Primer, kit and method for identifying Cobitidae fish

Abstract

The invention discloses a primer, a kit and a method for identifying Cobitidae fish. The identification primers include a first primer pair and a second primer pair, the upstream and downstream of the first primer pair respectively have nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.2, and the upstream and downstream of the second primer pair respectively have nucleotide sequences shown in SEQ ID NO.3 and SEQ ID NO.4. The method for identifying Cobitidae fish includes: S1: obtaining DNA of a Cobitidae fish sample; S2: performing PCR amplification on the DNA obtained in S1 with the first primer pair to obtain a first PCR amplification product; S3: performing nested PCR amplification with the second primer pair, using the first PCR amplification product as a template to obtain a second PCR amplification product; S4: performing agarose gel electrophoresis on the second PCR amplification product obtained in S3, cutting and purifying the gel, and obtaining a purified second PCR amplification product; and S5: sequencing the purified second PCR amplification product, comparing the mitochondrial gene sequence of the known species of Cobitidae fish in the NCBI database according to the base sequence of the sequencing result, and confirming the species of the Cobitidae fish sample.

Description

Technical Field

[0001] The invention relates to the field of molecular biology, and in particular to a primer, a kit and a method for identifying Cobitidae fish. Background Art

[0002] Cobitidae is a family of Cypriniformes in the class Actinopterygii. It is a small freshwater bottom-dwelling fish that is omnivorous. Most scholars tend to divide it into three subfamilies, namely, the subfamily of ray-finned fish, the subfamily of sand loach, and the subfamily of flower loach. The distribution of Cobitidae fish in my country is also very wide. There are many water systems in southern my country, so Cobitidae is more concentrated in the south. There are about 18 genera and 100 species and subspecies of Cobitidae in my country. The appearance of Cobitidae members is very different from their Cypriniformes counterparts. Most of them have elongated laterally flattened or cylindrical bodies and flat heads. The mouth is small and located in the lower position. The upper jaw is formed by the premaxilla, with a row of hypopharyngeal teeth and no keratin pads. The eyes are very small, and some fish species have spines under the eyes. There are also 3-6 pairs of whiskers, 2 pairs of snout whiskers, 1 or 2 pairs of mouth whiskers, and 1 pair of chin whiskers and nasal whiskers or none.

[0003] There are many species of Cobitidae fish, and it is difficult to accurately identify Cobitidae fish during their hatching period. The traditional method for early resource identification of Cobitidae fish is morphological method, which has large errors and causes inaccurate results of fishery resource surveys. Summary of the invention

[0004] In view of the problem of large error in morphological identification methods, the present invention provides a primer, a kit and a method for identifying Cobitidae fish. The method can accurately measure the target fragment sequence of the species to be tested, thereby accurately identifying the species.

[0005] In the first aspect, the present invention provides a primer for identifying Cobitidae fish, the identification primers comprising a first primer pair and a second primer pair, the upstream and downstream of the first primer pair respectively having nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.2, and the upstream and downstream of the second primer pair respectively having nucleotide sequences shown in SEQ ID NO.3 and SEQ ID NO.4.

[0006] In a second aspect, the present invention provides a kit for identifying Cobitidae fishes, the kit comprising the above primers.

[0007] Preferably, the kit further comprises PCR buffer, Taq DNA polymerase, dNTPs, and MgCl2.

[0008] In a third aspect, the present invention provides a method for identifying Cobitidae fish, and the specific steps are as follows: S1: obtaining DNA of a Cobitidae fish sample; S2: performing PCR amplification on the DNA obtained in S1 using a first primer pair to obtain a first PCR amplification product; S3: performing nested PCR amplification using a second primer pair to obtain a second PCR amplification product; S4: performing agarose gel electrophoresis on the second PCR amplification product obtained in S3, cutting and purifying the gel, and obtaining a purified second PCR amplification product; and S5: sequencing the purified second PCR amplification product, and comparing the mitochondrial gene sequences of known Cobitidae fish species in the NCBI database based on the base sequence of the sequencing result to confirm the species of the Cobitidae fish sample.

[0009] Preferably, the first PCR amplification product obtained in S2 is diluted 10 times.

[0010] Preferably, the reaction system for PCR amplification is: 3 μl of 10×PCR buffer, 2 μl of 2.5 mmol / L dNTP, 3 μl of MgCl2, 1 μl of upstream primer, 1 μl of downstream primer, 0.5 μl of Taq polymerase, 2 μl of DNA template, and 12.5 μl of ddH2O.

[0011] Preferably, the reaction procedure of PCR amplification in S2 is: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 57°C for 60 s, extension at 72°C for 30 s, 35 cycles; extension at 72°C for 10 min.

[0012] Preferably, the reaction procedure of nested PCR amplification in S3 is: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 61°C for 60 s, extension at 72°C for 30 s, 35 cycles; extension at 72°C for 10 min.

[0013] Preferably, the gel in S4 is 1.5% agarose gel.

[0014] In summary, the present invention has the following beneficial effects: (1) the primers of the present invention can be used to accurately and efficiently identify the target fragment sequence of Cobitidae fish, exclude non-specific amplified fragments, and accurately identify the type of the sample to be tested; (2) the primers of the present invention can be used to identify Cobitidae fish during their hatching period, which greatly improves the efficiency of Cobitidae fish identification and shortens the identification time. DETAILED DESCRIPTION

[0015] The present invention will be further described below with reference to specific embodiments. It will be understood by those skilled in the art that the purpose of providing these embodiments is merely to illustrate the implementable modes of the present invention so that those skilled in the art can better understand the present invention, and it is not intended to limit the present invention to these specific embodiments.

[0016] Example 1 Primer Design

[0017] Primers were designed based on the common mitochondrial sequence of Cobitidae fish.

[0018] First primer pair:

[0019] 1F: 5'-ACAGACCTACCGAACTTGGTGATA-3' (SEQ ID NO.1)

[0020] 1R: 5'-ATAGAAACTGACCTGGATTNCTCC-3' (SEQ ID NO. 2).

[0021] Second primer pair:

[0022] 2F: 5'-CTGATCCAACATCGAGGTCGTAAA-3' (SEQ ID NO.3)

[0023] 2R: 5'-AAGCAGCCANCTGAACAGAAAGCG-3' (SEQ ID NO. 4).

[0024] Example 2 Identification method

[0025] The primers designed in Example 1 were used to identify the fish of the family Cobitidae.

[0026] S1: Extraction of DNA from Cobitidae fish: 1. Take the fin rays preserved in alcohol, cut about 1g of fin rays into a centrifuge tube, wash them twice with distilled water, 1 hour each time, and put the fin rays into a 55℃ oven after washing until the alcohol is completely evaporated; 2. Pipette 10μl proteinase K and 500μl HOM buffer into the centrifuge tube, shake it manually and put the centrifuge tube into a 55℃ oven until the tissue is completely lysed and the liquid becomes transparent; 3. After the tissue is completely lysed, take 300μl 4.5mol / L Add NaCl and 200μl chloroform to the centrifuge tube, vortex gently to mix, and fully lyse for 15 minutes; 4.13000 rpm, centrifuge for 12 minutes, take 700μl of supernatant, and add it to a new 1.5ml centrifuge tube; 5. Add 490μl of pure isopropanol solution to the supernatant, vortex gently to mix it, and place it in a 4℃ refrigerator for more than 30 minutes; 6. Add 490μl of pure isopropanol solution to the supernatant, vortex gently to mix it, and place it in a 4℃ refrigerator for more than 30 minutes; 7. Add 75% alcohol solution at 4℃ to the centrifuge tube and let it stand for 20 minutes; 8.13000 rpm, centrifuge for 12 minutes, DNA is deposited at the bottom of the centrifuge tube, remove the supernatant, open the centrifuge tube lid and invert it on a clean paper to dry; 9. Add about 50μl of distilled water to the center of the bottom of the centrifuge tube, 13000 rpm, centrifuge for 2 minutes, get the DNA stock solution, and store at 4℃;

[0027] S2: Perform PCR amplification on the DNA extracted from S1 using the first primer pair to obtain a first PCR amplification product, wherein the upstream and downstream of the first primer pair have nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. The reaction procedure of PCR amplification is: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 57°C for 60 s, extension at 72°C for 30 s, 35 cycles; extension at 72°C for 10 min;

[0028] S3: The first PCR amplification product obtained in S2 was diluted 10 times, and a second PCR amplification product was obtained by nested PCR amplification using the second primer pair, wherein the upstream and downstream of the first primer pair have the nucleotide sequences shown in SEQ ID NO.3 and SEQ ID NO.4, respectively. The reaction procedure of the nested PCR amplification is: 94°C pre-denaturation for 3 min; 94°C denaturation for 30 s, 61°C annealing for 60 s, 72°C extension for 30 s, 35 cycles; 72°C extension for 10 min;

[0029] S4: electrophoresing the second PCR amplification product obtained in S3 using 1.5% agarose gel, cutting and purifying the gel to obtain a purified second PCR amplification product;

[0030] S5: Connecting the purified second PCR amplification product obtained in S4 to a vector and transforming it into Escherichia coli for cloning, and selecting a single clone for sequencing; and

[0031] S6: Based on the base sequence of the sequencing result, the mitochondrial gene sequence of known species of Cobitidae fish is compared in the NCBI database to confirm the species of the sample.

[0032] Example 3

[0033] S1: Extract DNA from samples: 1. During the natural reproduction detection of Yangtze River fish in 2020, 3 fish species suspected to be of the family Cobitidae were caught; 2. Take the fin rays preserved in alcohol, cut about 1g of fin rays into a centrifuge tube, wash them with distilled water twice, 1 hour each time, and put the fin rays in a 55℃ oven after washing until the alcohol is completely evaporated; 3. Pipette 10μl proteinase K and 500μl HOM buffer into the centrifuge tube, shake it manually and put the centrifuge tube in a 55℃ oven until the tissue is completely lysed and the liquid becomes transparent; 4. After the tissue is completely lysed, take 300μl 4.5mol / L Add NaCl and 200μl chloroform to the centrifuge tube, vortex gently to mix, and fully lyse for 15 minutes; 5.13000 rpm, centrifuge for 12 minutes, take 700μl of supernatant, and add it to a new 1.5ml centrifuge tube; 6. Add 490μl of pure isopropanol solution to the supernatant, vortex gently to mix it, and place it in a 4℃ refrigerator for more than 30 minutes; 7. Add 490μl of pure isopropanol solution to the supernatant, vortex gently to mix it, and place it in a 4℃ refrigerator for more than 30 minutes; 8. Add 75% alcohol solution at 4℃ to the centrifuge tube and let it stand for 20 minutes; 9.13000 rpm, centrifuge for 12 minutes, DNA is deposited at the bottom of the centrifuge tube, remove the supernatant, open the centrifuge tube lid and invert it on a clean paper to dry; 10. Add about 50μl of distilled water to the center of the bottom of the centrifuge tube, 13000 rpm, centrifuge for 2 minutes, get the DNA stock solution, and store at 4℃;

[0034] S2: Perform PCR amplification on the DNA extracted from S1 using the first primer pair to obtain a first PCR amplification product, wherein the upstream and downstream of the first primer pair have nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. The reaction procedure of PCR amplification is: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 57°C for 60 s, extension at 72°C for 30 s, 35 cycles; extension at 72°C for 10 min;

[0035] S3: The first PCR amplification product obtained in S2 was diluted 10 times, and a second PCR amplification product was obtained by nested PCR amplification using the second primer pair, wherein the upstream and downstream of the first primer pair have the nucleotide sequences shown in SEQ ID NO.3 and SEQ ID NO.4, respectively. The reaction procedure of the nested PCR amplification is: 94°C pre-denaturation for 3 min; 94°C denaturation for 30 s, 61°C annealing for 60 s, 72°C extension for 30 s, 35 cycles; 72°C extension for 10 min;

[0036] S4: electrophoresing the second PCR amplification product obtained in S3 using 1.5% agarose gel, cutting and purifying the gel to obtain a purified second PCR amplification product;

[0037] S5: Connecting the purified second PCR amplification product obtained in S4 to a vector and transforming it into Escherichia coli for cloning, and selecting a single clone for sequencing; and

[0038] S6: Based on the base sequence of the sequencing result, the mitochondrial gene sequence of known species of Cobitidae fish is compared in the NCBI database to confirm the species of the sample.

[0039] The final results of the sequence alignment are respectively Parasand loach with spots, purple thin loach and Hanshui flat tail thin loach. These fry are artificially cultured, and after 20 days of culture, the fry are fully developed and subjected to traditional morphological identification. After traditional morphological identification, these three suspected Cobitidae fish are Parasand loach with spots, purple thin loach and Hanshui flat tail thin loach, which are consistent with the identification results of the method provided in this application. The method provided in this application can obtain the identification results in 2 days, with high accuracy of identification, and shortens the identification time, greatly improving the efficiency of Cobitidae fish identification.

[0040] The above description is only an explanation of the present invention and is not a limitation of the present invention. After reading this specification, a person skilled in the art may make non-creative modifications to the present embodiment as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A method for identifying Cobitidae fish, characterized in that: The specific steps are as follows: S1: Obtain DNA from samples of Cobitidae fish; S2: performing PCR amplification on the DNA obtained in S1 using the first primer pair to obtain a first PCR amplification product; S3: performing nested PCR amplification with the second primer pair, using the first PCR amplification product as a template, to obtain a second PCR amplification product; S4: performing agarose gel electrophoresis on the second PCR amplification product obtained in S3, cutting and purifying the gel, and obtaining a purified second PCR amplification product; S5: Sequencing the purified second PCR amplification product, and comparing the base sequence of the sequencing result with the mitochondrial gene sequence of known species of Cobitidae fish in the NCBI database to confirm the species of the Cobitidae fish sample; The upstream and downstream of the first primer pair have nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.2, respectively, and the upstream and downstream of the second primer pair have nucleotide sequences shown in SEQ ID NO.3 and SEQ ID NO.4, respectively. The first PCR amplification product obtained in S2 is diluted 10 times. The reaction system for PCR amplification was: 3 μl of 10×PCR buffer, 2 μl of 2.5 mmol / L dNTP, 3 μl of MgCl2, 1 μl of upstream primer, 1 μl of downstream primer, 0.5 μl of Taq polymerase, 2 μl of DNA template, 12.5 μl of ddH2O, The reaction procedure of PCR amplification in S2 was as follows: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 57°C for 60 s, extension at 72°C for 30 s, 35 cycles; extension at 72°C for 10 min, The reaction program of nested PCR amplification in S3 was as follows: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 61°C for 60 s, extension at 72°C for 30 s, 35 cycles; extension at 72°C for 10 min; First primer pair: 1F: 5'-ACAGACCTACCGAACTTGGTGATA-3' (SEQ ID NO.1) 1R: 5'-ATAGAAACTGACCTGGATTNCTCC-3' (SEQ ID NO.2), Second primer pair: 2F: 5'-CTGATCCAACATCGAGGTCGTAAA-3' (SEQ ID NO.3) 2R: 5'-AAGCAGCCANCTGAACAGAAAGCG-3' (SEQ ID NO. 4).

2. The method according to claim 1, characterized in that The gel described in S4 is 1.5% agarose gel.