CN109112190B

CN109112190B - Corbicula fluminea mitochondrial genome enrichment extraction and identification method

Info

Publication number: CN109112190B
Application number: CN201811050921.3A
Authority: CN
Inventors: 张彤晴; 殷稼雯; 唐晟凯; 李大命; 刘小维; 刘燕山; 谷先坤
Original assignee: Freshwater Fisheries Research Institute of Jiangsu Province
Current assignee: Freshwater Fisheries Research Institute of Jiangsu Province
Priority date: 2018-09-10
Filing date: 2018-09-10
Publication date: 2021-09-17
Anticipated expiration: 2038-09-10
Also published as: CN109112190A

Abstract

The invention provides a method for enriching and extracting corbicula fluminea mitochondrial whole genome DNA and identifying corbicula fluminea through mitochondrial genes. The enrichment and extraction method provided by the invention can successfully obtain the sufficient mitochondrial genome DNA of corbicula fluminea. The primer pair Mit1 of the nad2-cox3 gene is designed according to the sequence of corbicula fluminea mitochondria, and the primer pair Mit2 designed according to the cob gene fragment, wherein the two primer pairs provided by the invention have strong specificity, can specifically amplify the sequence of corbicula fluminea mitochondria, but cannot amplify the gene fragment of other species mitochondria, has the advantages of good amplification performance, good discrimination, less generated byproducts and the like, and can identify the corbicula fluminea species.

Description

Corbicula fluminea mitochondrial genome enrichment extraction and identification method

Technical Field

The invention belongs to the technical field of molecular biology, and relates to a corbicula fluminea mitochondrial genome enrichment extraction and identification method, in particular to a method for specifically identifying corbicula fluminea mitochondria by using a specific primer pair.

Background

Corbicula sp also called Corbicula, Rapana, crab, etc., belonging to the order of Bispidae, true-petalobranchiales, Corbicula. Corbicula fluminea is a bivalve aquatic organism with strong environmental adaptability and is widely distributed in China, Korea, Japan and south-east Asia. In inland waters of China, corbicula fluminea is caved in the surface layer of sediment of water bottom, takes plankton as food, grows fast, has strong reproductive capacity, and is also suitable for artificial breeding. The corbicula fluminea meat is rich in nutrition, can be eaten fresh or used as a medicine, has the effects of appetizing, improving eyesight, promoting urination and the like, and is an important freshwater economic aquatic organism in China. At present, the research work on corbicula fluminea focuses on aspects of environmental adaptability, biomass, diversity and the like, but the research on molecular biology of corbicula fluminea is relatively less, and the research on mitochondrial complete genome or nuclear genome of corbicula fluminea is blank, so that the research on wider molecular biology is extremely important.

Mitochondria (mitochondria) have genomes that are independent of the nuclear chromosome, and are considered "semi-autonomous organelles". The mitochondrial genome generally follows maternally inheritance, with little recombination occurring, and the paternal mitochondria are degraded during fertilization or lost during replication. Because the mitochondria genome is small, the structure is simple, the evolution speed is fast, and the like, the mitochondria become an important object for researching the origin evolution of species and the genetic differentiation of groups. The size and structure of mitochondrial genome of different species are greatly different in the process of biological evolution, so that the complete sequence study of mitochondrial genome becomes strong evidence for molecular phylogeny.

With the rapid advance of high-throughput sequencing technology, a large amount of related science and technology development is driven, and large-scale genome sequencing becomes a milestone in the scientific development history and becomes the basis of biological research in various fields. Mitochondrial whole genome sequencing, which first requires obtaining mitochondrial DNA with higher purity and total amount, is also a demand for more and more research. The methods for obtaining mitochondrial DNA on the present day include proteinase K method, alkaline lysis method, various kits, etc., but all have disadvantages, such as low purity of the obtained mitochondria, complicated operation, high cost, etc.

The corbicula fluminea mitochondria is separated and enriched by adopting differential centrifugation to obtain sufficient high-purity mitochondrial DNA, and the defects of other mitochondrial DNA extraction methods are overcome.

The mitochondrial research method for corbicula fluminea, which has been disclosed so far, is based on the amplification of a partial region of a conserved gene of a homologous species. For example, the genetic diversity and population structure analysis of corbicula fluminea mitochondria are studied by amplifying fragments of the COI gene. According to the invention, two pairs of specific primer pairs are designed according to a mitochondrial genome sequence, so that corbicula fluminea mitochondrial gene fragments can be specifically amplified, and molecular species identification can be carried out on corbicula fluminea.

Disclosure of Invention

The invention aims to overcome the defects that the extraction purity of the mitochondrial genome is low, the total amount is small and the requirements of subsequent experiments are difficult to meet in the prior art, and provides a mitochondrial genome enrichment extraction and identification method, which is used for identifying species in molecular biology through amplification of mitochondrial gene fragments.

The specific technical scheme of the invention is as follows:

a corbicula fluminea mitochondrial genome enrichment extraction and identification method comprises the following steps:

s1: the differential centrifugation method of the sample to be identified crudely extracts mitochondria, and then extracts the mitochondrial genome DNA of the sample to be identified from the crudely extracted mitochondria;

s2: designing a specific primer pair Mit1 according to corbicula fluminea mitochondrial nad2-cox3 gene, and designing a specific primer pair Mit2 according to corbicula fluminea mitochondrial cob gene;

s3: performing PCR amplification by using mitochondrial genome DNA of a sample to be identified, which is extracted by S1, as a template and adopting a primer pair Mit1 and/or a primer pair Mit 2;

s4: carrying out agarose gel electrophoresis identification and sequencing identification on the PCR amplification product obtained from the S3;

the agarose gel electrophoresis is identified as whether the size of an agarose gel electrophoresis strip is consistent with the known molecular weight of a corresponding gene amplified by S3 by comparison, the molecular weight of the corbicula fluminea mitochondrial nad2-cox3 gene is 580bp, the molecular weight of the corbicula fluminea mitochondrial cob gene is 360bp, and if the agarose gel electrophoresis strip has the same size as the corresponding gene amplified by S3, the sample to be identified is corbicula fluminea;

the sequencing is identified as whether the sequencing result is consistent with the known sequence of the corresponding gene amplified by S3 by comparison, the known sequence of the corbicula fluminea mitochondrial nad2-cox3 gene is shown as SEQ ID No.1, the known sequence of the corbicula fluminea mitochondrial cob gene is shown as SEQ ID No.2, and if the sequencing result is consistent with the known sequence, the sample to be identified is corbicula fluminea.

Further, the centrifugal force combination of the crude mitochondria obtained by the differential centrifugation method of the sample to be identified in S1 is as follows: centrifuging the muscle grinding filtrate of the sample to be identified for 20 minutes by adopting 3000g, and taking the supernatant; centrifuging at 15000g for 20 min to obtain precipitate; adding 3000g of suspension buffer solution into the precipitate, centrifuging for 10 minutes, taking the supernatant, carrying out enzyme digestion on DNase I, centrifuging for 20 minutes at 18000g, and collecting the precipitate to obtain a crude mitochondria.

Further, the S1 includes the following steps:

1) taking 5g of fresh and alive sample muscle to be identified, thoroughly grinding the muscle in a precooled glass homogenizer, filtering the ground sample by using a filter membrane of 1um, and centrifuging the collected filtrate for 20 minutes by using 3000g of centrifugal force;

2) transferring the centrifuged supernatant into a new centrifuge tube, centrifuging for 20 minutes at 15000g, collecting the precipitate, adding 1ml of suspension buffer solution into the precipitate, and slightly suspending the precipitate by using a brush;

3) centrifuging 3000g of the suspension for 10 minutes, taking the supernatant, adding 2ug of DNase I into each gram of fresh weight, carrying out enzyme digestion at 35 ℃ for 2 hours, adding 0.5mol/L EDTA, terminating the enzyme digestion reaction, centrifuging 18000g for 20 minutes, and collecting the precipitate to obtain a crude mitochondria;

4) adding 1/10 volume of 10% SDS and 1/100 volume of 30mg/ml RNase into the crude mitochondria, and carrying out water bath at 50 ℃ for 30 minutes;

5) adding 1/150 volume of 25mg/ml proteinase K, and water bath at 37 ℃ for 30 minutes;

6) after the reaction solution was cooled to room temperature, 200ul of saturated sodium acetate was added to a final concentration of 0.8mol/L, and an equal volume of phenol was added at a volume ratio of 25:24: 1: chloroform: the isoamyl alcohol mixed solution is carefully and slowly mixed, and is centrifuged for 15 minutes at 10000g under the condition of 4 ℃ so as to separate solid from liquid;

7) and (3) lightly taking the supernatant, adding equal volume of phenol with the volume ratio of 25:24: 1: chloroform: the isoamyl alcohol mixed solution is carefully and slowly mixed, and is centrifuged for 15 minutes at 10000g under the condition of 4 ℃;

8) repeating the step 7) until the middle white protein layer disappears,

9) taking the supernatant, adding 1/10 volumes of 3mol/L sodium acetate and 2 volumes of precooled absolute ethyl alcohol, slowly mixing, and standing overnight at-20 ℃; centrifuging the next day, collecting DNA, washing with 75% ethanol for 2-3 times, and air drying at room temperature; adding ultrapure water or TE with proper amount for dissolution, and storing at-80 ℃ for later use.

Further, the specific primer pair Mit1 in S2 is:

Mit1F:5’-ATGGTTTAACGGCTGCGATTGAA-3’(SEQ ID No.3)，Mit1R:5’-CCAACATCGAGGTAGCAAACTTTCT-3’(SEQ ID No.4)；

the specific primer pair Mit2 is as follows:

Mit2F:5’-ATCTTACGGGTACTACGAACAAACG-3’(SEQ ID No.5)，Mit2R:5’-ACACCTCTACGGACGCCTCT-3’(SEQ ID No.6)。

further, the PCR reaction system in S3 is: 5 XFastpfu Buffer 4ul, 2ul2.5mM dNTPs, Forward Primer (5. mu.M) and Reverse Primer (5. mu.M) each 0.8ul, Fastpfu Polymerase 0.4ul, Template DNA 10ng, ultra pure water to 20 ul; the PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 50-60 ℃ for 30s, extension at 72 ℃ for 45s, 27 cycles, and final extension at 72 ℃ for 10 min.

Further, the annealing temperature in S3 was 55 ℃.

In the identification process, one pair of a primer pair Mit1 and a primer pair Mit2 can be adopted to amplify and detect mitochondrial genome DNA of a sample to be identified, namely only nad2-cox3 gene or only cob gene is amplified, the amplification product is subjected to subsequent experiments such as 1% agarose gel electrophoresis, the size of a strip of the amplification product of the sample to be identified is compared with the known size of the amplified nad2-cox3 or cob gene, the amplification product is subjected to ABI3730 sequencing, the sequencing result is compared with the known nucleotide sequence of the amplified nad2-cox3 or cob gene, and whether the sample to be identified is corbicula fluminea can be determined. Or two pairs of primer pairs can be adopted, namely mitochondrial genome DNA of a sample to be identified is taken as a template, nad2-cox3 gene and cob gene are respectively amplified, and the two amplification products are respectively subjected to subsequent experiments such as 1% agarose gel electrophoresis and ABI3730 sequencing for identification, so that the reliability is improved.

Compared with the prior art, the invention has the beneficial effects that:

1. the differential centrifugation method adopted by the invention is used for obtaining crude mitochondria and further extracting corbicula fluminea mitochondrial genome DNA, and an optimal centrifugal force combination is researched, the obtained mitochondria have more total amount and better quality, and the extracted mitochondria DNA has the advantages of high purity, good DNA integrity, more total amount, repeatable experiment and the like, and is the basis for carrying out subsequent experiments such as high-throughput sequencing, PCR amplification and the like. In other mitochondrial DNA extraction methods, the obtained DNA fragments are seriously degraded and contain a large amount of impurities, so that the next experiment cannot be carried out.

2. According to the invention, a primer pair Mit1 of nad2-cox3 gene is designed according to the sequence of mitochondria, a primer pair Mit2 is designed according to cob gene fragments, and the corbicula fluminea mitochondria nad2-cox3 and cob genes are used for identification, so that the stability and the reproducibility are good. The two pairs of primers provided by the invention have strong specificity, the primer pair can specifically amplify a mitochondrial sequence of corbicula fluminea, but cannot amplify the gene fragment of mitochondria of cyprinid fish, benthic organism hyriopsis cumingii and cerussa cristata, and the primers have the advantages of good amplification performance, good discrimination, less generated byproducts, strong specificity, high specificity and high sensitivity, and have very important significance for identifying the intraspecific level of corbicula fluminea, the research of molecular system geography and the protection of population genetic diversity. Species identification can be carried out on corbicula fluminea.

3. The identification method is simple and highly reliable, and can accurately identify the molecular species of corbicula fluminea.

Drawings

FIG. 1 is a gel diagram of a corbicula fluminea mitochondrial DNA electrophoresis obtained by the differential centrifugation method in example 1,

the first lane from the left is DL2000 standard score number marker; the second lane is corbicula fluminea mitochondrial DNA.

FIG. 2 agarose gel electrophoresis picture of PCR product amplified by each sample in example 2 using primer pair Mit 1.

The left side is as follows in sequence: the first lane (M) is DL2000 Normal score number marker; the second lane (1) is a mitochondrial gene fragment of corbicula fluminea; the third to eighth lanes (2-7) are silver gobio and Beishi in turn

Pseudorasbora parva, parabramis pekinensis, hyriopsis cumingii and Trapa patina, the ninth lane was not loaded, and the tenth lane (CK) was blank control.

FIG. 3 agarose gel electrophoresis picture of PCR product amplified by each sample in example 3 using primer pair Mit 2.

The left side is as follows in sequence: the first lane (M) is DL2000 Normal score number marker; the second lane (8) is a mitochondrial gene fragment of corbicula fluminea; the third to eighth lanes (9-14) are silver gobio and Beishi in sequence

Detailed Description

In order to make the technical means, experimental steps and effects of the present invention easier to understand, the present invention is further described below with reference to specific embodiments.

Example 1 differential centrifugation method for extracting Corbicula fluminea mitochondrial genome DNA

Taking 5g of fresh corbicula fluminea collected in Jiangsu of China, taking muscle of the corbicula fluminea, thoroughly grinding the corbicula fluminea in a precooled glass homogenizer, filtering the ground sample by using a filter membrane of 1um, and centrifuging the collected filtrate for 20 minutes by using 3000g of centrifugal force. Transferring the centrifuged supernatant into a new centrifuge tube, centrifuging at 15000g for 20 minutes, collecting the precipitate, adding 1ml of suspension buffer into the precipitate, and gently suspending the precipitate by using a brush. Centrifuging the suspension for 10 minutes at 3000g, taking the supernatant, adding DNase I at 2ug per gram of fresh weight, carrying out enzyme digestion for 2 hours at 35 ℃, adding EDTA at 0.5mol/L, terminating the enzyme digestion reaction, centrifuging for 20 minutes at 18000g, and collecting the precipitate, namely the crude mitochondria.

To the crude mitochondria was added 1/10 volume of 10% SDS and 1/100 volume of 30mg/ml RNase, and water bath was carried out at 50 ℃ for 30 minutes. 1/150 volumes of 25mg/ml proteinase K were added and the mixture was incubated in a water bath at 37 ℃ for 30 minutes. After the reaction solution was cooled to room temperature, 200ul of saturated sodium acetate was added to a final concentration of 0.8mol/L, and an equal volume of phenol in a volume ratio of 25:24: 1: chloroform: the isoamyl alcohol mixed solution is carefully and slowly mixed, and is centrifuged for 15 minutes at 10000g under the condition of 4 ℃ so as to separate solid from liquid. And (3) lightly taking the supernatant, adding equal volume of phenol with the volume ratio of 25:24: 1: chloroform: the isoamyl alcohol mixture was carefully and slowly mixed and centrifuged at 10000g for 15 minutes at 4 ℃. This procedure was repeated until the middle white protein layer disappeared. The supernatant was taken, added with 1/10 volumes of 3mol/L sodium acetate and 2 volumes of pre-cooled absolute ethanol, and mixed gently overnight at-20 ℃. The next day, DNA was collected by centrifugation, washed 2-3 times with 75% ethanol, and air-dried at room temperature. Adding ultrapure water or TE with proper amount for dissolution, and storing at-80 ℃ for later use.

Detecting the extracted mitochondrial DNA by 1% agarose gel electrophoresis, wherein the agarose gel electrophoresis detection step comprises:

1. 1g of agarose was weighed using a balance, gently poured into a conical flask measuring 100ml of 0.5XTBE buffer, and the mouth of the conical flask was capped with tinfoil.

2. And (3) putting the covered conical flask into a microwave oven to be heated for 1 minute and 30 seconds, wearing heat-proof gloves when the solution is boiled, and carefully shaking the conical flask to fully and uniformly dissolve the agarose. If the small particles are not dissolved, the mixture is placed in a microwave oven for 20 seconds and is gently shaken until the agarose is completely dissolved.

3. The solution was cooled to 50-60 deg.C, 3.5ul of DNAgreen dye was added to the solution and mixed well.

4. The agarose solution was poured into a gel mold and then a comb was inserted at the appropriate location. The gel was allowed to set at room temperature for about 30 minutes.

5. An appropriate amount of sample was taken and carefully added to the sample tank using a micropipette.

6. After the sample is added, the electrophoresis tank cover is closed, and the power supply is immediately switched on. The control voltage is kept at 110V, and the current is above 40 mA. Electrophoresis was stopped when the band moved to about 2cm (about 40min) from the gel front.

7. And taking a picture under a gel imager for observation.

The detection result shows that the strip is clear and has no degradation, and the genome DNA enrichment extraction method can successfully extract the genome DNA of corbicula fluminea from corbicula fluminea. (FIG. 1).

Comparative example

In order to compare the influence of different centrifugal forces on the mitochondrial yield, 6 different centrifugal force combinations were designed, as follows:

TABLE 1 different combinations of centrifugal forces

Serial number	Separation of disrupted tissue (g)	Isolated mitochondria (g)	Enriched mitochondria (g)	Separation and extraction effect
					Example 1	3000	15000	18000	Has high mitochondria content and purity
Comparative example 1	2200	15000	18000	High amount of mitochondria but not pure
					Comparative example 2	3000	15000	15000	Has low mitochondria content and is impure
Comparative example 3	2200	12000	18000	The amount of mitochondria is very small
					Comparative example 4	2200	12000	15000	The amount of mitochondria is very small
Comparative example 5	3000	12000	15000	The amount of mitochondria is very small

The result shows that compared with other centrifugal force combinations, the total amount and the purity of the obtained mitochondria are high under the centrifugal force condition, and the mitochondrial DNA with high purity, good DNA integrity and high total amount can be further extracted.

Example 2 amplification and identification of primer pair Mit1

The research is divided into corbicula fluminea groups and cyprinid fishes: silver gobio group and Bass

Group, pseudopekinensis group, pekinete group, hyriopsis cumingii group and cercospora fusca group.

The procedure for enriching and extracting mitochondrial genome DNA of the sample to be identified is the same as in example 1.

Mitochondrial genome DNA of each sample to be identified is taken as a template, and a primer pair Mit1 for amplifying corbicula fluminea mitochondrial nad2-cox3 gene fragments is adopted: mit1F: 5'-ATGGTTTAACGGCTGCGATTGAA-3' (SEQ ID No.3) and Mit1R: 5'-CCAACATCGAGGTAGCAAACTTTCT-3' (SEQ ID No.4) were subjected to PCR amplification.

Before PCR amplification, the reagents were thawed on ice, the concentration of the primers was diluted to 5u mol/L each, and the total reaction system for PCR was 20 ul. The reaction system is as follows: 5 XFastpfu Buffer 4ul, 2ul2.5mM dNTPs, Forward Primer (5. mu.M) and Reverse Primer (5. mu.M) each 0.8ul, Fastpfu Polymerase 0.4ul, Template DNA 10ng, and ultrapure water to 20 ul.

The PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 45s, 27 cycles, and final extension at 72 ℃ for 10 min.

The PCR reaction was set to CK, which is ultrapure water containing no DNA, as a negative control.

The amplification products were detected by electrophoresis on a 1% agarose gel. The procedure of agarose gel electrophoresis detection was the same as in example 1.

The detection results are shown in fig. 2, which are, in order from the left: the first lane is DL2000 standard score marker; the second lane is mitochondrial gene fragment of corbicula fluminea; the third to the eighth lanes are silver gobio and Bashi in turn

The results show that the corbicula fluminea has clear bands, and the corresponding bands cannot be amplified in the cyprinid fish, the hyriopsis cumingii and the patina periwinkle, so that the primer pair can be proved to be capable of specifically amplifying the mitochondrial nad2-cox3 gene sequence of the corbicula fluminea, but cannot amplify the gene fragment in the mitochondria of the cyprinid fish, the benthic organism hyriopsis cumingii and the patina periwinkle.

The amplified product is subjected to ABI3730 sequencing, and the sequencing result of the amplified product ABI3730 of corbicula fluminea group is as follows:

CGANGGTTGGCGGTATTTTATAACCATTAAGGGGAACTTGGCGGTTAAACCGATGATCCTCGTAATACTTTACCCTAAGTTTTCTTTGCATACCGCCGTCATTAGGTGTACCTAAAGGGGAAAGATTGCAGCGAGATATCATTTATTTAATATGTGAAGAAGTTCAGGTAGACGTGTAGGGTTTCTTAGGGGCTTAGCTGAGTTACAAATGTAAAGCAATTATGGATCATAAAATGAAATATTTATGTGAAAGCGTACTTATTAGTAAATTTTAAAGAGAGAAATTTTGAATTGAGTAAATAAAATGTGTACAAATCGCCCGGCGCCCTCGAATATTTAAATTGAGGTAAGTCGTAACATAGTAATGCTAGTAAAAACTGGTGTTGTGAAGAGTAAACTAAGTAAAGTTTTCTGGTTCATGCCCAGAGTATAGAGAAAACATCTCTCTCTTTTAATTTCATCCTGTAGTATAAAAAGTATATTAAGTTGCAACCTTAATGGTGTGATATTTCTCCAGGGTTTAAAAGAGGCGCNAAAAAAGAGGGTGTA

the results show that the sequence obtained by the corbicula fluminea group is completely consistent with the gene sequence of corbicula fluminea mitochondria nad2-cox3 shown in SEQ ID NO.1 obtained by the laboratory high-throughput sequencing, and prove that the primer pair can specifically amplify the mitochondrial sequence of corbicula fluminea, but cannot amplify the gene segment of mitochondria of cyprinidae fishes, benthos hyriopsis cumingii and cercospora chaloticus. The species identification of corbicula fluminea can be carried out by combining the primer with ABI3730 sequencing detection.

Example 3 amplification and identification of primer pair Mit2

Mitochondrial genome DNA of each sample to be identified is taken as a template, and a primer pair Mit2 for amplifying corbicula fluminea mitochondrial cob gene fragment is adopted: mit2F: 5'-ATCTTACGGGTACTACGAACAAACG-3' (SEQ ID No.5) and Mit2R: 5'-ACACCTCTACGGACGCCTCT-3' (SEQ ID No.6) were subjected to PCR amplification.

The detection results are shown in fig. 3, which are, in order from the left: the first lane is DL2000 standard score marker; the second lane is mitochondrial gene fragment of corbicula fluminea; the third to the eighth lanes are silver gobio and Bashi in turn

The results show that the corbicula fluminea has clear strips, and the corresponding strips cannot be amplified by the cyprinid fish, the hyriopsis cumingii and the patina conus are blank controls, so that the primer pair can be proved to be capable of specifically amplifying the mitochondrial cob gene sequence of the corbicula fluminea, but cannot amplify the gene fragment of the mitochondria of the cyprinid fish, the benthic organism hyriopsis cumingii and the patina conus.

GTAGCATATAATTTGCCCTTTATTGGGGGAGAGAATGAATGGTTTGACGGTAAAAAAGCTGTTTTAAAAATAATTAAAGAAGTTAACTTTTAAGTGAAAAGGCTTAAGTTTTTATAAAAGACGAGAAGACCCCGTCGAGCTTAATTAGGATAGCTTTATTTAAAAGATATCTAAAATTTTATTGGGGCAATAGAAAATGAAAAGAATCATTTTTTTATAGAATAAGGATCCAGTTTTGACTGAAAAAAGCAAAAGCTACCGCGGGGATAACAGGGTAATTTTTTCTGAGAGTTCATATTTAAGAGAAAGTTTGCTACCTCGATGTTGGA

the result shows that the sequence obtained by the corbicula fluminea group is completely consistent with the gene sequence of corbicula fluminea mitochondria cob shown in SEQ ID NO.2 obtained by the laboratory high-throughput sequencing. The primer pair is proved to be capable of specifically amplifying a mitochondrial sequence of Corbicula fluminea, but the gene segment can not be amplified for mitochondria of Cyprinus carpioides, Hyriopsis cumingii and Trapa paticola. The species identification of corbicula fluminea can be carried out by combining the primer with ABI3730 sequencing detection.

The differential centrifugation method adopted by the invention for extracting Corbicula fluminea mitochondrial genome DNA is the basis for carrying out subsequent experiments such as high-throughput sequencing, PCR amplification and the like. The mitochondrial genome DNA extracted by the method has the advantages of high purity, good DNA integrity, more total amount, repeatable experiment and the like.

The foregoing has described the general principles, essential features, and advantages of the invention. It will be appreciated by those skilled in the art that the present invention is not limited by the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, and that modifications or alterations may be made thereto without departing from the spirit and scope of the present invention.

Sequence listing

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Claims

1. A corbicula fluminea mitochondrial genome enrichment extraction and identification method is characterized by comprising the following steps:

the agarose gel electrophoresis is identified as whether the size of an agarose gel electrophoresis strip is consistent with the known molecular weight comparison of the corresponding gene amplified by S3, the molecular weight of the nad2-cox3 gene is 580bp, the molecular weight of the cob gene is 360bp, and if the agarose gel electrophoresis strip has the same size as the corresponding gene amplified by S3, the sample to be identified is corbicula fluminea;

the sequencing is identified as whether the sequencing result is consistent with the known sequence comparison of the corresponding gene amplified by S3, the known sequence of the nad2-cox3 gene is shown as SEQ ID NO.1, the known sequence of the cob gene is shown as SEQ ID NO.2, if the sequencing result is consistent with the known sequence, the sample to be identified is corbicula fluminea,

the specific primer pair Mit1 in S2 is:

Mit1F:5’-ATGGTTTAACGGCTGCGATTGAA-3’(SEQ ID No.3)，

Mit1R:5’-CCAACATCGAGGTAGCAAACTTTCT-3’(SEQ ID No.4)；

the specific primer pair Mit2 is as follows:

Mit2F:5’-ATCTTACGGGTACTACGAACAAACG-3’(SEQ ID No.5)，

Mit2R:5’-ACACCTCTACGGACGCCTCT-3’(SEQ ID No.6)。

2. the method for enriching, extracting and identifying corbicula fluminea mitochondrial genome according to claim 1, wherein the centrifugal force combination of the sample to be identified for the crude mitochondria by the differential centrifugation method in S1 is as follows: centrifuging 3000g of muscle grinding filtrate of a sample to be identified for 20 minutes, and taking supernatant; centrifuging at 15000g for 20 min to obtain precipitate; adding 3000g of suspension buffer solution into the precipitate, centrifuging for 10 minutes, taking the supernatant, carrying out enzyme digestion on DNase I, centrifuging for 20 minutes at 18000g, and collecting the precipitate to obtain a crude mitochondria.

3. The method for enriching, extracting and identifying corbicula fluminea mitochondrial genome according to claim 1 or 2, wherein the step of S1 comprises the following steps:

8) repeating the step 7) until the middle white protein layer disappears,

4. The method for enriching, extracting and identifying corbicula fluminea mitochondrial genome according to claim 1, wherein the PCR reaction system in S3 is as follows: 5 XFastpfu Buffer 4ul, 2ul2.5 mdNTPs, 5 uM Forward Primer and 5 uM Reverseprimer are 0.8ul respectively, Fastpfu Polymerase is 0.4ul, Template DNA is 10ng, and ultrapure water is supplemented to 20 ul; the PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 50-60 ℃ for 30s, extension at 72 ℃ for 45s, 27 cycles, and final extension at 72 ℃ for 10 min.

5. The method for enriching, extracting and identifying corbicula fluminea mitochondrial genome according to claim 4, wherein the annealing temperature is 55 ℃.