Procambarus clarkii mitochondrial gene specific amplification primers and application thereof

Abstract

The invention relates to a group of specific amplification primers for mitochondrial genes of procambarus clarkia and application thereof, and belongs to the technical field of molecular biology, and the mitochondrial genes of the procambarus clarkia comprise cytochrome c oxidase I subunit genes and cytochrome b genes. The specific amplification primer of the CO I gene comprises a CO I gene upstream primer and a CO I gene downstream primer, wherein the base sequences of the CO I gene upstream primer and the CO I gene downstream primer are respectively shown as SEQ NO.1 and SEQ NO.2; the specific amplification primer of the Cytb gene comprises a Cytb gene upstream primer and a Cytb gene downstream primer of which the base sequences are respectively as shown in SEQ NO.3 and SEQ NO.4. The invention further discloses a kit for detecting the Cytb gene. According to the amplification method of the mitochondrial gene of the procambarus clarkia, PCR (Polymerase Chain Reaction) amplification is carried out on a DNA (Deoxyribonucleic Acid) template solution of the procambarus clarkia by adopting amplification primers of a mitochondrial COI gene and a Cytb gene of the procambarus clarkia. The procambarus clarkia mitochondrial gene specific amplification primer has high specificity, a single target gene can be stably amplified, the length of the obtained procambarus clarkia mitochondrial COI gene segment reaches about 1000 bp, and the length of the obtained procambarus clarkia mitochondrial Cytb gene segment reaches about 800 bp.

Description

Technical Field

[0001] This invention relates to the field of molecular biology technology, and in particular to a set of primers for specific amplification of mitochondrial genes in Procambarus clarkii and their applications. Background Technology

[0002] Red swamp crayfish (Procambarus clarkii) Procambarus clarkii Crayfish, commonly known as freshwater crayfish, belongs to the genus *Prorocentrum* of the family Craniopoda in the order Malacostraca. Originally from North America, it was introduced to Nanjing, my country from Japan in the 1930s. Due to its high reproductive capacity and low water quality requirements, it is now widely distributed in many provinces along the Yangtze River. Crayfish is a high-protein, low-fat aquatic food rich in minerals and bioactive substances, and has become a popular consumer food in recent years. The scale of crayfish farming continues to expand. In 2023, my country's crayfish farming area reached 29.5 million mu (approximately 1.67 million hectares), with a yield of 3.161 million tons, representing year-on-year increases of 5.36% and 9.35%, respectively. Crayfish production accounted for 9.26% of the total freshwater aquaculture output in my country, ranking fourth among freshwater aquaculture species, after grass carp, silver carp, and bighead carp.

[0003] Existing technologies disclose the use of SSR molecular markers to analyze differences between different aquaculture populations of *Procambarus clarkii*, such as CN202011234812.4, "An SSR molecular marker for distinguishing specific geographical populations of *Procambarus clarkii* and its application." The present invention is characterized by: extracting genomic DNA from *Procambarus clarkii* populations from 14 geographical regions in China; designing *Procambarus clarkii* SSR primers based on *Procambarus clarkii* genome sequencing data; using the designed SSR primers to perform PCR amplification on collected samples to obtain the target fragment; detecting whether the target fragment has been amplified using agarose gel electrophoresis; further detecting the banding pattern of the PCR products using polyacrylamide gel electrophoresis; and performing statistical analysis of the banding patterns of the populations; and comparing the genotype distribution differences between samples using information from allele A or homozygous allele B contained at the PCM2076 locus, thereby enabling genotyping of *Procambarus clarkii* from different geographical populations. CN201510032853.8 discloses a method for preparing SSR primers for the red swamp crayfish (Procambarus clarkii), comprising five steps: 1. Extracting genomic DNA from the abdominal muscle of the red swamp crayfish; 2. Hybridizing the probe with the target fragment: hybridizing a biotin-labeled probe with the target fragment to obtain a hybridization product; 3. Enriching the hybridization product using magnetic beads; 4. Sequencing positive clones and designing primers based on microsatellite flanking sequences to amplify the genomic DNA; and 5. Detecting the SSR primers. This invention enables simple and rapid screening of microsatellite sequences throughout the entire genome of the red swamp crayfish, and allows for the development of a large number of SSR primers in a short time, providing a technical means for the germplasm conservation and genetic improvement of the red swamp crayfish. However, to date, no technical solution has been reported for amplifying the mitochondrial genes of the red swamp crayfish for its geographical population typing, species conservation, and analysis.

[0004] Compared to nuclear inheritance, the mitochondrial genome is characterized by rapid evolution, high abundance, a simple inheritance pattern, and a conserved, compact structure. Furthermore, mitochondrial inheritance exhibits maternal inheritance. Its mitochondrial genes primarily include the cytochrome c oxidase I subunit (…). CO I) Genes and cytochrome b gene ( Cytb These two genes, with their appropriate length and evolutionary rate, play a crucial role in species identification, phylogenetic studies, and population genetic diversity analysis.

[0005] Currently, there is a lack of primers with high specificity and high amplification efficiency for these two mitochondrial genes in the red swamp crayfish (Procambarus clarkii). Existing universal primers often suffer from non-specific amplification, primer dimers, or amplification failure. Therefore, developing primers specific to the red swamp crayfish is of great significance for improving detection accuracy and research efficiency. Summary of the Invention

[0006] The technical problem to be solved by this invention is to overcome the shortcomings of the prior art and provide a specific primer for amplifying the mitochondrial gene of Procambarus clarkii with high amplification efficiency, and to use the primer to amplify the mitochondrial gene of Procambarus clarkii, thereby improving the accuracy and efficiency of detecting the genetic characteristics and germplasm resources of Procambarus clarkii.

[0007] One of the technical solutions adopted by this invention to solve its technical problem is: A set of primers for specific amplification of the mitochondrial genes of *Procambarus clarkii*, the mitochondrial genes of *Procambarus clarkii* including the cytochrome c oxidase I subunit (… CO I) Genes and cytochrome b gene ( Cytb ) gene, the CO The specific amplification primers for gene I include the base sequence shown in SEQ NO. 1. CO The upstream primer and base sequence of gene I are shown in SEQ NO. 2. CO I gene downstream primer; the CO The primers for gene-specific amplification include base sequences as shown in SEQ NO.3. Cytb The upstream primer and base sequence of the gene are shown in SEQ NO.4. Cytb Downstream primers for genes.

[0008] Wherein: the sequence shown in SEQ NO.1 is: CO IF: 5'-GGAGTTGGAACAGGATGGACT-3'; The sequence shown in SEQ NO.2 is: CO IR: 5'-ACAAATCCTAACACAGCTACCA-3'; The sequence shown in SEQ NO.3 is: Cytb F: 5'-TCGAGACGTAAACTATGGGTGAC-3'; The sequence shown in SEQ NO.4 is: Cytb R: 5'-GGCGTGCTCCAATTCATGTT-3'.

[0009] Among them, the Cytb The upstream primer for the I gene corresponds to positions 355-375 in the mitochondrial genome sequence of the red swamp crayfish (JN991197) in GenBank. CO The downstream primer for gene I corresponds to positions 1400-1421 in the mitochondrial genome sequence of *Procambarus clarkii*, numbered JN991197 in GenBank; CO The upstream primer for the gene corresponds to positions 213-235 in the mitochondrial genome sequence of *Procambarus clarkii*, numbered JN991197 in GenBank. CytbThe downstream primer of the gene corresponds to the 1027-1046 sites in the mitochondrial genome sequence of Procambarus clarkia numbered as JN991197 in GenBank.

[0010] The mitochondrial genome of the Procambarus clarkia Cytb I gene and CO The gene amplification primer is used for PCR amplification of the DNA template solution of the Procambarus clarkia; and the fragment length of the I gene obtained after the PCR amplification is 1000-1100 bp; and the fragment length of the Cytb gene is 800-900 bp. Cytb I gene and

[0011] Another technical solution adopted by the present application to solve its technical problems is: The amplification method of the mitochondrial genome of the Procambarus clarkia adopts the mitochondrial genome of the Procambarus clarkia CO I gene and CO The gene amplification primer is used for PCR amplification of the DNA template solution of the Procambarus clarkia.

[0012] The amplification method of the mitochondrial genome of the Procambarus clarkia comprises the following steps: S1, obtaining a known complete sequence of the mitochondrial genome of the Procambarus clarkia, and locating the positions of the Cytb I gene and CO gene in the complete sequence; S2, primer design and synthesis: comparing the seven mitochondrial genome sequences of the Procambarus clarkia downloaded from GenBank by using MEGA software, observing by naked eyes and combining with primer design software primer3 Plus to design and evaluate the upstream and downstream primers in the 100-200 base pair conservative regions of the 5' and 3' ends of the C0I gene and the Cytb gene.

[0013] S3, DNA extraction: extracting DNA by using the marine animal tissue genomic DNA extraction kit (centrifugal column type) DP324 kit of Tian Gen Biochemical Technology (Beijing) Co., Ltd., detecting the integrity of the extracted DNA by using 1.2% agarose gel electrophoresis, detecting the concentration and purity by using a microspectrophotometer, and then diluting with sterilized water to 100 ng / μL for subsequent PCR amplification experiments, and storing the DNA template solution in a -20C refrigerator for standby; S4, amplifying the target gene: PCR amplifying the Cytb I gene and CO gene fragments of the Procambarus clarkia by using the primers synthesized in S2; S5, purifying and recovering the PCR amplification product obtained in S4, and performing forward and reverse bidirectional sequencing by using the same primers for amplification to obtain the target fragment.

[0014] S5 further comprises: using 1.2% agarose gel electrophoresis to detect the amplified product, and screening at least one set of amplification primers with single band and high amplification efficiency.

[0015] The PCR amplification reaction adopts a Touchdown program: 95 DEG C pre-denaturation for 5 min; then 95 DEG C denaturation for 30 s, 56-62 DEG C annealing for 30 s, 72 DEG C extension for 30 s, 10 cycles of repeating, and decreasing 1 DEG C each time; then 95 DEG C denaturation for 30 s, 52 DEG C annealing for 30 s, 72 DEG C extension for 30 s, 25 cycles of repeating; finally, 72 DEG C extension for 20 min.

[0016] In an exemplary embodiment, the PCR amplification system comprises sterilized water, 10x buffer, dNTP, MgCl2, upstream primer, downstream primer, Taq enzyme and DNA template, and specifically, the PCR amplification system is 50 muL, comprising: 31 muL of sterilized water, 5 muL of 10x buffer, 4 muL of 2.5 mmol / L dNTP, 3 muL of 25 mmol / L MgCl2, 1 muL of 10 mu mol / L upstream primer, 1 muL of 10 mu mol / L downstream primer, 0.2 muL of 5 U / muL Taq enzyme and 5 muL of 100 ng / muL DNA template solution.

[0017] In an exemplary embodiment, the DNA template solution is extracted by using a kit (such as: marine animal tissue genomic DNA extraction kit (centrifugal column type) DP324 of Tianguan Biochemical Technology (Beijing) Co., Ltd., or individual kit of Procambarus clarkii of Jiangsu Shufeng Aquatic Seed Industry Co., Ltd.), and is diluted to 100 ng / muL by using sterilized water.

[0018] Another technical solution adopted by the present application to solve the technical problems is: The application of the specific amplification primer of the mitochondrial gene of Procambarus clarkii in preparing a detection and analysis kit for the germplasm or regional population of Procambarus clarkii.

[0019] The beneficial effects of the set of specific amplification primers of the mitochondrial gene of Procambarus clarkii are: The specific amplification primer of the Procambarus clarkia mitochondrial gene is designed based on the mitochondrial genome of the Procambarus clarkia (such as GenBank accession number JN991197), can effectively avoid cross reaction with other species or non-target gene regions, has high specificity, and has the characteristics of single band and high amplification efficiency; the amplification primer can stably amplify a single and bright expected band under standard PCR conditions, without primer dimer or non-specific product, and has high amplification efficiency; the amplification primer can increase the length of the obtained mitochondrial COI gene fragment of the Procambarus clarkia to about 1000 bp, and the length of the Cytb gene fragment to about 800 bp; and can better meet the needs of subsequent mitochondrial genome research of the Procambarus clarkia and genetic diversity research of the Procambarus clarkia population.

[0020] The specific amplification primer of the Procambarus clarkia mitochondrial gene can efficiently amplify the Cytb I gene and CO gene in the mitochondrial gene of the Procambarus clarkia, and the amplification primer sequence is intermittent and accurately corresponds to the specific site of the Cytb I gene and CO gene of the Procambarus clarkia, which significantly improves the amplification accuracy and amplification efficiency of the Cytb I gene and CO gene; and the amplification primer can be used alone or in combination, and is suitable for conventional Taq DNA polymerase and commercial PCR reagent kits.

[0021] The specific amplification primer of the Procambarus clarkia mitochondrial gene can obtain specific mitochondrial genes, thereby improving the detection and analysis accuracy and efficiency of the Procambarus clarkia germplasm or regional population, and is suitable for mitochondrial gene typing in molecular identification (DNA barcoding), genetic diversity evaluation, population structure analysis, phylogenetic reconstruction and host interaction research (such as white spot syndrome virus) of the Procambarus clarkia, and has wide application. BRIEF DESCRIPTION OF DRAWINGS

[0022] Cytb Fig. 1 is a gel electrophoresis diagram of the amplification products of the mitochondrial genes of the Procambarus clarkia in Example 2 of the present application amplified by using three pairs of different primers; Figure 1 I gene; wherein D11, D12 and D13 are three parallel samples of the amplification products obtained by using the primers in Comparative Example 1; D21, D22 and D23 are three parallel samples of the amplification products obtained by using the primers in Comparative Example 2; and S11, S12 and S13 are three parallel samples of the amplification products obtained by using the primers in Example 1; CO Fig. 2 is a gel electrophoresis diagram of the amplification products of the mitochondrial genes of the Procambarus clarkia in Example 2 of the present application amplified by using three pairs of different primers; Figure 2Gel electrophoresis diagram of gene.

[0023] Wherein: D11, D12, D13 are three parallel samples of amplification products obtained by using primers in Comparative Example 1; D21, D22, D23 are three parallel samples of amplification products obtained by using primers in Comparative Example 2; S11, S12, S13 are three parallel samples of amplification products obtained by using primers in Example 1; Cytb The amplification product of the mitochondrial gene of the Procambarus clarkii in Example 3 is amplified by using the primers in Example 1 Figure 3 Gel electrophoresis diagram of gene I; Wherein the culture sites of the Procambarus clarkii: M: maker (molecular weight marker), S1: Honghu, S2: Panyanghu, S3: Egypt, S4: Dongtinghu, S5: Nanzhou Town of Nanchuan, S6: Qingsuzui Town of Nanchuan, S7: Sanshenhu Town of Nanchuan, S8: Maochaojie Town of Nanchuan, S9: Sijihong Town of Yuanjiang, S10: Shihu Town of Yuanjiang, S11: Nandadian Town of Yuanjiang, S12: Caoweizhen of Yuanjiang; CO The amplification product of the mitochondrial gene of the Procambarus clarkii in Example 3 is amplified by using the primers in Example 1 Figure 4 Gel electrophoresis diagram of gene.

[0024] Wherein the culture sites of the Procambarus clarkii: M: maker (molecular weight marker), S1: Honghu, S2: Panyanghu, S3: Egypt, S4: Dongtinghu, S5: Nanzhou Town of Nanchuan, S6: Qingsuzui Town of Nanchuan, S7: Sanshenhu Town of Nanchuan, S8: Maochaojie Town of Nanchuan, S9: Sijihong Town of Yuanjiang, S10: Shihu Town of Yuanjiang, S11: Nandadian Town of Yuanjiang, S12: Caoweizhen of Yuanjiang. DETAILED DESCRIPTION

[0025] The application will be further described below in combination with the drawings and examples.

[0026] DNA extraction kit: marine animal tissue genomic DNA extraction kit (centrifugal column type) DP324 of Tiangen Biosciences (Beijing) Co., Ltd.

[0027] Comparative Example 1

[0028] A set of mitochondrial gene specific amplification primers of the Procambarus clarkii, wherein the mitochondrial gene of the Procambarus clarkii comprises cytochrome c oxidase I subunit (COI) Cytb I) gene and cytochrome b gene (Cyt b) gene, wherein the specific amplification primer of the COI gene comprises the base sequence of CO Cytb I) gene and cytochrome b gene (Cyt b) gene, wherein the specific amplification primer of the COI gene comprises the base sequence of​CO I F1 5'-TAACGGGTGGAGTTAGGTCA-3' of the cytochrome c oxidase I subunit (COI) gene and a primer specific for amplifying the cytochrome b gene (Cytb) having a base sequence of CO I F2 5'-GAACTGCTATTGCTCATGCGG-3' of the cytochrome c oxidase I subunit (COI) gene and a primer specific for amplifying the cytochrome b gene (Cytb) having a base sequence of CO I R1 5'-CCGTTCCAAACACAGCCCCT-3' of the cytochrome c oxidase I subunit (COI) gene and a primer specific for amplifying the cytochrome b gene (Cytb) having a base sequence of CO I R2 5'-AGCGCAGGAGGATAAGAATGC-3' of the cytochrome c oxidase I subunit (COI) gene and a primer specific for amplifying the cytochrome b gene (Cytb) having a base sequence of CO I F1 5'-TAACGGGTGGAGTTAGGTCA-3' of the cytochrome c oxidase I subunit (COI) gene and a primer specific for amplifying the cytochrome b gene (Cytb) having a base sequence of Cytb I F2 5'-GAACTGCTATTGCTCATGCGG-3' of the cytochrome c oxidase I subunit (COI) gene and a primer specific for amplifying the cytochrome b gene (Cytb) having a base sequence of Cytb I R1 5'-CCGTTCCAAACACAGCCCCT-3' of the cytochrome c oxidase I subunit (COI) gene and a primer specific for amplifying the cytochrome b gene (Cytb) having a base sequence of Cytb I R2 5'-AGCGCAGGAGGATAAGAATGC-3' of the cytochrome c oxidase I subunit (COI) gene and a primer specific for amplifying the cytochrome b gene (Cytb) having a base sequence of

[0029] Comparative Example 2

[0030] A set of primers specific for amplifying the cytochrome c oxidase I subunit (COI) gene and the cytochrome b gene (Cytb) of the Procambarus clarkia mitochondrion, the cytochrome c oxidase I subunit (COI) gene and the cytochrome b gene (Cytb) including a base sequence of Cytb I F1 5'-TAACGGGTGGAGTTAGGTCA-3' of the cytochrome c oxidase I subunit (COI) gene and a primer specific for amplifying the cytochrome b gene (Cytb) having a base sequence of CO I F2 5'-GAACTGCTATTGCTCATGCGG-3' of the cytochrome c oxidase I subunit (COI) gene and a primer specific for amplifying the cytochrome b gene (Cytb) having a base sequence of Cytb I R1 5'-CCGTTCCAAACACAGCCCCT-3' of the cytochrome c oxidase I subunit (COI) gene and a primer specific for amplifying the cytochrome b gene (Cytb) having a base sequence of CO I R2 5'-AGCGCAGGAGGATAAGAATGC-3' of the cytochrome c oxidase I subunit (COI) gene and a primer specific for amplifying the cytochrome b gene (Cytb) having a base sequence of CO I F1 5'-TAACGGGTGGAGTTAGGTCA-3' of the cytochrome c oxidase I subunit (COI) gene and a primer specific for amplifying the cytochrome b gene (Cytb) having a base sequence of CO I F2 5'-GAACTGCTATTGCTCATGCGG-3' of the cytochrome c oxidase I subunit (COI) gene and a primer specific for amplifying the cytochrome b gene (Cytb) having a base sequence of CO I R1 5'-CCGTTCCAAACACAGCCCCT-3' of the cytochrome c oxidase I subunit (COI) gene and a primer specific for amplifying the cytochrome b gene (Cytb) having a base sequence of CO I R2 5'-AGCGCAGGAGGATAAGAATGC-3' of the cytochrome c oxidase I subunit (COI) gene and a primer specific for amplifying the cytochrome b gene (Cytb) having a base sequence of I F1 5'-TAACGGGTGGAGTTAGGTCA-3' of the cytochrome c oxidase I subunit (COI) gene and a primer specific for amplifying the cytochrome b gene (Cytb) having a base sequence of Cytb I F2 5'-GAACTGCTATTGCTCATGCGG-3' of the cytochrome c oxidase I subunit (COI) gene and a primer specific for amplifying the cytochrome b gene (Cytb) having a base sequence of Cytb I R1 5'-CCGTTCCAAACACAGCCCCT-3' of the cytochrome c oxidase I subunit (COI) gene and a primer specific for amplifying the cytochrome b gene (Cytb) having a base sequence of Cytb I R2 5'-AGCGCAGGAGGATAAGAATGC-3' of the cytochrome c oxidase I subunit (COI) gene and a primer specific for amplifying the cytochrome b gene (Cytb) having a base sequence of

[0031] Example 1

[0032] Referring to Cytb and Figure 1 A set of primers specific for amplifying the cytochrome c oxidase I subunit (COI) gene and the cytochrome b gene (Cytb) of the Procambarus clarkia mitochondrion, the cytochrome c oxidase I subunit (COI) gene and the cytochrome b gene (Cytb) including a base sequence of Figure 2 I F1 5'-TAACGGGTGGAGTTAGGTCA-3' of the cytochrome c oxidase I subunit (COI) gene and a primer specific for amplifying the cytochrome b gene (Cytb) having a base sequence of CO I F2 5'-GAACTGCTATTGCTCATGCGG-3' of the cytochrome c oxidase I subunit (COI) gene and a primer specific for amplifying the cytochrome b gene (Cytb) having a base sequence of CytbThe specific amplification primer of the I gene comprises a base sequence as shown in SEQ NO. 1 CO The upstream primer of the I gene comprises a base sequence as shown in SEQ NO. 2 CO The downstream primer of the I gene; the specific amplification primer of the CO The specific amplification primer of the I gene comprises a base sequence as shown in SEQ NO. 3 Cytb The upstream primer of the I gene comprises a base sequence as shown in SEQ NO. 4 Cytb The downstream primer of the I gene.

[0033] The sequence shown in SEQ NO. 1 is COIF: 5'-GGAGTTGGAACAGGATGGACT-3'; The sequence shown in SEQ NO. 2 is COIR: 5'-ACAAATCCTAACACAGCTACCA-3'; The sequence shown in SEQ NO. 3 is: Cytb F: 5'-TCGAGACGTAAACTATGGGTGAC-3'; The sequence shown in SEQ NO. 4 is: Cytb R: 5'-GGCGTGCTCCAATTCATGTT-3'.

[0034] The upstream primer of the I gene corresponds to the 355-375 sites in the mitochondrial genome sequence of Procambarus clarkia in GenBank numbered as JN991197, Cytb The downstream primer of the I gene corresponds to the 1400-1421 sites in the mitochondrial genome sequence of Procambarus clarkia in GenBank numbered as JN991197; the specific amplification primer of the CO The upstream primer of the I gene corresponds to the 213-235 sites in the mitochondrial genome sequence of Procambarus clarkia in GenBank numbered as JN991197, CO The downstream primer of the I gene corresponds to the 1027-1046 sites in the mitochondrial genome sequence of Procambarus clarkia in GenBank numbered as JN991197. Cytb

[0035] Example 2

[0036] An amplification method of a mitochondrial gene of Procambarus clarkia, which adopts the mitochondrial genome of Procambarus clarkia as described above Cytb I gene and CO The DNA template solution of Procambarus clarkia is subjected to PCR amplification by using the amplification primer of the I gene and the amplification primer of the S1, obtaining a known full sequence of a mitochondrial genome of Procambarus clarkia, and locating the I gene and the Cytb I gene and CO ​The location of the gene; specifically: landing in the National Center for Biotechnology Information (NCBI, http: / / www.ncbi.nlm.nih.gov / ) of the United States, downloading the existing whole sequence of the mitochondrial genome of Procambarus clarkii, and locating the positions of the COI gene and the Cytb gene.

[0037] S2, primer design and synthesis: the 7 mitochondrial genome sequences of Procambarus clarkii downloaded from GenBank were aligned using MEGA software, and the upstream and downstream primers were designed and evaluated by naked eye observation and combined with primer design software primer3 Plus in the 100-200 base pair conserved regions at the 5' and 3' ends of the COI gene and the Cytb gene. When designing primers, try to avoid the appearance of hairpin structures, primer dimers, etc.; Three groups of primers were designed and synthesized by Wuhan Tianyihuiyuan Biotechnology Co., Ltd.

[0038] Among them Cytb The 3 groups of primers of the I gene are respectively: The CO I primer shown in Comparative Example 1: CO I F1 5'-TAACGGGTGGAGTTAGGTCA-3' CO I R1 5'-CCGTTCCAAACACAGCCCCT-3' The CO I primer shown in Comparative Example 2: CO I F2 5'-GAACTGCTATTGCTCATGCGG-3' CO I R2 5'-AGCGCAGGAGGATAAGAATGC-3' The CO I primer shown in Example 1: CO I F: 5'-GGAGTTGGAACAGGATGGACT-3', CO I R: 5'-ACAAATCCTAACACAGCTACCA-3'; Among them CO The 3 groups of primers of the gene are respectively: The primer shown in Comparative Example 1: Cytb Cytb F1 5'-TTCGACGTAAACTATGGGTGACTAT-3 Cytb R1 5'-CTTTGCTCCAATTCATGTTAGT-3' The primer shown in Comparative Example 2: Cytb Cytb ​​F2 5'-GGACACATTTGTCGAGACGT-3' Cytb R2 5'-CCATTCTACTGGGCGTGCTC-3' Example 1 shown Cytb Primers: Cytb F: 5'-TCGAGACGTAAACTATGGGTGAC-3, Cytb R: 5'-GGCGTGCTCCAATTCATGTT-3'; S3. DNA Extraction: DNA was extracted from individuals of *Procambarus clarkii* from the aquaculture site in Sanxianhu Town, Nanxian County using the Marine Animal Tissue Genomic DNA Extraction Kit (Centrifugal Column Type) DP324 from Tiangen Biotech (Beijing) Co., Ltd. The integrity of the extracted DNA was detected by 1.2% agarose gel electrophoresis, and the concentration and purity were detected by micro-spectrophotometer. The DNA was then diluted with sterile water to 100 ng / μL for subsequent PCR amplification experiments. The DNA template solution was frozen at -20°C for later use. S4. Amplification of the target gene: Using primers synthesized in S2, PCR amplification was performed on individual red swamp crayfish. Cytb I gene and CO Gene fragments; The specific procedure is as follows: In a 50 μL PCR reaction system, synthesized primers are used to amplify the individual red swamp crayfish. Cytb I and CO The PCR reaction system for the gene fragment included: 31 μL sterile water (adjustable according to the amount of DNA template used), 5 μL 10X buffer, 4 μL dNTP (2.5 mmol / L), 3 μL MgCl2 (25 mmol / L), 1 μL each of forward and reverse primers (10 μmol / L), 0.2 μL Taq enzyme (5 U / μL), and 5 μL DNA template solution (100 ng / μL) (adjustable according to the extraction quality of the DNA template). To improve product specificity, the PCR reaction used a Touchdown program: 95℃ pre-denaturation for 5 min; then 95℃ denaturation for 30 s, 56-62℃ annealing for 30 s, and 72℃ extension for 30 s, repeated 10 times, decreasing the temperature by 1℃ each time; then 95℃ denaturation for 30 s, 52℃ annealing for 30 s, and 72℃ extension for 30 s, repeated 25 times; finally, 72℃ extension for 20 min.

[0039] S5. The amplified products were detected by 1.2% agarose gel electrophoresis. CytbThe gel electrophoresis samples of the I gene from left to right are the extension products of the primers shown in Example 1, Control Example 1 and Control Example 2, each sample has three parallel samples, and one gel electrophoresis sample hole is left between adjacent samples CO The electrophoretogram of the I gene fragment is shown in CO ; Figure 1 The gel electrophoresis samples of the I gene from left to right are the extension products of the primers shown in Example 1, Control Example 1 and Control Example 2, each sample has three parallel samples, and one gel electrophoresis sample hole is left between adjacent samples Cytb The electrophoretogram of the I gene fragment is shown in Cytb ; Figure 2 and Figure 1 It can be seen from the above that the primers in Control Examples 1 and 2 do not effectively amplify the mitochondrial DNA of the Procambarus clarkii, and the primers shown in Example 1 effectively amplify the mitochondrial DNA of the Procambarus clarkii Figure 2 I gene and CO gene, and after the primers shown in Example 1 effectively amplify the mitochondrial DNA of the Procambarus clarkii Cytb I gene and CO gene, a single band of the gene fragment is obtained.

[0040] It can be seen from the above experiment that the primers shown in Example 1 can effectively amplify the mitochondrial DNA of the Procambarus clarkii Cytb I gene, CO gene, and the amplification efficiency is high, and the obtained product is single and bright, without primer dimer or non-specific product. In order to verify whether the above primers are suitable for amplifying the mitochondrial DNA of the Procambarus clarkii from different breeding places, the applicant further develops the amplification experiment of the mitochondrial DNA of the Procambarus clarkii from 12 different breeding places by using the above primers Cytb I gene, CO gene. Cytb I gene, CO gene.

[0041] Example 3

[0042] A method for amplifying the mitochondrial DNA of the Procambarus clarkii, which adopts the mitochondrial DNA amplification primers of the Procambarus clarkii shown in the above Cytb I gene and CO gene to perform PCR amplification on the DNA template solution of the Procambarus clarkii; and specifically includes the following steps: S1, obtaining the known complete sequence of the mitochondrial genome of the Procambarus clarkii, and locating the Cytb I gene and COThe location of the genes; specifically: landing in the United States National Center for Biotechnology Information (NCBI, http: / / www.ncbi.nlm.nih.gov / ), downloading the existing whole sequence of the mitochondrial genome of Procambarus clarkii, and locating the positions of the COI gene and the Cytb gene therein.

[0043] S2, primer design and synthesis: the designed primers are as described in Example 1, and are synthesized by Wuhan Tianyihuiyuan Biotechnology Co., Ltd.

[0044] S3, DNA extraction: the DNA of Procambarus clarkii from 12 different breeding sites, i.e., Honghu Lake, Panyang Lake, Egypt, Dongting Lake, Nanzhou Town, Qingshuzui Town, Sanchenhu Town, Maoqiao Street Town, Sijihong Town, Sihushan Town, Nandadaocan Town, Caoweizhen Town of Yuanjiang, is extracted by using the marine animal tissue genomic DNA extraction kit (centrifugal column type) DP324 kit of Tian Gen Bio-Engineering Co., Ltd. (Beijing), and the integrity of the extracted DNA is detected by 1.2% agarose gel electrophoresis, and the concentration and purity are detected by a microspectrophotometer, and then the DNA template solution is diluted to 100 ng / μL with sterilized water for subsequent PCR amplification experiments, and the DNA template solution is stored in a -20C refrigerator for standby; S4, amplification of target genes: the primers synthesized in S2 are used to amplify the Cytb I gene and CO gene fragments of Procambarus clarkii individuals; Specifically: in a 50 μL PCR reaction system, the primers synthesized are used to amplify the Cytb I and CO gene fragments of Procambarus clarkii individuals, and the PCR reaction system includes: sterilized water 31 μL (which can be appropriately adjusted according to the amount of DNA template), 10X buffer 5 μL, dNTP (2.5 mmol / L) 4 μL, MgC12 (25 mmol / L) 3 μL, upper and lower stream primers (10 μmol / L) 1 μL each, Taq enzyme (5 U / μL) 0.2 μL, DNA template solution (100 ng / μL) 5 μL (which can be appropriately adjusted according to the extraction quality of the DNA template). In order to improve the specificity of the product, the PCR reaction adopts the Touchdown program: 95°C pre-denaturation for 5 min; then 95°C denaturation for 30 s, 56-62°C annealing for 30 s, 72°C extension for 30 s, repeated for 10 cycles, and reduced by 1°C each time; then 95°C denaturation for 30 s, 52°C annealing for 30 s, 72°C extension for 30 s, repeated for 25 cycles; finally, 72°C extension for 20 min.

[0045] S5, the amplified product is detected by 1.2% agarose gel electrophoresis, and the mitochondrial CytbElectrophoresis diagram of the I gene fragment is shown below. CO As shown, the mitochondria of the red swamp crayfish Figure 3 Electrophoresis diagram of gene fragments as shown Cytb As shown. The PCR amplification product obtained from S4 was purified and recovered (sent to Wuhan Tianyi Huiyuan Biotechnology Co., Ltd.), and bidirectional sequencing was performed using the same primers as for amplification to obtain the target fragment.

[0046] PCR amplification Figure 4 The target fragment designed with primers for gene I is approximately 1000 bp in length. After sequencing, and by deleting inaccurate bases at the beginning and end of the sequence, a sequence of approximately 1000 bp can be obtained for subsequent analysis. CO The target fragment length of the gene primer design is approximately 800 bp; the amplification of S1-S12... Cytb I gene obtained after sequencing CO Representative nucleotide sequences of gene I are shown in SEQ NO.5~SEQ NO.16, and the amplification of S1-S12 are as follows. CO Genes obtained after sequencing Cytb Representative nucleotide sequences of the gene are shown in SEQ NO.17-28. Comparative analysis of the amplified product's nucleotide sequence with the NCBI database revealed that the amplified product is indeed the desired mitochondrial gene from the red swamp crayfish. Cytb I gene and CO Gene.

[0047] As can be seen from the above, the primers for specific amplification of mitochondrial genes in *Procambarus clarkii* involved in this invention can target the mitochondrial genes of *Procambarus clarkii* cultured in different locations, including... Cytb I gene and CO Cytb Efficient gene amplification to obtain bright, single target gene fragments can help in the molecular identification of different species of Procambarus clarkii, assessment of genetic diversity, monitoring and analysis of population structure, phylogenetic reconstruction, and mitochondrial genotyping in the study of pathogen (such as white spot syndrome virus) and host interaction.

[0048] Therefore, the applicant proposed the application of a set of primers for the specific amplification of mitochondrial genes in Procambarus clarkii in the preparation of a kit for the detection and analysis of Procambarus clarkii germplasm or regional populations.

[0049] This invention is not limited to the specific embodiments described above, and various modifications and variations are possible. Any modifications, equivalent substitutions, or improvements made to the above embodiments based on the technical essence of this invention should be included within the scope of protection of this invention.

Claims

1. A set of specific primers for amplification of Procambarus clarkia mitochondrial genes, characterized in that, The mitochondrial genes of the red swamp crayfish include the cytochrome c oxidase I subunit (… CO I) Genes and cytochrome b gene ( Cytb ) gene, the CO The specific amplification primers for gene I include the base sequence shown in SEQ NO.

1. CO The upstream primer and base sequence of gene I are shown in SEQ NO.

2. CO I gene downstream primer; the Cytb The primers for gene-specific amplification include base sequences as shown in SEQ NO.

3. Cytb The upstream primer and base sequence of the gene are shown in SEQ NO.

4. Cytb Downstream primers for genes.

2. The Procambarus clarkia mitochondrial gene-specific primer of claim 1, wherein, The CO The upstream primer of the COI gene corresponds to positions 355-375 in the mitochondrial genome sequence of Procambarus clarkia in GenBank with the number JN991197, CO The downstream primer of the COI gene corresponds to positions 1400-1421 in the mitochondrial genome sequence of Procambarus clarkia in GenBank with the number JN991197; the Cytb The upstream primer of the COI gene corresponds to positions 213-235 in the mitochondrial genome sequence of Procambarus clarkia in GenBank with the number JN991197, Cytb The downstream primer of the COI gene corresponds to positions 1027-1046 in the mitochondrial genome sequence of Procambarus clarkia in GenBank with the number JN991197.

3. The Procambarus clarkia mitochondrial gene-specific primer according to claim 1 or 2, characterized by The procambarus clarkia mitochondrion CO I gene and Cytb Gene amplification primers for PCR amplification of a DNA template solution of procambarus clarkia; Obtained after PCR amplification CO The fragment length of the I gene is 1000bp-1100bp; the fragment length of the Cytb gene is 800bp-900bp.

4. A method of amplifying a Procambarus clarkia mitochondrial gene, characterized by, The DNA template solution of the Procambarus clarkia is subjected to PCR amplification by using the Procambarus clarkia mitochondrial gene-specific amplification primer according to any one of claims 1-3, wherein the Procambarus clarkia mitochondrial gene-specific amplification primer comprises CO an I gene amplification primer, and Cytb a gene amplification primer.

5. The method for amplifying the mitochondrial gene of *Procambarus clarkii* as described in claim 4, characterized in that, The amplification method of the Procambarus clarkia mitochondrial gene comprises the following steps: S1, obtaining a known whole sequence of a mitochondrial genome of a procambarus clarkii, and locating positions of CO I gene and Cytb genes; S2, primer design and synthesis: the downloaded seven mitochondrial genome sequences of Procambarus clarkia from GenBank are aligned by MEGA software, the upstream and downstream primers are designed by naked eye observation and combined with primer design software primer3 Plus in the 100-200 base pair conservative regions of the 5' and 3' ends of the COI gene and the Cytb gene, and the primers are evaluated; S3, DNA extraction; S4, amplifying the target gene: the primers synthesized in S2 are used to amplify the individual of Procambarus clarkii CO I gene and Cytb gene fragment; S5, the PCR amplification product obtained in S4 is purified and recovered, and the same primers as amplification are used for forward and reverse bidirectional sequencing to obtain the target fragment.

6. The method of claim 5, wherein the primer set is: 5'-GTTTATACCGTGCTGGTTG-3' (SEQ ID NO: 1) and 5'-GTTTATACCGTGCTGGTTG-3' (SEQ ID NO: 2). In S4, the PCR amplification reaction adopts the Touchdown program: 95℃ pre-denaturation for 5min; then 95℃ denaturation for 30s, 56-62℃ annealing for 30s, 72℃ extension for 30s, 10 cycles of repetition, 1℃ reduction each cycle; then 95℃ denaturation for 30s, 52℃ annealing for 30s, 72℃ extension for 30s, 25 cycles of repetition; finally 72℃ extension for 20min.

7. The method for amplifying the mitochondrial gene of *Procambarus clarkii* as described in claim 5, characterized in that, The PCR amplification system is 50μL, including: 31μL of sterile water, 5μL of 10×buffer, 4μL of 2.5mmol / L dNTP, 3μL of 25mmol / L MgCl2, 1μL of 10μmol / L upstream and downstream primers respectively, 0.2μL of 5U / μL Taq enzyme, and 5μL of 100ng / μL DNA template solution.

8. The method of claim 5, wherein the Procambarus clarkia mitochondrial gene is amplified by the primer set of SEQ ID NO: 1 and SEQ ID NO:

2. In S3, the DNA template solution is extracted by a kit and diluted with sterile water to 100ng / μL, and the DNA template solution is stored in a-20C refrigerator for standby.

9. The method of claim 5 to 8 for amplifying the Procambarus clarkii mitochondrial gene, wherein, S5 further comprises: the amplified product is detected by 1.2% agarose gel electrophoresis, and at least one set of amplification primers with single band and high amplification efficiency is screened out.

10. The application of the Procambarus clarkia mitochondrial gene specific amplification primer in the preparation of a Procambarus clarkia germplasm or regional population detection analysis kit according to any one of claims 1-3.

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