Cotton rat species specific primer designed based on mitochondrial D-loop region

Abstract

The invention relates to the technical field of biology, in particular to a cotton rat species specific primer designed based on a mitochondrial D-loop region. The specific primer comprises a forward primer, a reverse primer, a forward primer, a reverse primer and a reverse primer, wherein the forward primer is GGACATAGGAAGGAAG; and a reverse primer: TAACTATAAGGCTAGGACC. The kit is characterized in that the kit is used for detecting the disease resistance of the human The specific primer provided by the invention can be specifically combined with a target sequence of a cotton rat mitochondrial D-loop region, a DNA fragment of the region is amplified through a PCR reaction, the fragment covers the complete D-loop region of a cotton rat mitochondrial genome, and species specificity identification of cotton rats can be realized through sequencing and sequence comparison. Meanwhile, a detection product for identifying cotton rat species can be prepared based on the primer sequence.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a cotton rat species-specific primer designed based on a mitochondrial D-loop region. Background Art

[0002] Mitochondrial DNA (mtDNA) is a molecular marker widely used in evolutionary biology, population genetics and forensic research. Due to its unique maternal inheritance mode, high mutation rate and relatively simple genome structure, mitochondrial DNA is of great significance in species identification and kinship studies.

[0003] In the mitochondrial genome, the D-loop region (control region) is one of the most variable regions, and its nucleotide sequence and length variation provide rich information between different species. Compared with the nuclear genome, the mitochondrial genome sequence is shorter and contains highly variable regions and conserved regions, making it an ideal choice for classification and identification of species at different evolutionary levels. The high variability of the D-loop region exists not only in inter-species differences, but also in intra-species individual variation, and its reliability for species identification has been verified by many studies.

[0004] Cotton rat (Sigmodon hispidus) is a common experimental animal, and its mitochondrial genome has been fully published in the NCBI database (NC_035572.1). In order to accurately identify cotton rats and their subspecies, it is necessary to develop primers that can specifically amplify the D-loop region of cotton rat mitochondria. Summary of the invention

[0005] The present invention aims to disclose a cotton rat species-specific primer designed based on the mitochondrial D-loop region.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] A cotton rat species-specific primer designed based on the mitochondrial D-loop region, the primer comprising the following sequence:

[0008] Forward primer: GGACATCAGGAAGGAAAGA;

[0009] Reverse primer: TAACTATAAGGCTAGGACC.

[0010] The present invention also provides the use of the cotton rat species-specific primers as described above in amplifying the complete D-loop region of the cotton rat mitochondrial genome.

[0011] Furthermore, the application method of the application comprises the following steps:

[0012] S1: Extraction of genomic DNA from cotton rats:

[0013] S2: The extracted genomic DNA was amplified using cotton rat species-specific primers to obtain a complete D-loop region sequence fragment;

[0014] S3: D-loop region sequence fragments were detected by agarose gel electrophoresis;

[0015] S4: performing nucleic acid sequence determination on the samples that pass the test;

[0016] S5: The determined sequence was compared with the mitochondrial D-loop region sequence of closely related species of cotton rats to construct a phylogenetic tree and complete the species-specific identification of cotton rats.

[0017] Furthermore, in step S5, the cotton rat-related species include mice, rats, hamsters, guinea pigs, voles, dormice, and squirrels.

[0018] Furthermore, the D-loop region sequence fragment in step S2 includes a fragment from tRNA-Pro to 12S rRNA on mitochondria.

[0019] The present invention also provides the use of the cotton rat species-specific primers described above in preparing a detection product for identifying cotton rat species.

[0020] Furthermore, the detection product includes a kit and a probe.

[0021] The present invention has the following beneficial effects:

[0022] 1. The primers designed by the present invention can specifically bind to and amplify the target fragment of the D-loop region of cotton rat mitochondria, and the amplification results are highly accurate and reliable. It is not only suitable for the identification of cotton rat species, but also can be applied to the fields of evolutionary biology, population genetics and forensic medicine, providing a reliable molecular tool for related scientific research.

[0023] 2. The amplified product can be directly sequenced and analyzed without further subcloning, which has high experimental efficiency, reduces experimental materials and steps, and saves costs.

[0024] 3. The design combines the information of conserved regions of the mitochondrial genome of various rodents, and has good cross-species universal characteristics. It can be studied and further designed for the research and identification of other related species. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0026] Figure 1It is a schematic diagram of Primer software design of the present invention;

[0027] Figure 2 It is the PCR amplification result diagram of the present invention;

[0028] Figure 3 This is an overview of the alignment results between the amplified sequence and the cotton rat mitochondrial genome sequence (NC_035572.1);

[0029] Figure 4 This is the paiwise diagram of the alignment results between the amplified sequence and the cotton rat mitochondrial genome sequence (NC_035572.1);

[0030] Figure 5 This is the phylogenetic tree diagram of the sequences obtained by this method and the closely related species of cotton rat. DETAILED DESCRIPTION

[0031] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0032] Embodiment 1:

[0033] Embodiment 1 provides a specific primer for the D-loop region of cotton rat mitochondria, and the design process of the primer includes the following steps:

[0034] According to the mitochondrial genome sequence of cotton rat on NCBI (NC_035572.1 Sigmodon hispidus mitochondrion, complete genome), a fragment containing complete coverage of the D-loop region was selected as the target fragment for primer design. The intended amplified fragment included a fragment of about 1163 bp from tRNA-Pro to 12S rRNA on the mitochondria.

[0035] like Figure 1 As shown, primers were designed using Primer software, upstream and downstream primers were searched separately, and hairpin structures, primer dimers and primer pairs with few mismatches were selected as test primers for experimental testing, and finally a pair of ideal primers was obtained, and the length of the amplified fragment was about 1163bp.

[0036] The primer sequences are as follows:

[0037] F: GGACATCAGGAAGGAAAGA (as shown in SEQ ID NO: 1)

[0038] R:TAACTATAAGGCTAGGACC (as shown in SEQ ID NO: 2)

[0039] The target sequence is as follows:

[0040] GGACATCAGGAAGGAAAGA GGTCCTAGCCTTATA GTTA (as shown in SEQ ID NO:3)

[0041] Embodiment 2:

[0042] Example 2 The cotton rat species-specific primers designed based on the mitochondrial D-loop region in Example 1 were applied to the amplification of the cotton rat mitochondrial D-loop region to verify the feasibility of the primers. The specific verification process includes:

[0043] Genomic DNA was extracted from cotton rat blood samples, and the target fragment was amplified using designed primers, followed by agarose gel electrophoresis, gel excision, and sequencing.

[0044] PCR amplification results are as follows Figure 2 shown.

[0045] The D-loop region of cotton rat mitochondria was sequenced. The sequencing results were spliced ​​to obtain the following sequence:

[0046] >MSD-loop

[0047]

[0048] The determined sequence was compared using NCBI's Blast, and the comparison results showed that the similarity with the cotton rat mitochondrial genome sequence in the database (NC_035572.1 Sigmodon hispidus mitochondrion, complete genome) was 97.26%.

[0049] The specific comparison results are as follows: Figure 3 and Figure 4 shown.

[0050] The sequences of this study and the sequences of closely related rodent species downloaded from GenBank mainly include NC011638.1 yellow-breasted rat (Rattus tanezumi1), NC 012374.1 black house mouse (Rattus rattus), NC 012389.1 Burmese rat (Rattus exulans), AC 000022.2 Wistar rat Rattus noregicus strain Wistar, NC012461.1 New Guinea rat (Rattus praetor), NC005089.1 house mouse (Mus musculus), NC006914.1 Western European house mouse (Mus musculus.domesticus), NC 012387.1 small house mouse (Mus musculus castaneus), NC 006915.1 Japanese mouse (Mus musculus molossinus), NC 010339.1 Eastern European house mouse (Mus musculus musculus), NC_035572.1 cotton rat (Sigmodon hispidus1), NC_003041.1 field mouse (Microtus kikuchii), NC_007936.1 Chinese hamster (Cricetulus griseus), NC_013068.1 giant hamster (Tschershia triton), NC_001892.1 fat dormouse (Glis glis), NC_002658.1 giant vine mouse (Thryonomys swinderianus), NC_000884.1 guinea pig (Caviaporcellus), NC_009056.1 scaly-tailed squirrel (Anomalurus sp.GP2005) and NC005314 African jumping mouse (Jaculus) were aligned using MEGA 6.0, and the phylogenetic tree was established using the maximum likelihood method. The results showed that the cotton rat D-loop sequence obtained by this method can be well distinguished from closely related species.

[0051] Specific results such as Figure 5 As shown in the figure. The D-loop region is the region with the largest variation in nucleic acid sequence and length in the entire mitochondrial genome, and is the main reason for the variation in mitochondrial genome length. The copy number varies not only between species, but also between individuals within a species, and its reliability for species identification has been proven by many studies. Therefore, compared with the huge and complex eukaryotic genome, the mitochondrial genome has a simple structure, a shorter sequence, and contains both conserved and highly variable regions, so it is suitable for the classification and identification of species at different levels of evolution.

[0052] In summary, the method of amplifying and sequencing the mitochondrial D-loop region proved that the amplified sample had the highest similarity with the cotton rat (Sigmodon hispidus), reaching 97.26%, and the cotton rat D-loop sequence obtained by this method can be used for the identification of cotton rat species through phylogenetic tree analysis.

[0053] Embodiment three:

[0054] Example 3 provides another application of the cotton rat species-specific primers designed based on the mitochondrial D-loop region in Example 1, specifically using it in the preparation of detection products for identifying cotton rat species, and the detection products include but are not limited to kits, probes, etc.

[0055] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A cotton rat species-specific primer designed based on the mitochondrial D-loop region, characterized in that: The primers include the following sequences: Forward primer: GGACATCAGGAAGGAAAGA; Reverse primer: TAACTATAAGGCTAGGACC.

2. Use of the cotton rat species-specific primers as claimed in claim 1 in amplifying the complete D-loop region of the cotton rat mitochondrial genome.

3. The use according to claim 2, characterized in that The application method of the application comprises the following steps: S1: Extraction of genomic DNA from cotton rats: S2: The extracted genomic DNA was amplified using cotton rat species-specific primers to obtain a complete D-loop region sequence fragment; S3: D-loop region sequence fragments were detected by agarose gel electrophoresis; S4: performing nucleic acid sequence determination on the samples that pass the test; S5: The determined sequence was compared with the mitochondrial D-loop region sequence of closely related species of cotton rats to construct a phylogenetic tree and complete the species-specific identification of cotton rats.

4. The use according to claim 3, characterized in that: In step S5, the cotton rat-related species include mice, rats, hamsters, guinea pigs, voles, dormice, and squirrels.

5. The use according to claim 3, characterized in that: The D-loop region sequence fragment in step S2 includes the fragment from tRNA-Pro to 12S rRNA on mitochondria.

6. Use of the cotton rat species-specific primers according to claim 1 in preparing a detection product for identifying cotton rat species.

7. The use according to claim 6, characterized in that: The detection product includes a test kit and a probe.

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