Universal primers and amplification method for amplifying the whole mitochondrial genome of Acaridae

Abstract

The present invention provides universal primers and a method for amplifying the whole mitochondrial genome of mites from the family Acaridae. This invention, for the first time, provides a set of universal primers for amplifying the whole mitochondrial genome of mites from the family Acaridae. By dividing the mitochondrial genome into six overlapping fragments, conventional PCR amplification, Sanger sequencing, and sequence splicing are performed to rapidly obtain the complete mitochondrial genome sequence of mites from the family Acaridae. This method requires relatively low instrumentation and analytical equipment, making it easy to implement. Furthermore, the entire mitochondrial genome sequence can be obtained from the total DNA of a single individual, avoiding interference from the genomic DNA of multiple individuals. This method has been validated in the amplification of the mitochondrial genomes of six species of mites from the family Acaridae. This method is of great significance for obtaining more complete mitochondrial sequences of mites from the family Acaridae, further inferring the phylogenetic relationships between the genera of the family Acaridae, and the phylogenetic relationships between the families of the family Acaridae. It also provides a reference for studying the evolution and phylogenetic relationships of the Acari.

Description

Technical Field

[0001] The invention belongs to the field of molecular biotechnology, and particularly relates to a group of universal primers for amplifying the full mitochondrial sequence of Acaridae mites and an amplification method thereof. Background Art

[0002] The family Acaridae, belonging to the subclass Acariformes, order Sarcoptiformes, and order Astigmatina, is closely related to the quality of stored goods and human health, and holds significant economic and medical significance. Acaridae is the largest free-living group of Astigmatina, comprising over 500 known species in 79 genera. Common species include Acarus siro, Tyrophagus putrescentiae, Aleuroglyphus ovatus, Caloglyphus berlesei, Rhizoglyphus robini, and Thyreophagus entomophagus.

[0003] Mitochondrial genomes, characterized by their relatively small size, maternal inheritance, and high mutation rate, are widely used in research on population genetics, molecular evolution, comparative and evolutionary genomics. Currently, only 10 complete mitochondrial sequences of Acaridae mites have been released into GenBank. As a key group within the aspiracle family, more complete mitochondrial genome sequences of Acaridae mites are needed to be amplified and sequenced.

[0004] Studies have found that the mitochondrial genomes of all sequenced mites are closed circular double-stranded DNA, but they are more compact, especially in some species of the order True Acarina, which are generally 13 to 15 kilobases in length. Currently, the main sequencing strategies include shotgun high-throughput sequencing, direct sequencing of traditional PCR amplification products, and methods for deriving mitochondrial genome sequences from transcriptome data.

[0005] However, the sequencing cost required for shotgun high-throughput sequencing methods is high, the biological information splicing process is relatively complex, and mitochondrial pseudogenes derived from genomic DNA are easily mixed in, and control regions containing repeated fragments due to short read lengths are difficult to splice completely. In addition, because the mite body is tiny, about 0.5 mm in size, the total DNA obtained from a single individual is not enough to complete the library construction required for high-throughput sequencing. The primer walking method for obtaining mitochondrial genome sequences based on traditional PCR uses a large number of primers and has low amplification efficiency. If the number of primers is reduced, the difficulty of amplifying large fragments using long PCR will gradually increase. It is affected by the dual effects of primer universality and amplification efficiency, and it is often necessary to design specific primers for specific groups, or to redesign primers based on the measured fragments to amplify unknown regions, which greatly reduces the efficiency of obtaining mitochondrial genome sequences. The method of obtaining mitochondrial genome sequences from transcriptome data is expensive and also needs to be combined with traditional PCR methods to obtain the complete mitochondrial genome. However, the existing technology does not disclose universal primers for the complete mitochondrial genome sequence of the Acaridae family. Summary of the Invention

[0006] The purpose of the present invention is to provide universal primers for amplifying the whole mitochondrial genome of the Acaridae family. By comparing the mitochondrial genomes of the Acaridae family, conserved regions with minimal sequence variation are screened out, and 6 pairs of universal primers for the mitochondrial genomes of the Acaridae family are designed in the conserved regions. The amplified fragments are 1.2Kb to 3.8Kb in length, and there is an overlapping region of 100 to 600bp between the amplified products, covering the entire mitochondrial genome. There is no need to design specific primers for the Acaridae family, and the rapid amplification of the mitochondrial genome of a single individual can be achieved, so that the mitochondrial genome information of the Acaridae family can be obtained efficiently and at low cost.

[0007] Another object of the present invention is to provide an amplification method for amplifying the whole mitochondrial genome of Acaridae mites using universal primers.

[0008] The specific technical solutions of the present invention are as follows:

[0009] The present invention provides universal primers for amplifying the whole mitochondrial genome of Acaridae mites, including six pairs of universal primers corresponding to the Cox1-Cox3, Cox3-rrnL, rrnS-Nad4, Nad4-Nad5, Nad5-Cytb, and Cytb-Cox2 regions. In practical applications, sequence fragments of the corresponding regions can be amplified according to specific needs. Specifically, they are:

[0010] First pair of primers:

[0011] Cox1-F, as shown in SEQ ID NO: 1: 5'-CBCCTTTYCCWGCYCAYACT-3';

[0012] Cox3-R, as shown in SEQ ID NO: 2: 5'-TGDBAMCCYTCRTAACAACCC-3';

[0013] Second pair of primers:

[0014] Cox3-F, as shown in SEQ ID NO: 3: 5'-GTCCTTGRCCTATYTTDACTTCT-3';

[0015] rrnL-R, as shown in SEQ ID NO: 4: 5'-TAACTAATGATYTCGCTACCT-3';

[0016] The third pair of primers:

[0017] rrnS-F, as shown in SEQ ID NO: 5: 5'-ACATATCGCCCGTCRCTCTT-3';

[0018] Nad4-R, as shown in SEQ ID NO: 6: 5'-CGAAAAATMCCRTAACCCCC-3';

[0019] The fourth pair of primers:

[0020] Nad4-F, as shown in SEQ ID NO: 7: 5'-TGGTTCTATRATTYTKGCTGGGGT-3;

[0021] Nad5-R, as shown in SEQ ID NO: 8: 5'-AAGAAGAAACMGGAGTAGGAGC-3';

[0022] Fifth pair of primers:

[0023] Nad5-F, as shown in SEQ ID NO: 9: 5'-AGBTCTAGWTTRGAYTCTGG-3';

[0024] Cytb-R, as shown in SEQ ID NO: 10: 5'-CCMTGRGGRCAAATATCT-3';

[0025] Sixth pair of primers:

[0026] Cytb-F, as shown in SEQ ID NO: 11: 5'-GADGANCCTGKYTTGTGGAGT-3';

[0027] Cox2-R, as shown in SEQ ID NO: 12: 5'-ARACAAGAAAGYTCYCCYATT-3';

[0028] The amplification method of universal primers for amplifying the whole mitochondrial genome of Acaridae provided by the present invention is specifically as follows: using the above 6 pairs of universal primers, a 50 μl PCR system and PCR amplification conditions to amplify the target fragment.

[0029] The 50 μl PCR system specifically includes: 10 μl 5× Buffer, 4 μl dNTP Mix, 4 μl upstream primer, 4 μl downstream primer, 2 μl GXL enzyme, 4 μl DNA template, and 22 μl double-distilled water.

[0030] The PCR amplification conditions specifically include: pre-denaturation at 98°C for 3 minutes; denaturation at 98°C for 10 seconds, annealing at 48-55°C for 15 seconds, extension at 68°C for 1 minute 40 seconds, amplification for 30-35 cycles, and final extension at 68°C for 10 minutes.

[0031] Among them, the first pair of primers was used to amplify the region Cox1-Cox3, with an annealing temperature of 50°C and a product length of 1800-2100 bp;

[0032] The second pair of primers was used to amplify the region Cox3-rrnL, with an annealing temperature of 50°C and a product length of 2800-3100 bp;

[0033] The third pair of primers was used to amplify the region rrnS-Nad4, with an annealing temperature of 52°C and a product length of 3500-3800 bp;

[0034] The fourth pair of primers was used to amplify the region Nad4-Nad5, with an annealing temperature of 50°C and a product length of 1200-1500 bp;

[0035] The fifth primer pair was used to amplify the region Nad5-Cytb, with an annealing temperature of 48°C and a product length of 3300-3600 bp;

[0036] The sixth pair of primers was used to amplify the region Cytb-Cox2, with an annealing temperature of 50°C and a product length of 2300-2600 bp.

[0037] After amplifying the mitochondrial genomes of the thick-legged acarid mite, the elliptical acarid mite, the putrescent tyrophagous mite, the fan-zhang tyrophagous mite, the wood-loving mite and the insectivorous tyrophagous mite using the above amplification method, the PCR products were sequenced. The sequencing results were spliced using DNAMAN and compared with the corresponding full sequences in the Genbank database. It was found that the sequences were basically consistent, further confirming that the universal primers of the present invention can indeed effectively amplify the entire mitochondrial genome of the Acaridae family.

[0038] This invention provides, for the first time, a set of universal primers for amplifying the complete mitochondrial genome of Acaridae mites. By dividing the mitochondrial genome into six overlapping fragments, conventional PCR amplification, Sanger sequencing, and sequence splicing can rapidly obtain complete mitochondrial genome sequences of Acaridae mites. This method requires minimal instrumentation and analytical equipment, making it easy to implement. The complete mitochondrial genome sequence can be obtained from the total DNA of a single individual, avoiding interference from genomic DNA from multiple individuals. The method has been validated in amplifying the mitochondrial genomes of six Acaridae species. This method is of great significance for obtaining more complete mitochondrial sequences of Acaridae mites, further inferring the relationships between genera within the Acaridae family, and the phylogenetic relationships between aspiracle families. It also provides a reference for studying the evolution and phylogenetic relationships of the Acari.

[0039] Compared with the existing technology, the universal primers for amplifying the whole mitochondrial genome of the Acaridae family provided by the present invention require fewer primer pairs, can achieve rapid amplification of the mitochondrial genome of a single individual, and the amplified fragments are of moderate size. The universal primers for amplifying the whole sequence of the mitochondrial genome of the Acaridae family provided by the present invention have overlapping regions between adjacent amplified products, and the splicing accuracy is high. There is no need to design specific primers for the Acaridae family mites. The whole sequence information of the mitochondrial genome of the Acaridae family can be quickly obtained by Sanger sequencing. There is no need to involve a large amount of complex bioinformatics software and high-throughput sequence analysis processes, which saves time and greatly reduces costs. It has great application prospects in the sequencing of mitochondrial genomes of mites. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Schematic diagram of the design positions of the six pairs of universal primers of the present invention;

[0041] Figure 2 The electrophoresis diagram of the amplification of the powder mite by the primers SEQ ID NO.1 and SEQ ID NO.2 of the present invention;

[0042] Figure 3 The electrophoresis diagram of the amplification of the powder mite by the primers SEQ ID NO.3 and SEQ ID NO.4 of the present invention;

[0043] Figure 4 The electrophoresis diagram of the amplification of the powder mite by the primers SEQ ID NO.5 and SEQ ID NO.6 of the present invention;

[0044] Figure 5 The electrophoresis diagram of the amplification of the powder mite by the primers SEQ ID NO.7 and SEQ ID NO.8 of the present invention;

[0045] Figure 6The electrophoresis diagram of the amplification of the powder mite by the primers SEQ ID NO.9 and SEQ ID NO.10 of the present invention;

[0046] Figure 7 This is the electrophoresis diagram of the amplification of the powder mite by the primers SEQ ID NO.11 and SEQ ID NO.12 of the present invention;

[0047] in, Figure 2-Figure 7 Among them, 1 is the wood mite of Burke, 2 is the insectivorous narrow mite, 3 is the thick-legged flour mite, 4 is the putrescent tyrophagous mite, 5 is the fanzhang tyrophagous mite, 6 is the elliptical flour mite, and 7 is the 15000bp Maker. DETAILED DESCRIPTION

[0048] The following will provide a clear and complete description of the technical solutions in the embodiments of the present invention, in conjunction with the accompanying drawings. Obviously, the described embodiments are only a portion of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are also within the scope of protection of the present invention.

[0049] Example 1

[0050] Universal primers for amplification of the whole mitochondrial genome of Acaridae mites, specifically designed as follows:

[0051] 1) Isolation, identification and DNA extraction of individual Acaridae mites

[0052] The acarid samples used in the examples were all collected from storage environments in Wuhu. The acarid mites in the samples were isolated and cultured in pure culture. Ten or more adult mites were selected from the pure culture, washed with distilled water, dried, and fixed with a sealing fluid. Slide specimens were prepared and morphologically identified as Acaridae under a high-power microscope, and the specimens were registered. Ten or more adult mites were then selected from the pure culture, soaked in anhydrous ethanol, and stored in a refrigerator at 4°C for later use. A single mite was selected and crushed, and a lysis solution was prepared with NaCl (1 mol / L), Tris-HCl (pH = 8, 1 mol / L), and EDTA (pH = 8, 1 mol / L). The crushed mite was then added to a lysis solution, and proteinase K was added for PCR amplification to obtain the whole-genomic DNA of the Acaridae mite sample. The extracted whole-genomic DNA was stored at -20°C for later use.

[0053] 2) Design of universal primers for amplification of mitochondrial genomes of Acaridae mites:

[0054] All currently published complete mitochondrial genome sequences of Acaridae mites were downloaded from the Genbank database, as shown in Table 1. DNAMAN software was first used to align the 13 evenly distributed, long PCGs and two rRNA genes within the mitochondrial genome. Eight highly conserved genes with high gene identity and appropriate intergenic spacing were selected: Cox1, Cox2, Cox3, rrnS, rrnL, Nad4, Nad5, and Cytb. These genes were then further aligned using the MEME online program to identify conserved sequence regions with minimal base variation.

[0055] Table 1 The present invention uses the complete mitochondrial genome of the family Acaridae

[0056] Species Genbank ID Size(bp) Reference Aleuroglyphus ovatus KC700022 14328 Sun et al. 2014 Caloglyphus berlesei KF499016 14273 Sun et al. 2014 Robin root mite Rhizoglyphus robini MF596168 14244 Xue et al. 2018 Tyrophagus putrescentiae MK393792 14156 Fang et al. 2020 Tyrophagus fanetzhangorum MN560776 14257 Su et al. 2020 Scatoglyphus polytremetus MT133686 13966 Fang et al. 2021 Thyreophagus entomophagus OK166751 14242 Fang et al. 2022 Acarus siro OK040814 14402 Fang et al. 2022

[0057] According to the above comparison results, Primer Premier 5 was used to design universal primers in the conserved 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 1 to 4 Kb. At the same time, degenerate sites were introduced according to the frequency of base occurrence at each site shown by MEME to increase the universality of the primers. Finally, the primers were brought into each full sequence by Oligo7 for screening and adjustment to ensure that the primers met the basic design principles and that there was an overlap of more than 100 bp between the amplified bands so that subsequent experiments could perform sequence splicing. The synthesized primers were used to amplify the mitochondrial genome of the Acaridae family to detect its universality and amplification efficiency in the Acaridae family. In the process, multiple pairs of primers were designed, and the experimental results showed that the 6 pairs of universal primers of the present invention were the best, as shown in Table 2. They correspond to the Cox1-Cox3, Cox3-rrnL, rrnS-Nad4, Nad4-Nad5, Nad5-Cytb and Cytb-Cox2 regions, respectively. Figure 1 shown.

[0058] Table 2 Common primer sequences for mitochondrial genomes of Acaridae mites

[0059]

[0060] 3) Using the synthesized primers, we amplified, sequenced, and assembled the mitochondrial genomes of the Acaridae family to test their universality and amplification efficiency within the family.

[0061] 3-1) PCR amplification was performed using the six primer pairs described above, using the genome of a single Acaridae mite as a template. The PCR reaction system consisted of: 10 μl 5× Buffer, 4 μl dNTP Mix, 4 μl upstream primer, 4 μl downstream primer, 2 μl GXL enzyme, 4 μl DNA template, and 22 μl double-distilled water. PCR amplification conditions included: pre-denaturation at 98°C for 3 min, denaturation at 98°C for 10 s, annealing at 48-55°C for 15 s, extension at 68°C for 1 min 40 s, 30-35 cycles, and a final extension at 68°C for 10 min. The PCR product was electrophoresed on a 1% agarose gel. The gel electrophoresis pattern is shown below. Figure 2-Figure 7 As shown, the size of the obtained product was close to the target band.

[0062] 3-2) Sequencing and analyzing the amplified fragments, comparing the sequencing peaks, and after the base sequences of the six fragments are verified to be correct, splicing is performed; using the oval powder mite Aleuroglyphus ovatus as an example;

[0063] The sequencing results of the amplification using the first pair of primers SEQ ID NO.1-SEQ ID NO.2 are shown in SEQ ID NO.13.

[0064] The second pair of primers SEQ ID NO.3-SEQ ID NO.4 was used for amplification and sequencing, and the result is shown in SEQ ID NO.14.

[0065] The sequencing results of amplification using the third pair of primers SEQ ID NO.5-SEQ ID NO.6 are shown in SEQ ID NO.15.

[0066] The amplification and sequencing results using the fourth pair of primers SEQ ID NO.7-SEQ ID NO.8 are shown in SEQ ID NO.16.

[0067] The sequencing results of the amplification using the fifth pair of primers SEQ ID NO.9-SEQ ID NO.10 are shown in SEQ ID NO.17.

[0068] The sequencing results of the amplification using the sixth pair of primers SEQ ID NO.11-SEQ ID NO.12 are shown in SEQ ID NO.18.

[0069] 3-3) The six fragments were segmented and spliced using DNAMAN software. Since the universal primers used to design the amplified products had overlapping regions of 100 to 600 bp, the amplified fragments were aligned to identify duplicate sequences. After removing these duplicate sequences, a complete mitochondrial genome sequence was obtained. The result of segmented splicing of the six fragments amplified from the oval powdery mite Aleuroglyphus ovatus is shown in SEQ ID NO. 19 (SEQ ID NO: 19).

[0070] 3-4) The spliced results were compared with the complete sequences of Acaridae mites in the Genbank database. As shown in Table 1, the similarity reached over 99.85%, further confirming that the universal primers of the present invention can effectively amplify the complete mitochondrial genome of Acaridae mites.

[0071] Table 1 Comparison of the splicing results of this application with the complete sequences of Acaridae in the Genbank database

[0072]

[0073]

[0074] The above is a clear and complete description of the technical solutions in the embodiments of the present invention. The described embodiments are only a part of the embodiments of the present invention. It should be understood that the terms described in the present invention are only for describing specific implementation methods, and are not used to limit the present invention rather than all embodiments, and are not used to limit this application. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field by adjusting and optimizing the technical solutions of this example without making creative work should fall within the scope of protection of the present invention.

Claims

1. A method for amplifying the whole mitochondrial genome of Acaridae using universal primers for non-diagnostic purposes, characterized in that: Six pairs of universal primers were used to amplify the target fragments using a 50 μl PCR system under PCR amplification conditions; The universal primers for amplifying the whole mitochondrial genome of the family Acaridae include 6 pairs of universal primers corresponding to the Cox1-Cox3, Cox3-rrnL, rrnS-Nad4, Nad4-Nad5, Nad5-Cytb and Cytb-Cox2 regions, respectively. Specifically, First pair of primers: Cox1-F, the sequence of which is shown in SEQ ID NO: 1; Cox3-R, the sequence of which is shown in SEQ ID NO: 2; Second pair of primers: Cox3-F, the sequence of which is shown in SEQ ID NO: 3; rrnL-R, the sequence of which is shown in SEQ ID NO:4; The third pair of primers: rrnS-F, the sequence of which is shown in SEQ ID NO:5; Nad4-R, the sequence of which is shown in SEQ ID NO:6; The fourth pair of primers: Nad4-F, the sequence of which is shown in SEQ ID NO:7; Nad5-R, the sequence of which is shown in SEQ ID NO:8; Fifth pair of primers: Nad5-F, the sequence of which is shown in SEQ ID NO:9; Cytb-R, the sequence of which is shown in SEQ ID NO: 10; Sixth pair of primers: Cytb-F, the sequence of which is shown in SEQ ID NO: 11; Cox2-R, the sequence of which is shown in SEQ ID NO: 12; The 50 μl PCR system specifically includes: 10 μl 5× Buffer, 4 μl dNTP Mix, 4 μl upstream primer, 4 μl downstream primer, 2 μl GXL enzyme, 4 μl DNA template, and 22 μl double-distilled water; The PCR amplification conditions specifically include: pre-denaturation at 98°C for 3 minutes; denaturation at 98°C for 10 seconds, annealing at 48-55°C for 15 seconds, extension at 68°C for 1 minute 40 seconds, amplification for 30-35 cycles, and final extension at 68°C for 10 minutes.

2. The amplification method according to claim 1, characterized in that The first pair of primers was used to amplify the region Cox1-Cox3, the annealing temperature was 50°C, and the product length was 1800-2100 bp.

3. The amplification method according to claim 1, characterized in that The second pair of primers amplified the Cox3-rrnL region with an annealing temperature of 50°C and a product length of 2800-3100 bp.

4. The amplification method according to claim 1, characterized in that The third pair of primers was used to amplify the region rrnS-Nad4, with an annealing temperature of 52°C and a product length of 3500-3800 bp.

5. The amplification method according to claim 1, characterized in that The fourth pair of primers was used to amplify the region Nad4-Nad5, with an annealing temperature of 50°C and a product length of 1200-1500 bp.

6. The amplification method according to claim 1, characterized in that The fifth pair of primers was used to amplify the region Nad5-Cytb, with an annealing temperature of 48°C and a product length of 3300-3600 bp.

7. The amplification method according to claim 1, characterized in that The sixth pair of primers was used to amplify the Cytb-Cox2 region, with an annealing temperature of 50°C and a product length of 2300-2600 bp.

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