CN105838802B - The primer and method of molecular identificalion threadfin and more squama threadfins - Google Patents

Abstract

The present invention discloses a primer for molecular identification of S. chinensis and S. serrata Including primers F2 and R2, the nucleotide sequence of primer F1 is shown in SEQ ID NO: 1, the nucleotide sequence of primer R1 is shown in SEQ ID NO: 2, and the nucleotide sequence of primer F2 is shown in SEQ ID NO: As shown in 3, the nucleotide sequence of primer R2 is shown in SEQ ID NO:4. The primers can identify the four-fingered horse bay fish and the polysquamous four-fingered horse bay fish at the molecular level. The invention also discloses a method for molecular identification of the four-fingered slug and the polysquamous four-fingered squirrel, which is fast, convenient, accurate and reliable.

Description

Molecular identification of the primers and methods of S.

technical field

The invention belongs to the technical field of molecular identification of closely related species, and in particular relates to a primer and a method for molecular identification of four-fingered squirrel and polysquamous four-fingered squirrel.

Background technique

Eleutheronema tetradactylum (Eleutheronema tetradactylum) and E. rhadinum are two common species of Eleutheronema (Eleutheronema) in the family Polynemoidea (Mugiliformes) , Mayou, with high fat content, tender meat, rich protein, high edible value and economic value, is a valuable high-end marine fish. In my country, the artificial breeding and aquaculture of maba is still in its infancy, and the finished fish mainly relies on fishing. With the rising demand for high-end and precious seafood in recent years, the natural fishery resources of maba are suffering from overfishing and are increasingly depleted. The amount obtained is difficult to meet the market demand.

There are many species in the family Squirrel that have similar external shapes, insignificant identification features, and easy confusion. Morphological taxonomy is mainly based on the number of free filamentous fin rays below the pectoral fins, such as four-fingered squid, six-finger squid, and multi-fingered squid. Bay and so on. Four-fingered and polysquamous four-fingered horses, both belonging to the genus Four-fingered, have 4 free filamentous rays below the pectoral fins. Except for the color of the pectoral fin, the number of lateral line scales and the vomer tooth plate, other morphological features are different. The characteristics are almost the same and are often mistaken for the same fish. The two fishes are not only very similar in morphology, but also in similar habitats, often mixed in the same waters, and it is more difficult to identify the small larvae and juveniles, which makes further research on their early life history and their replenishment dynamics. Difficulties are not conducive to the protection and management of resources. Therefore, there is an urgent need for a quick, convenient, accurate and reliable method for distinguishing the four-fingered mussels and the scaly scaly mussels.

SUMMARY OF THE INVENTION

The first object of the present invention is to provide a primer for molecular identification of S. chinensis and S. chinensis, which can differentiate between S. chinensis and S. chinensis at the molecular level.

The purpose of the present invention is also to provide a method for molecular identification of the four-fingered slug and the polysquamous scaly, which is fast, convenient, accurate and reliable.

The first object of the present invention is achieved by the following technical solutions: a primer for molecular identification of S. chinensis and S. chinensis, the primers include primer pair 1 and primer pair 2, and the primer pair 1 includes primers F1 and R1, and the primer pair 2 includes primers F2 and R2, wherein the nucleotide sequence of primer F1 is shown in SEQ ID NO: 1, and the nucleotide sequence of primer R1 is shown in SEQ ID NO: 2, The nucleotide sequence of primer F2 is shown in SEQ ID NO:3, and the nucleotide sequence of primer R2 is shown in SEQ ID NO:4.

Specifically, the nucleotide sequence of each primer from 5'-3' is as follows:

F1: 5'-GCTCCTCCTCAGAGTTTCG-3';

R1: 5'-TCAATGATAATGCCCACCA-3';

F2: 5'-GGAAAGCGTGACATAGT-3';

R2: 5'-CAGTGAAGTCCAGGTAAT-3'.

The second object of the present invention is achieved through the following technical solutions: a method for molecular identification of the four-fingered horse shark and the polysquamous four-fingered cockroach, comprising the following steps:

(1) Design the above-mentioned primer pair 1 and primer pair 2;

(2) Extracting the DNA of the four-fingered horse bay or the polysquamous four-fingered horse bay;

(3) using the DNA in step (2) as a template, using primer pair 1 and primer pair 2 to carry out PCR amplification, after the amplification reaction is completed, carry out electrophoresis detection, the electrophoresis results show that there are a specific line at the vicinity of 469bp and 606bp respectively Sex DNA-specific bands, the sample tested at this time is the four-fingered horse bay fish, if there is no band near 469bp or 606 bp, it is the polysquamous four-fingered horse bay fish.

In the above-mentioned method for molecularly discriminating the four-fingered mackerel and the polysquamous four-fingered mackerel:

In step (2), it is preferred to use the phenol-chloroform organic solvent method to extract the DNA of the four-fingered horse bayu or the polysquamous four-fingered bay fish.

The phenol chloroform organic solvent method is the conventional phenol chloroform organic solvent method, and the concrete method can refer to the documents Sambroo J, Fritsch EF, Maniatis T.MOLECULAR CLONING: A Laboratory Manual 2nd ed.ColdSpring Harbor Laboratory Press, 1989.

When using primer pair 1 and primer pair 2 for PCR amplification in step (3), the 20 μL PCR reaction system preferably contains 2 μL of DNA, 10 μL of primer pair 1 or primer pair 2, and 2 μL of PCR buffer premixed with Taq, dNTP, and MgCl ₂ , ddH ₂ O 6 μL.

When using primer pair 1 and primer pair to carry out PCR amplification in step (3), primer pair 1 and primer pair 2 can be used for PCR amplification respectively, or primer pair 1 and primer pair 2 can be used for PCR amplification simultaneously. Amplification with a pair of primers showed a band of 469 bp (primers F1 and R1) or 606 bp (primers F2 and R2) in S. tetradactyla, while no band appeared in S. chinensis. If two pairs of primers are used to amplify at the same time, two bands of 469bp (primers F1 and R1) and 606bp (primers F2 and R2) will appear at the same time in S.

When primer pair 1 is used for PCR amplification in step (3), the recommended PCR reaction program is: firstly 95°C for 5 min, then 32 cycles of 94°C for 30s, 66°C for 30s, 72°C for 45 minutes, and then 72°C for 10min; using primers When performing PCR amplification on 2, the recommended PCR reaction program is: first 95°C for 5 minutes, then 94°C for 30s, 56°C for 30s, 72°C for 45 seconds, and then 72°C for 10 minutes.

When performing electrophoresis detection in step (3), it is preferable to use agarose gel electrophoresis detection with a mass percentage of 1.5%, and add Redsafe to observe under ultraviolet light.

The present invention has the following advantages:

(1) the present invention has designed the primer of the molecular identification of the four-fingered horse bay and the polysquamous four-fingered horse for the first time.

(2) The method of the present invention is a new method established at home and abroad for the first time at the molecular level to identify the four-fingered squid and the polysquamous four-fingered squirrel. It uses four specific primers to complete a PCR reaction and can identify Four-fingered and scaly four-fingered squid;

(3) Compared with the morphological identification method, the method of the present invention does not need to rely on the professional knowledge of fish taxonomy, and more importantly, it can not only identify the finished fish of the four-fingered squid and the polysquamous four-fingered squid, but also can identify Their larvae and juveniles, fish eggs and even processed seafood are characterized by simplicity, speed, accuracy, stability, objectivity and fairness, and economy and practicality.

Description of drawings

Fig. 1 is the characteristic spectrum band of the four-fingered horse bayu amplified by primers F1 and R1 in Example 2, wherein M is the DNA marker (DL2000), S is the four-fingered horse bayu, and D is the polysquamous four-fingered horse bay fish;

Fig. 2 is the characteristic spectrum band of the four-fingered horse bayu amplified by primers F2 and R2 in Example 2, wherein M is the DNA marker (DL2000), S is the four-fingered horse bayu, and D is the polysquamous four-fingered horse bay fish;

Fig. 3 is the electrophoresis detection result of the four-fingered horse bay fish amplified by primers F1 and R1 in Example 3, wherein M is a DNA marker (DL2000), and 1-4 are the four-fingered horse bay fish samples;

FIG. 4 is the electrophoresis detection result of the four-fingered horse bay fish amplified by primers F2 and R2 in Example 3, wherein M is a DNA marker (DL2000), and d1-d4 are the polysquamous four-fingered horse bay fish samples.

Detailed ways

The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

Example 1

The primers provided in this example for molecular identification of S. chinensis and S. chinensis include primer pair 1 and primer pair 2, primer pair 1 includes primers F1 and R1, and primer pair 2 includes primers F2 and R2, wherein The nucleotide sequence of primer F1 is shown in SEQ ID NO: 1, the nucleotide sequence of primer R1 is shown in SEQ ID NO: 2, the nucleotide sequence of primer F2 is shown in SEQ ID NO: 3, and the nucleotide sequence of primer R2 is shown in SEQ ID NO: 3. The nucleotide sequence is shown in SEQ ID NO:4.

The nucleotide sequence of each primer 5'-3' is as follows:

F1: 5'-GCTCCTCCTCAGAGTTTCG-3';

R1: 5'-TCAATGATAATGCCCACCA-3';

F2: 5'-GGAAAGCGTGACATAGT-3';

R2: 5'-CAGTGAAGTCCAGGTAAT-3'.

The primer pair 1 and the primer pair 2 in the present invention are obtained in the following manner: first, use a random primer of 10 bp as a primer to carry out PCR amplification on S. ), the specific fragments amplified by random primers are screened out, cloned and sequenced, and then the 10-20bp sequence at one end is added to the original 10bp random primer sequence to design primers, and PCR amplification is performed again to screen out primers that can produce specific bands. (as shown above).

Example 2

According to professional morphological identification, a number of known species of the four-fingered mackerel and polysquamous four-fingered mackerel were selected, their DNA was extracted by conventional phenol-chloroform organic solvent method, and the genome was amplified by PCR using 4 kinds of primers. .

The 20 μL PCR reaction system contains 2 μL of DNA, 10 μL of primers (F1/R1, F2/R2); 2 μL of PCR buffer premixed with Taq, dNTP, and MgCl ₂ , and 6 μL of ddH ₂ O.

The PCR reaction program was 95°C 5min, [94°C 30sec→66°C (primer F1, R1) or 56°C (primer F2, R2) 30sec→72°C 45sec] × 32 cycles→72°C 10min.

After the PCR reaction was completed, 5 μL of the product was taken and detected by 1.5% agarose gel electrophoresis, and observed under ultraviolet light after adding Redsafe.

The results showed that a specific DNA band with size of 469bp (primers F1 and R1) and 606bp (primers F2 and R2) appeared at the same time in multiple samples of S.

On the contrary, there were no specific DNA bands with sizes of 469bp (primers F1 and R1) and 606bp (primers F2 and R2) in multiple samples of the serrata, as shown in Figure 1-2, indicating that when primers When the pair 1 and primer pair 2 were amplified by PCR respectively, when specific bands appeared at the same time at 469bp (primers F1 and R1) and 606bp (primers F2 and R2), it was a sample of S. Refers to the mabfish sample.

Example 3

The method for molecular identification of the four-fingered mackerel and the polysquamous four-fingered mackerel provided by the present embodiment comprises the following steps:

(1) Design the above-mentioned primer pair 1 and primer pair 2, and refer to Example 1 for the specific process;

(2) each selects the four-fingered squid or the polysquamous four-fingered squid of 8 mature individuals for use, adopts the conventional phenol chloroform organic solvent method to extract the DNA of the four-fingered squid or the polysquamous four-fingered squid;

(3) Using the DNA in step (2) as a template, using primer pair 1 and primer pair 2 to carry out PCR amplification respectively.

The 20 μL PCR reaction system contains 2 μL of DNA, 10 μL of primers (F1/R1, F2/R2); 2 μL of PCR buffer (buffer) premixed with Taq, dNTP, and MgCl ₂ , and 6 μL of ddH ₂ O.

The PCR reaction program was 95°C 5min, [94°C 30sec→66°C (primer F1, R1) or 56°C (primer F2, R2) 30sec→72°C 45sec] × 32 cycles→72°C 10min.

After the PCR reaction was completed, 5 μL of the product was taken and detected by agarose gel electrophoresis with a mass percentage of 1.5%, and observed under ultraviolet light after adding Redsafe.

The results showed that the 4 samples identified as four-fingered horse bayfish all had a specific DNA band of 469 bp (primers F1 and R1) or 606 bp (primers F2 and R2) at the same time, indicating that the samples were four-fingered horse bayfish. As shown in Figure 3-4.

The 4 samples identified by morphological identification as the four-fingered horse bay fish do not have bands, indicating that the samples are the multi-scale four-fingered horse bay fish. This result is completely consistent with the results of morphological identification, as shown in Figure 3-4.

The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, The simplification should be an equivalent replacement manner, which is included in the protection scope of the present invention.

Claims (6)

1. a primer for molecular identification of four-fingered horse shark and polysquamous four-fingered horse shark, is characterized in that: described primer comprises primer pair 1 and primer pair 2, and described primer pair 1 comprises primer F1 and R1, and described primer Pair 2 includes primers F2 and R2, wherein the nucleotide sequence of primer F1 is shown in SEQ ID NO: 1, the nucleotide sequence of primer R1 is shown in SEQ ID NO: 2, and the nucleotide sequence of primer F2 is shown in SEQ ID NO: 2 ID NO: 3, the nucleotide sequence of primer R2 is shown in SEQ ID NO: 4. 2. a kind of method for molecular identification of four-fingered squirrel and polyscaly four-fingered squirrel, it is characterized in that comprising the following steps: (1) design primer pair 1 and primer pair 2 in claim 1; (2) Extracting the DNA of the four-fingered horse bay or the polysquamous four-fingered horse bay; (3) using the DNA in step (2) as a template, using primer pair 1 and primer pair 2 to carry out PCR amplification, after the amplification reaction is completed, carry out electrophoresis detection, the electrophoresis results show that there are a specific line at the vicinity of 469bp and 606bp respectively Sex DNA-specific bands, the sample tested at this time is the four-fingered horse bay fish, if there is no band near 469bp or 606 bp, it is the polysquamous four-fingered horse bay fish. 3. the method for Molecular Identification of four-fingered squid and polysquamous squid according to claim 2, is characterized in that: in step (2), adopt phenol chloroform organic solvent method to extract squamous squid or polysquamous four-fingered squid The DNA of the mackerel. 4. the method for molecular identification of four-fingered horse shark according to claim 2 and polysquamous four-fingered horse shark, is characterized in that: when adopting primer pair 1 and primer pair 2 to carry out PCR amplification in step (3), 20 μL PCR reaction The system contains 2 μL of DNA, 10 μL of primer pair 1 or 2, 2 μL of PCR buffer premixed with Taq, dNTP, and MgCl ₂ , and 6 μL of ddH ₂ O. 5. the method for Molecular Identification of four-fingered mackerel according to claim 2 and polysquamous four-fingered mackerel, it is characterized in that: when adopting primer pair 1 to carry out PCR amplification in step (3), PCR reaction program is: firstly 95°C for 5 min, followed by 32 cycles of 94°C for 30s, 66°C for 30s, 72°C for 45, and then 72°C for 10min; when using primer pair 2 for PCR amplification, firstly 95°C for 5min, then 94°C for 30s, 56°C for 30s, 72°C 45 for a total of 32 cycles, and then 72°C for 10 min. 6. the method for molecular identification of four-fingered horse slug and polysquamous four-fingered squirrel according to claim 2, it is characterized in that: when carrying out electrophoresis detection in step (3), adopt mass percentage composition to be 1.5% agarose Detection by gel electrophoresis, and observation under UV light after adding Redsafe.