophilous insects, more complete mitochondrial genome sequences of the family of necrophilous insects still need to be amplified and sequenced.

[0004] The study found that the mitochondrial genomes of the family of beetles that have been sequenced are all closed circular double-stranded DNA. The main sequencing strategies currently include shotgun high-throughput sequencing methods, methods based on direct sequencing of traditional PCR amplification products, and methods for obtaining mitochondrial genome sequences from transcriptome data. However, the sequencing cost required for shotgun high-throughput sequencing methods is high, the bioinformatics splicing process is relatively complex, and mitochondrial pseudogenes from genomic DNA are easily mixed in, and control regions containing repetitive fragments due to short read lengths are difficult to splice completely. The primer walking method based on traditional PCR to obtain mitochondrial genome sequences uses a large number of primers and has low amplification efficiency. The method of obtaining mitochondrial genome sequences from transcriptome data using second-generation high-throughput sequencing is difficult to guarantee the success rate and accuracy of amplification for control regions with more simple repetitive sequences in the genome due to the limitation of the amplified fragment library (150-400bp), and the analysis process requires corresponding bioinformatics expertise; although the third-generation single-molecule sequencing can obtain the entire mitochondrial genome read length through a single sequencing, it has strict requirements on the DNA quality of insect samples, and the sequencing cost is high, and it also needs to be combined with traditional PCR methods to obtain a complete mitochondrial genome. In addition, the prior art does not disclose universal primers for the complete sequence of the mitochondrial genome of the family Betygrinidae. Summary of the invention

[0005] Based on the above technical problems, the present invention compares the mitochondrial genome of the family of beetles, screens out the conservative region with the smallest sequence variation, and designs 6 pairs of universal primers for the mitochondrial genome of the family of beetles in the conservative region. The amplified fragment is 3Kb to 4.6Kb long, and there is an overlapping region of 100 to 1200bp between the amplified products, covering the entire mitochondrial genome. It is not necessary to design specific primers for the family of beetles, and the rapid amplification of the mitochondrial genome of a single individual can be achieved, so that the mitochondrial genome information of the family of beetles can be obtained efficiently and at low cost. The purpose is to provide universal primers for the full sequence of the mitochondrial genome of the family of beetles, and a method and application thereof for amplifying the same.

[0006] The present invention protects universal primers for the complete sequence of the mitochondrial genome of the family of beetles, and the universal primers include 6 pairs of primers corresponding to the Nad2-Cox2, Cox1-Nad3, Cox3-Nad4, Nad4-Cytb, Cytb-rrnS and rrnL-Nad2 regions, respectively. In practical applications, sequence fragments of the corresponding regions can be amplified according to specific needs. The 6 pairs of universal primers are specifically:

[0007] First pair of primers:

[0008] TM-J211, whose sequence number is shown in SEQ ID.NO.1;

[0009] C2-N3390, whose sequence number is shown in SEQ ID.NO.2;

[0010] Second pair of primers:

[0011] C1-J2196, whose sequence number is shown in SEQ ID.NO.3;

[0012] N3-N5729, whose sequence number is shown in SEQ ID.NO.4;

[0013] The third pair of primers:

[0014] C3-J5526, whose sequence number is shown as SEQ ID.NO.5;

[0015] N4-N8728, whose sequence number is shown in SEQ ID.NO.6;

[0016] The fourth pair of primers:

[0017] N4-J8109, whose sequence number is shown in SEQ ID.NO.7;

[0018] CB-N11337, whose sequence number is shown as SEQ ID.NO.8;

[0019] Fifth pair of primers:

[0020] CB-J10942, whose sequence number is shown as SEQ ID.NO.9;

[0021] SR-N14748, whose sequence number is shown as SEQ ID.NO.10;

[0022] The sixth pair of primers:

[0023] LR-J14612, whose sequence number is shown as SEQ ID.NO.11;

[0024] N2-N303, whose sequence number is shown as SEQ ID.NO.12.

[0025] Furthermore, the sequences of the six pairs of universal primers for the complete mitochondrial genome sequence of the Eurycomycetes are as follows:

[0026] The first pair of primers and their sequences:

[0027] TM-J211, 5'-AAGCTDNTGGGTTCATACCCC-3';

[0028] C2-N3390, 5'-TATTCRTADGWTCARTAYCATTG-3';

[0029] The second pair of primers and their sequences:

[0030] C1-J2196, 5'-TGATTYTTYGGNCAYCCWGAAGT-3';

[0031] N3-N5729, 5'-TTDGGRTCAAAHCCRCATTC-3';

[0032] The third pair of primers and their sequences:

[0033] C3-J5526, 5'-TCTATYTAYTGATGAGGWAGNT-3';

[0034] N4-N8728, 5'-AARTCDTTRATTGCNTATTCWTC-3';

[0035] The fourth pair of primers and their sequences:

[0036] N4-J8109, 5'-TATCTTTGRYYCCACAAAYCAAT-3';

[0037] CB-N11337, 5'-AARTAYCATTCDGGYTGRATRTG-3';

[0038] The fifth pair of primers and their sequences:

[0039] CB-J10942, 5'-TGRGGNCAAATATCHTTYTGAGG-3';

[0040] SR-N14748, 5'-TTTGTGCCAGCAGCAGYWGCGGTTA-3';

[0041] The sixth pair of primers and their sequences:

[0042] LR-J14612, 5'-AAAATAATAGGGTATCTAATCCTAGTTT-3'; N2-N303, 5'-AGTCCTATTCAYATWCCTATYCA-3'.

[0043] The present invention also protects a method for amplifying the full sequence of the mitochondrial genome of the family of beetles using universal primers, the specific steps of which include the following:

[0044] Step 1, collection, identification and DNA extraction of the family of beetles: the collected specimens of the family of beetles are soaked in anhydrous ethanol and stored in a refrigerator at -20°C for standby use, a single beetle is selected for dissection, 30 mg of chest muscle is taken, 1 mol / L NaCl, pH=8, 1 mol / L Tris-HCl, pH=8, 1 mol / L EDTA are used to prepare a lysis solution, and then added to the insect muscle, proteinase K is added to extract the whole genome DNA, and the extracted whole genome DNA is stored at -20°C for standby use;

[0045] Step 2, design of universal primers for mitochondrial genome amplification of the family of Eurypteridae: download all the currently published complete sequences of the mitochondrial genome of the family of Eurypteridae from the Genbank database; first, use Geneious software to compare 13 PCGs and 2 rRNA genes with uniform distribution and long length in the mitochondrial genome, and select 8 highly conserved genes with high gene identity and appropriate gene interval, namely Cox1, Cox2, Cox3, rrnS, rrnL, Nad4, Nad5 and Cytb genes, to find out the The conservative sequence region with the smallest base variation; then, according to the comparison results, Geneious software was used to design universal primers in the conservative sequence region. In order to ensure the high efficiency and amplification efficiency of the universal primers, the length of the amplified band was limited to 3-4.6Kb. At the same time, degenerate sites were introduced according to the frequency of base occurrence at each site to increase the universality of the primers; finally, the primers were brought into each full sequence through Oligo7 for screening and adjustment to ensure that the primers met the basic design principles and there was an overlap of more than 100-1200bp between the amplified bands, so that the subsequent experiments could be used for sequence splicing;

[0046] Step 3, using the designed and synthesized primers to amplify, sequence and splice the mitochondrial genome of the family of beetles, and compare and detect its universality and amplification efficiency in the family of beetles;

[0047] Step 3.1, PCR amplification: PCR amplification was performed using the designed 6 pairs of primers and the extracted genome of a single Eurycoma beetle as a template. The PCR reaction system was: 25 μl 2×KOD One TM PCR Master Mix, 3 μl upstream primer, 3 μl downstream primer, 2 μl DNA template, 17 μl double distilled water; PCR amplification conditions: 98°C pre-denaturation for 3 min, 96°C denaturation for 15 s, 50°C annealing for 30 s, 68°C extension for 3 min, amplification for 35-40 cycles, and finally 68°C extension for 10 min; PCR products were electrophoresed on 1% agarose gel;

[0048] Step 3.2, PCR amplified fragment sequencing: sequencing the base sequences of the 6 amplified fragments;

[0049] Step 3.3, splicing the PCR amplified fragments after sequencing: the sequencing results of the 6 fragments after verification are segmented and spliced ​​using Geneious software. Since there is an overlapping region of 100 to 1200 bp between the amplified products when designing universal primers, the amplified fragments can be compared to find the repeated sequence; after removing the repeated sequence, the complete mitochondrial genome sequence is finally obtained;

[0050] Step 3.4, compare the complete mitochondrial genome sequence after splicing: compare the splicing result with the complete sequence of the family Betaeidae in the Genbank database.

[0051] Furthermore, the conserved sequence region amplified when the first pair of universal primers are designed is Nad2-Cox2, and the length of the amplified band is limited to 3000-3300 bp.

[0052] Furthermore, the conserved sequence region amplified by the second pair of universal primers is designed to be Cox1-Nad3, and the length of the amplified band is limited to 3500-3700 bp.

[0053] Furthermore, the conserved sequence region amplified when the third pair of universal primers is designed is Cox3-Nad4, and the length of the amplified band is limited to 3100-3400 bp.

[0054] Furthermore, the amplified conserved sequence region when the fourth pair of universal primers is designed is Nad4-Cytb, and the amplified band length is limited to 3200-3500 bp.

[0055] Furthermore, the conserved sequence region amplified when the fifth pair of universal primers is designed is Cytb-rrnS, and the length of the amplified band is limited to 3800-4000bp.

[0056] Furthermore, the amplified conserved sequence region when the sixth universal primer is designed is rrnL-Nad2, and the length of the amplified band is limited to 3600-4600bp.

[0057] The present invention also protects the application of universal primers for the complete sequence of the mitochondrial genome of the family Betygrinidae insects. The universal primers are applied in amplifying and sequencing the complete sequence of the mitochondrial genome of the family Betygrinidae insects.

[0058] Compared with the existing technology, the present invention has the following beneficial effects:

[0059] The present invention can quickly obtain the full sequence information of the mitochondrial genome of the family Pocillopidae by dividing the mitochondrial genome into 6 overlapping fragments, performing ordinary PCR amplification, Sanger sequencing and sequence splicing. The invention has low requirements on operating instruments and analysis equipment, is easy to implement, requires a small number of primer pairs, and the total DNA of a single individual can obtain the full sequence of the entire mitochondrial genome, avoiding interference from genomic DNA of multiple individuals. The invention has been verified in the amplification of the mitochondrial genomes of 5 species of Pocillopidae, and is of great significance for obtaining more full mitochondrial sequences of the family Pocillopidae, further inferring the relationship between the genera of the family Pocillopidae and the phylogenetic relationship between the families of the superfamily Paederoidea, and also provides a reference for studying the evolution and phylogenetic relationship of the suborder Polyphaga. The universal primers provided for amplifying the full sequence of the mitochondrial genome of the Funeridae insects have overlapping regions between adjacent amplification products, and the splicing accuracy is high, and there is no need to design specific primers for the Funeridae insects; the universal primers provided for amplifying the full sequence of the mitochondrial genome of the Funeridae insects have overlapping regions between adjacent amplification products, and the splicing accuracy is high, and there is no need to design specific primers for the Funeridae insects; the full sequence information of the mitochondrial genome of the Funeridae insects can be quickly obtained through Sanger sequencing, and there is no need to involve a large number of complex bioinformatics software and high-throughput sequence analysis processes, which saves time and greatly reduces costs, and has great application prospects in the sequencing of the mitochondrial genome of the Funeridae insects. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 Design position map of 6 pairs of universal primers of the present invention;

[0061] Figure 2 The electrophoresis diagram of the amplification of Z. quwangensis (ZG1-6), Z. oligis (ZE1-6) and Z. limnis (ZF1-6) by 6 pairs of universal primers of the present invention;

[0062] Figure 3 This is the electrophoresis diagram of the amplification of the red-breasted burial beetle (ZK1-6) and the two-spotted burial beetle (ZC1-6) by the six pairs of universal primers of the present invention. DETAILED DESCRIPTION

[0063] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0064] Example 1

[0065] The method for amplifying the complete sequence of the mitochondrial genome of the family of beetles comprises the following specific steps:

[0066] 1. Collection, identification and DNA extraction of the family of burial insects

[0067] All samples of the family of burials were collected in Xining, Qinghai Province. The collected specimens of the family of burials were soaked in anhydrous ethanol and stored in a refrigerator at -20°C for later use. The identification of the family of burials was referred to the Chinese burial beetle. A single burial beetle was selected for dissection. 30 mg of the chest muscle was prepared with 1 mol / L NaCl, pH=8, 1 mol / L Tris-HCl, pH=8, and 1 mol / L EDTA to prepare a lysis solution, which was then added to the insect muscle. After adding proteinase K, the whole genome DNA was extracted, and the extracted whole genome DNA was stored at -20°C for later use.

[0068] 2. Design of universal primers for amplification of mitochondrial genomes of Morcellidae insects

[0069] All the published complete mitochondrial genome sequences of the family of Pseudocercidae were downloaded from the Genbank database, as shown in Table 1. First, 13 PCGs and 2 rRNA genes with even distribution and long length in the mitochondrial genome were compared using Geneious software, and 8 highly conserved genes with high gene identity and appropriate gene intervals, namely Cox1, Cox2, Cox3, rrnS, rrnL, Nad4, Nad5 and Cytb genes, were selected to find the conserved sequence regions with the smallest base variation in the above genes.

[0070] Table 1 The complete mitochondrial genome of the family Betyphaidae used in this application

[0071] Species GenBank ID Size(bp) Nicrophorus nepalensis NC_059900 17299 Necrodes littoralis NC_056196 17830 Diamesus osculans NC_045874 19400 Necrophila americana NC_018352 16902 Thanatophilus sinuatus OK480618 18633

[0072] Then, according to the comparison results, Geneious software was used to design universal primers in the conserved sequence region. To ensure the high efficiency and amplification efficiency of the universal primers, the length of the amplified band was limited to 3-4.6 Kb (among which, the amplified conserved sequence region when the first pair of universal primers was designed was Nad2-Cox2, and the amplified band length was limited to 3000-3300 bp; the amplified conserved sequence region when the second pair of universal primers was designed was Cox1-Nad3, and the amplified band length was limited to 3500-3700 bp; the amplified conserved sequence region when the third pair of universal primers was designed was Cox3-Nad4, and the amplified band length was limited to 3100-3400 bp; the amplified conserved sequence region when the fourth pair of universal primers was designed was Nad4-Cytb, and the amplified band length was limited to The conserved sequence region when the fifth pair of universal primers is designed is Cytb-rrnS, and the length of the amplified band is limited to 3800-4000bp; the conserved sequence region when the sixth pair of universal primers is designed is rrnL-Nad2, and the length of the amplified band is limited to 3600-4600bp), and degenerate sites are introduced according to the frequency of base occurrence at each site to increase the universality of the primers; finally, the primers are brought into each full sequence through Oligo7 for screening and adjustment to ensure that the primers meet the basic design principles and there is an overlap of more than 100 to 1200bp between the amplified bands, so as to facilitate sequence splicing in subsequent experiments; the synthesized primers are used to amplify the mitochondrial genome of the Funerary Betaeidae to detect its universality and amplification efficiency in the Funerary Betaeidae. During the process, multiple pairs of primers were designed. The experimental results showed that the 6 pairs of universal primers in this application were the best, as shown in Table 2; they corresponded to the Nad2-Cox2, Cox1-Nad3, Cox3-Nad4, Nad4-Cytb, Cytb-rrnS and rrnL-Nad2 regions, respectively. The specific positions are shown in the attached Figure 1 As shown;

[0073] Table 2 Sequence information of six universal primer pairs for mitochondrial genome of Eurycomycetes

[0074]

[0075]

[0076] 3. Use the designed and synthesized primers to amplify, sequence and splice the mitochondrial genome of the Funerary Betidae insects, and compare and detect its universality and amplification efficiency in the Funerary Betidae.

[0077] PCR amplification

[0078] Six pairs of primers were designed and the genome of a single Eurycoma beetle was used as a template for PCR amplification. The PCR reaction system was: 25 μl 2×KOD One TMPCR Master Mix, 3 μl upstream primer, 3 μl downstream primer, 2 μl DNA template, 17 μl double distilled water; PCR amplification conditions: 98℃ pre-denaturation for 3 min, 96℃ denaturation for 15 s, 50℃ annealing for 30 s, 68℃ extension for 3 min, amplification for 35-40 cycles, and finally extension at 68℃ for 10 min; PCR products were electrophoresed on 1% agarose gel. The gel electrophoresis patterns of Thanatophilus sinuatus (Fabricius, 1775), Thanatophilus roborowskyi (Jakovlev, 1887), Necrodes littoralis (Linnaeus, 1758), Necrophila (Calosilpha) brunnicollis (Kraatz, 1877), and Ptomascopus splagiatus (Ménétriés, 1854) are shown in the attached figure. Figure 2-3 As shown, the product size is close to the target band; in the figure: the primers for amplifying fragment 1 are TM-J211 and C2-N3390; the primers for amplifying fragment 2 are C1-J2196 and N3-N5729; the primers for amplifying fragment 3 are C3-J5526 and N4-N8728; the primers for amplifying fragment 4 are N4-J8109 and CB-N11337; the primers for amplifying fragment 5 are CB-J10942 and SR-N14748; the primers for amplifying fragment 6 are LR-J14612 and N2-N303.

[0079] 3.2. Sequencing of PCR amplified fragments

[0080] The base sequences of the 6 amplified fragments were sequenced.

[0081] 3.3. Splicing of PCR amplified fragments after sequencing

[0082] After verification, the sequencing results of the 6 fragments were segmented and spliced ​​using Geneious software. Since there was an overlapping region of 100 to 1200 bp between the amplified products when the universal primers were designed, the amplified fragments could be compared to find the repeated sequences; after removing the repeated sequences, the complete mitochondrial genome sequence was finally obtained.

[0083] 3.4. Comparison of the complete mitochondrial genome sequence after assembly

[0084] The splicing results were compared with the complete sequences of the family of beetles in the Genbank database. As shown in Table 3, the similarity reached more than 80%, further confirming that the universal primers of the present invention can indeed effectively amplify the complete mitochondrial genome of the family of beetles.

[0085] Table 3 Comparison results between the splicing results of this application and the complete sequences of the family Betygrinidae in the Genbank database

[0086] Species GenBank ID Pre.Ident Nepalese burial beetle Nicrophorus nepalensis Hope, 1831 NC_059900 74.57％ Necrodes littoralis (Linnaeus, 1758) NC_056196 81.76％ Diamesus osculans (Vigors, 1825) NC_045874 77.76％

[0087] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. Universal primers for the complete sequence of mitochondrial genome of the family Betyphaidae, characterized in that: The universal primers include 6 pairs of primers, corresponding to Nad2-Cox2, Cox1-Nad3, Cox3-Nad4, Nad4-Cytb, Cytb-rrnS and rrnL-Nad2 regions, respectively. In practical applications, sequence fragments of the corresponding regions can be amplified according to specific needs. The 6 pairs of primers are specifically: First pair of primers: TM-J211, whose sequence number is shown in SEQ ID.NO.1; C2-N3390, whose sequence number is shown in SEQ ID.NO.2; Second pair of primers: C1-J2196, whose sequence number is shown in SEQ ID.NO.3; N3-N5729, whose sequence number is shown in SEQ ID.NO.4; The third pair of primers: C3-J5526, whose sequence number is shown as SEQ ID.NO.5; N4-N8728, whose sequence number is shown in SEQ ID.NO.6; The fourth pair of primers: N4-J8109, whose sequence number is shown in SEQ ID.NO.7; CB-N11337, whose sequence number is shown as SEQ ID.NO.8; Fifth pair of primers: CB-J10942, whose sequence number is shown as SEQ ID.NO.9; SR-N14748, whose sequence number is shown as SEQ ID.NO.10; The sixth pair of primers: LR-J14612, whose sequence number is shown as SEQ ID.NO.11; N2-N303, whose sequence number is shown as SEQ ID.NO.12; The insects of the family Morcellidae are the Nepalese mortuary beetle Nicrophorus nepalensis, the coastal mortuary beetle Necrodeslittoralis, the horizontally striped shield mortuary beetle Diamesus osculans, the American mortuary beetle Necrophila americana, and the curved mortuary beetle Thanatophilus sinuatus.

2. The method for amplifying the complete sequence of the mitochondrial genome of the family of Betelgeuse insects according to claim 1, characterized in that: The specific steps include the following: Step 1, collection, identification and DNA extraction of the family of beetles: the collected specimens of the family of beetles are soaked in anhydrous ethanol and stored in a refrigerator at -20°C for standby use, a single beetle is selected for dissection, 30 mg of chest muscle is taken, 1 mol / L NaCl, pH=8, 1 mol / L Tris-HCl, pH=8, 1 mol / L EDTA are used to prepare a lysis solution, and then added to the insect muscle, proteinase K is added to extract the whole genome DNA, and the extracted whole genome DNA is stored at -20°C for standby use; Step 2, design of universal primers for mitochondrial genome amplification of the family of Eurypteridae: download all the currently published complete sequences of the mitochondrial genome of the family of Eurypteridae from the Genbank database; first, use Geneious software to compare 13 PCGs and 2 rRNA genes with uniform distribution and long length in the mitochondrial genome, and select 8 highly conserved genes with high gene identity and appropriate gene interval, namely Cox1, Cox2, Cox3, rrnS, rrnL, Nad4, Nad5 and Cytb genes, to find out the The conservative sequence region with the smallest base variation; then, according to the comparison results, Geneious software was used to design universal primers in the conservative sequence region. In order to ensure the high efficiency and amplification efficiency of the universal primers, the length of the amplified band was limited to 3-4.6Kb. At the same time, degenerate sites were introduced according to the frequency of base occurrence at each site to increase the universality of the primers; finally, the primers were brought into each full sequence through Oligo7 for screening and adjustment to ensure that the primers met the basic design principles and there was an overlap of more than 100-1200bp between the amplified bands, so that the subsequent experiments could be used for sequence splicing; Step 3, using the designed and synthesized primers to amplify, sequence and splice the mitochondrial genome of the family of beetles, and compare and detect its universality and amplification efficiency in the family of beetles; Step 3.1, PCR amplification: PCR amplification was performed using the designed 6 pairs of primers and the extracted genome of a single Eurycoma beetle as a template. The PCR reaction system was: 25 μL 2×KOD One TM PCR Master Mix, 3 μL upstream primer, 3 μL downstream primer, 2 μL DNA template, 17 μL double distilled water; PCR amplification conditions: 98°C pre-denaturation for 3 min, 96°C denaturation for 15 s, 50°C annealing for 30 s, 68°C extension for 3 min, amplification for 35-40 cycles, and finally 68°C extension for 10 min; PCR products were electrophoresed on 1% agarose gel; Step 3.2, PCR amplified fragment sequencing: sequencing the base sequences of the 6 amplified fragments; Step 3.3, splicing of PCR amplified fragments after sequencing: The sequencing results of the 6 fragments after verification are segmented and spliced ​​using Geneious software. Since there is an overlapping region of 100 to 1200 bp between the amplified products when designing universal primers, the amplified fragments can be compared to find the repeated sequence; after removing the repeated sequence, the complete mitochondrial genome sequence is finally obtained; Step 3.4, compare the complete mitochondrial genome sequence after splicing: compare the splicing result with the complete sequence of the family Betaeidae in the Genbank database.

3. The method for amplifying the complete sequence of the mitochondrial genome of the family of Betelgeuse insects according to claim 2, characterized in that: The conserved sequence region amplified when the first pair of primers is designed is Nad2-Cox2, and the length of the amplified band is limited to 3000-3300 bp.

4. The method for amplifying the complete sequence of the mitochondrial genome of the family of Betelgeuse insects according to claim 2, characterized in that: The conserved sequence region amplified by the second pair of primers is designed to be Cox1-Nad3, and the length of the amplified band is limited to 3500-3700 bp.

5. The method for amplifying the complete sequence of the mitochondrial genome of the family of Betelgeuse insects according to claim 2, characterized in that: The amplified conserved sequence region when the third pair of primers is designed is Cox3-Nad4, and the length of the amplified band is limited to 3100-3400 bp.

6. The method for amplifying the complete sequence of the mitochondrial genome of the family of Betelgeuse insects according to claim 2, characterized in that: The amplified conserved sequence region when the fourth pair of primers is designed is Nad4-Cytb, and the length of the amplified band is limited to 3200-3500bp.

7. The method for amplifying the complete sequence of the mitochondrial genome of the family of Betelgeuse insects according to claim 2, characterized in that: The amplified conserved sequence region when the fifth pair of primers is designed is Cytb-rrnS, and the length of the amplified band is limited to 3800-4000bp.

8. The method for amplifying the complete sequence of the mitochondrial genome of the family of Betelgeuse insects according to claim 2, characterized in that: The amplified conservative sequence region when the sixth pair of primers is designed is rrnL-Nad2, and the length of the amplified band is limited to 3600-4600bp.

9. The use of universal primers for the complete sequence of the mitochondrial genome of the family Betygrinidae according to claim 1, characterized in that: The universal primers are used in full sequence amplification and sequencing of mitochondrial genomes of the family Necrophoridae; the Necrophoridae insects include the Nepalese burial beetle Nicrophorus nepalensis, the coastal burial beetle Necrodes littoralis, the horizontally striped shield burial beetle Diamesus osculans, the American funeral beetle Necrophila americana, and the curved burial beetle Thanatophilus sinuatus.

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