CN116555433B - Primer and kit for identifying Huang Qiji and flat sea bream and application - Google Patents
Primer and kit for identifying Huang Qiji and flat sea bream and application Download PDFInfo
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- 238000012408 PCR amplification Methods 0.000 claims description 26
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Abstract
The invention discloses a primer for identifying Huang Qiji porgy and flat porgy, which is primer 1 or primer 2, wherein the primer 1 comprises a forward primer 1F and a reverse primer 1R, and the nucleotide sequence of the forward primer 1F is shown as SEQ ID NO:1, the nucleotide sequence of the reverse primer 1R is shown as SEQ ID NO:2 is shown in the figure; the primer 2 comprises a forward primer 2F and a reverse primer 2R, wherein the nucleotide sequence of the forward primer 2F is shown as SEQ ID NO:3, the nucleotide sequence of the reverse primer 2R is shown as SEQ ID NO: 4. The invention also discloses a kit and a method for identifying the oplegnathus fasciatus and the flat porgy, and application of the primer, the kit and the method in identifying the Huang Qiji porgy and the flat porgy.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a primer, a kit and application for identifying Huang Qiji porgy and flat porgy.
Background
The fish mitochondrial genome DNA comprises: 1 major non-coding region (D-loop), 1 light chain replication initiation region (O L), 2 ribosomal RNA genes (12 rRNA and 16S rRNA), 22 transfer RNA genes (tRNA) and 13 protein-encoding genes. Pamaceraceae (Sparidae) is belonging to the class Osteichthyes, the order Perciformes, the order Percoides (Percoides). There are 34 species of 133 reported worldwide, widely distributed in temperate to tropical waters of the atlantic, indian and pacific. The fish of the Papilionaceae has delicious meat, rich nutrition and higher economic value, and occupies an important position in China and even world ocean fishery resources. Many species are rare economic fish and important mariculture subjects, such as Huang Qiji porgy (Acanthopagrus latus (Houttuyn, 1782)), and flat porgy [ Rhabdosargussarba ]1775)]Etc. In recent years, with the success of artificial breeding technology, the cultivation area of fish of the Papilionaceae family of China is continuously enlarged, and the cultivation variety is also continuously increased.
Since Huang Qiji sea bream and flat sea bream have many similarities in morphological characteristics, particularly in the fry stage, it is difficult to distinguish them by naked eyes, which brings about many inconveniences for fry screening and management in the fry farm. At present, CN115094145A discloses a acanthus praecox SNP marker and a screening method thereof, and CN109055571A discloses a Huang Qiji snapper microsatellite marker specific primer and application, but the two patents are mainly used for family identification, population genetic structure, genetic breeding, paternity test and the like of Huang Qiji snapper. In addition, CN103131762A discloses a molecular biological method for identifying the flat sea bream, but the primer is only suitable for identifying the flat sea bream, but not suitable for molecular identification of other sea bream fishes, and the primer design adopts a mode of combining other fish universal primers and flat sea bream specific primers. Therefore, a method capable of rapidly and effectively distinguishing two kinds of snapper fishes is developed, and the method has important significance for identifying snapper fish species in aquaculture.
Disclosure of Invention
The invention aims to provide a primer and a kit for identifying Huang Qiji sea bream and flat sea bream.
The invention also aims at providing a method for identifying the acanthus vannamei and the flat sea bream.
A final object of the invention is to provide the use of the above primers, kit and method for identifying Huang Qiji sea bream and flat sea bream.
The first object of the present invention can be achieved by the following technical means: a primer for identifying Huang Qiji porgy and flat porgy, wherein the primer is primer 1 or primer 2, the primer 1 comprises a forward primer 1F and a reverse primer 1R, and the nucleotide sequence of the forward primer 1F is shown as SEQ ID NO:1, the nucleotide sequence of the reverse primer 1R is shown as SEQ ID NO:2 is shown in the figure; the primer 2 comprises a forward primer 2F and a reverse primer 2R, wherein the nucleotide sequence of the forward primer 2F is shown as SEQ ID NO:3, the nucleotide sequence of the reverse primer 2R is shown as SEQ ID NO: 4.
Specific:
the nucleotide sequence of primer 1 comprises a forward primer 1F and a reverse primer 1R, and the nucleotide sequences are respectively:
forward primer 1F: TCTATGGTTTGCAGAGTTGC;
reverse primer 1R: ATTGAATCCTGATGGATGGC.
The nucleotide sequence of primer 2 comprises a forward primer 2F and a reverse primer 2R, and the nucleotide sequences are respectively:
forward primer 2F: AACAAGCGAGGACATCACTG;
reverse primer 2R: GATGTTGCACGTGGCGCTCA.
The invention also provides a kit for identifying Huang Qiji porgy and flat porgy, which comprises the primer.
The second object of the present invention can be achieved by the following technical means: a method for identifying oplegnathus fasciatus and oplegnathus fasciatus, comprising the following steps: and (3) taking genomic DNA of the sample to be detected as a template, carrying out PCR amplification by using the primer, carrying out electrophoresis on a PCR amplification product, and judging whether the sample to be detected is Huang Qiji sea bream or flat sea bream according to an electrophoresis result.
As a preferred embodiment of the present invention, when PCR amplification is performed using primer 1, when the electropherogram shows a target band of 477bp in size, it is identified as Huang Qiji porgy; the electropherogram shows a target band with the size of 572bp, and the flat sea bream is identified.
As another preferred embodiment of the present invention, when PCR amplification is performed using the primer 2, it is identified as Huang Qiji porgy when the electropherogram shows a band of size 494bp, and it is identified as flat porgy when the electropherogram shows a band of size 1025 bp.
Preferably, in PCR amplification, a PCR amplification system is used comprising: 90-110 ng of template DNA, 0.15 mu L of Taq enzyme, 0.15 mu L of upstream and downstream primers respectively, and filling deionized water to 10 mu L.
Preferably, in PCR amplification, the procedure for PCR amplification is: pre-denaturation at 95 ℃ for 5min; denaturation at 95 ℃ for 30s, annealing at 50-60 ℃ for 30s, and extension at 72 ℃ for 1min for 30 cycles; extending at 72 ℃ for 10min; preserving at 4 ℃.
The last object of the invention can be achieved by the following technical scheme: the primer, the kit or the method is applied to identifying Huang Qiji porgy and flat porgy.
The invention has the following advantages:
(1) According to the invention, any one of the two pairs of primers is utilized, and the PCR amplification method is adopted to rapidly and effectively identify the two snapper fishes, so that the reaction is stable and the repeatability is good;
(2) Compared with mitochondrial genome DNA bar code sequencing technology, the method has better intuitiveness;
(3) Compared with the traditional morphological identification technology, the method has the characteristics of high detection accuracy and strong objectivity;
(4) The primer or the kit provided by the invention has wide application prospect in identifying two kinds of snapper fishes or preparing the kit for identifying the two kinds of snapper fishes.
Drawings
The invention will be further described with reference to the accompanying drawings, in conjunction with examples.
FIG. 1 is a diagram showing the result of electrophoresis of PCR amplification products of a pair of primers 1 of example 3 for a pair of Huang Qiji sea bream and flat sea bream, wherein the left side 1-12 are 12 samples of the sea bream Acanthopagrus latus and the right side 1-12 are 12 samples of the flat sea bream Rhabdosargussarba;
FIG. 2 is a diagram showing the results of electrophoresis of PCR amplification products of 2 pairs of Huang Qiji sea bream and flat sea bream using the primer set in example 3, wherein the left side 1-12 are 12 samples of sea bream Acanthopagrus latus and the right side 1-12 are 12 samples of flat sea bream Rhabdosargussarba.
Detailed Description
The following detailed description of the present invention is provided in connection with specific embodiments so that those skilled in the art may better understand and practice the present invention. The following examples and figures are for illustrative purposes only and are not to be construed as limiting the invention. The reagents or materials used in the examples, unless otherwise specified, were all commercially available. Unless otherwise indicated, all laboratory instruments used are laboratory conventional.
Example 1
First, the DNA differential sequence of Huang Qiji sea bream and flat sea bream was identified, and amplification primers were designed and electrophoresed. The method comprises the following specific steps of:
(1) The Huang Qiji or flat porgy second generation genomic sequencing data was assembled into contig sequences using the soap de novo software, or the Huang Qiji or flat porgy third generation genomic sequencing data was assembled into contig sequences using canu2.2 (default parameters).
(2) Single copy ortholog sequence detection using blastn (version 2.13.0) alignment software, set maximum similarity to 1e -50 Under the condition that the minimum alignment length is 1000bp, the Huang Qiji sea bream and the flat sea bream are mutually aligned. When Huang Qiji sea bream is aligned to flat sea bream, huang Qiji sea bream sequences are aligned only to one sequence of flat sea bream, and the corresponding sequence of flat sea bream is aligned back to the sequence corresponding to Huang Qiji sea bream, which are optimal alignment in both directions. Satisfying the above conditions, is defined as a single copy orthologous sequence.
(3) The sequence pairs of the oplegnathus fasciatus and the oplegnathus fasciatus defined as single copy orthologous sequences were subjected to multiple sequence alignment by using multiple sequence alignment software such as mafft or muscle. Sequences flanked by conserved sequences (suggested that the conserved sequences are greater than 100bp in length and that the conserved sequences are more than 90% similar) and centered on the difference in fragment length are selected as identification sequences.
(4) In the conserved region of the molecular marker, primer5 software was used to design specific primers.
(5) And (3) verifying the specificity of the primer, adopting a blastn comparison strategy, comparing the designed primer back to the genome sequence, wherein the comparison is a default parameter, and if the comparison is only to a target sequence section, the primer is used for subsequent molecular marker development.
The final screened primer is primer 1 or primer 2, wherein the primer 1 comprises a forward primer 1F and a reverse primer 1R, and the nucleotide sequence of the forward primer 1F is shown as SEQ ID NO:1, the nucleotide sequence of the reverse primer 1R is shown as SEQ ID NO:2 is shown in the figure; primer 2 comprises a forward primer 2F and a reverse primer 2R, and the nucleotide sequence of the forward primer 2F is shown as SEQ ID NO:3, the nucleotide sequence of the reverse primer 2R is shown as SEQ ID NO: 4.
Specific:
the nucleotide sequence of primer 1 comprises a forward primer 1F and a reverse primer 1R, and the nucleotide sequences are respectively:
forward primer 1F: TCTATGGTTTGCAGAGTTGC (SEQ ID NO: 1);
reverse primer 1R: ATTGAATCCTGATGGATGGC (SEQ ID NO: 2).
The nucleotide sequence of primer 2 comprises a forward primer 2F and a reverse primer 2R, and the nucleotide sequences are respectively:
forward primer 2F: AACAAGCGAGGACATCACTG (SEQ ID NO: 3);
reverse primer 2R: GATGTTGCACGTGGCGCTCA (SEQ ID NO: 4).
The primer can also be developed into a kit for use.
Then, the genomic DNAs of 12 yellow-fin porgy and 12 flat porgy were used as templates, PCR amplification was performed using the above-mentioned primer 1 or primer 2, and the products were electrophoresed, and the type of the fish of the Papilionaceae family of the sample was verified based on the electrophoresis result.
In PCR amplification, the PCR amplification system adopted comprises: 90-110 ng of template DNA, 0.15 mu L of Taq enzyme, 0.15 mu L of upstream and downstream primers respectively, and filling deionized water to 10 mu L.
In PCR amplification, the procedure for PCR amplification was: pre-denaturation at 95 ℃ for 5min; denaturation at 95 ℃ for 30s, annealing at 50-60 ℃ for 30s, and extension at 72 ℃ for 1min for 30 cycles; extending at 72 ℃ for 10min; preserving at 4 ℃.
As shown in FIG. 1 (left side numbers 1-12 represent 12 individuals of yellow-fin sea bream, right side numbers 1-12 represent 12 individuals of flat sea bream), when PCR amplification was performed using the primer 1, when the electropherogram showed a target band (SEQ ID NO: 5) of about 477bp in size, it was identified as Huang Qiji sea bream; the electropherogram shows a target band (shown as SEQ ID NO: 6) with the size of about 572bp, and the flat sea bream is identified.
The target band sequence of the primer 1 amplified Huang Qiji porgy is as follows:
TCTATGGTTTGCAGAGTTGCGCCGTAAACATTAATTTTGGTAAGTGGGC
TGCTTAAGTCAATGTGAAGTATATAGCCAAAATGCTATGGTAACAGTTTCAA
CCAAAAACAAACAAAAGAAAAGGGTAAAATATTTCTCCTGATCTTGTTAAT
CTTATACANGAGTCTCGATGTAATTTTCTTTACTTTACTGTATTGAATATTACA
GTTTTCTGCTGTACCAACCCAATTTCCCTAGAGGGGCATTAAGGTTTAATCA
TATCCTCGACATAATACTAATCTAACATTAAGAAACTAAAATTAAACAAAAA
TGAACAATTTGATGATGATAACCACTGTGTATCAACATCAAATGACCAGTTT
GAAACATGTTAATTGATTCTCTCTGCAGGTCTTGAAGGTTTGATAGCGCTGC
TATTTATATGGCACAACTGTTCATTTGCTGACAAGGACAAATCGCCATCCAT
CAGGATTCAAT(shown in SEQ ID NO: 5).
The target band sequence of the primer 1 for amplifying the flat sea bream is as follows:
TCTATGGTTTGCAGAGTTGCGCCGCACATATTCATTCTGGTGATTGGCT
TGCTTAAGTCAATGTAAAAGTATATAGCCAAAATGCTCTGGTAAACATTTCA
ACCAAAAACAAACAAAAGAAAAGGCTAAAATATTTCTCCTGATCTTGTTAA
TTCTATACACTAGTCTAGAGGTCATTTTCTTCCTTTTACTGTATTGAGTATTAC
AGTTTTCTGCTGCACCAACCCAATTTCCCTAGGGGGGTATTAAGGTTTAATC
ATATCCTCGATATAATACTAATGTAGCATTTAGAAACTAAAATCAAGCAAAA
ATGAACAATGTGATGACCACTGCGTATCATCATCAAATGGCCAGTTTAAAAC
ATAATACTAATATATAATACTAATGTAGCATTTAGAAACTAAAATCAAGCAAA
AATGAACAATGTGATGACCACTGCGTATCATCATCAAATGGCCAGTTTAAA
ACATGTTAATAGATTCTCTCTGCAGGTCCTGAAAGTTTGATAGCGCTGCTAT
TCAACTGCTCATTTGCTGACAAGGACAAATGATCGCCATCCATCAGGATTC
AAT(shown in SEQ ID NO: 6).
As shown in FIG. 2 (left hand numbers 1-12 represent 12 individuals of yellow-fin sea bream, right hand numbers 1-12 represent 12 individuals of flat sea bream), when PCR amplification was performed using primer 2, when the electropherogram showed a band of interest of about 494bp in size (SEQ ID NO: 7), it was identified as Huang Qiji sea bream, and when the electropherogram showed a band of interest of about 1025bp in size (SEQ ID NO: 8), it was identified as flat sea bream.
The target band sequence of the primer 2 amplified Huang Qiji porgy is as follows:
AACAAGCGAGGACATCACTGCGGTCGGCAGAGAAAATAATCAGCACATTC
ATTGTTAATATAAACAAGAATCATTTGTTACAGCCCTCTGAGTCAAAGGAAC
CTTCTGAAATCTGAAGTACCTTGTATCTTTAATCCAATCTCAAACTTAATAGC
ACCTTTAAATCACACCTGTATTGATCAACTCTTGGCTTGTACTATTCTGTACA
TACTTGTTGTTATATAGATTTGATCTTCCGAGCCGTTTCCATCCATAAATGAT
CAAAAATAAAAGGTTTATCATTTTACCGTTTCCAGTGACATGAGAATATTAA
CCTTCGTGTGAATGTCATGTGATGTTTTCAACACACATGAAGATACATACAT
TTCTAAGGAATGCTGGATTGAATCAGCCGGAATAATGAAATAACAGGAGGT
CATTTTTTGCAGCGTGTTAGTGGTGCAGCTTCGCTTCAGAGCGCTGACGGG
GAAAACGTGAGCGCCACGTGCAACATC(shown in SEQ ID NO: 7).
The target band sequence of the primer 2 for amplifying the flat sea bream is as follows:
AACAAGCGAGGACATCACTGCAGTCGACAGGGAGAATAATCAGCACA
TCATTGCTCATATAAATAATAAGCGTTTGTTACAACCCTAAAATCACACTAA
ACGGCTGGACAAGTGGAGGTTGCTATTTAGGGTTAAAACTGATTACTGATG
TCCTGACAGTCACTTCATATTATTCACATAAAAATCAAGAGCTGCACTGAGG
TGTAGCTCCTGTAGTTTTAGGGCTCGACTGCGCTTAATCATGAAGTTTACTG
TTATATACAAGTTTCAGTATAACACCTTTTACTTTTTTATAGCTCATTTGTTTA
AATTTAAAAATTGTAATTGTCACCATTCATATGAAGCCGAATGTGTTACTAG
CTGTCCCTGTTTGCAGTGTAAACATGGACGTGCTTACAACTAAAACTGGAT
TACTTTGATACCGAAGAAAACTTAAAATCACAATGATTCTCAGGAAAGTTC
TGCAGGTGAAGGAACCGTCTGAAGTTATCCAAGTTATTTTTGACACGAAAT
GCTTCCTCATTTTATCATCTTCAACCAGAATCTTTGGATTTACACTTTTGGGT
GAATTGATCCTTCTGAATTCAATTGAATTCTAAGATAAGTTATTTGTCATTCA
TCATTTCTTTCCTCTTTCTTTCTTTCTTTCTTTCTTTCTTCATCTTGTATCTTTA
CACAAATTTCAAACTTCATATCACCTTTAAATCACACCTGTATTGATCAACT
CTTGGCTCGTACGATTCTGTACATACTGGGTGTTTTATAGCTTAGATCTTCCG
AGCCATCTCCATCCGTAAGTGATCAAAAATAAAAAGTTTCATCATTTTACCG
TTTCCAGCAACATGAGAATATTAACTGTCGTGTGACATTTTCAACACACATG
AAGATATGTACATTTCTAAAGAATCCTCTATTGAATCAGTCGGAATAATGAA
ATTACAGTAAGTCATTTTTTGCAGCGTGTTnGTGGTGCAGCGTCTCCTCAGA
GCGCTGACGGGGAAAACGTGAGCGCCACGTGCAACATC(shown in SEQ ID NO: 8).
The above embodiments are merely illustrative of the present invention, and the protective scope of the present invention is not limited to the above embodiments only. The object of the present invention can be achieved by a person skilled in the art based on the above disclosure, and any modifications and variations based on the concept of the present invention fall within the scope of the present invention, which is defined in the claims.
Claims (7)
1. The method for identifying the oplegnathus fasciatus and the oplegnathus fasciatus is characterized by comprising the following steps of: taking genomic DNA of a sample to be detected as a template, carrying out PCR amplification by using a primer, carrying out electrophoresis on a PCR amplification product, judging the sample to be detected as Huang Qiji porgy or flat porgy according to an electrophoresis result, wherein the primer is primer 1 or primer 2, the primer 1 comprises a forward primer 1F and a reverse primer 1R, and the nucleotide sequence of the forward primer 1F is shown as SEQ ID NO:1, the nucleotide sequence of the reverse primer 1R is shown in SEQ ID NO:2, the preparation method comprises the following steps of (1); the primer 2 comprises a forward primer 2F and a reverse primer 2R, wherein the nucleotide sequence of the forward primer 2F is shown as SEQ ID NO:3, the nucleotide sequence of the reverse primer 2R is shown in SEQ ID NO: 4.
2. The method for identifying Huang Qiji and flat bream according to claim 1, wherein: when the primer 1 is adopted for PCR amplification, if the electropherogram shows a target band with the size of 477bp, identifying the target band as Huang Qiji porgy; the electropherogram shows a target band with the size of 572bp, and the flat sea bream is identified.
3. The method for identifying Huang Qiji and flat bream according to claim 1, wherein: when the primer 2 is used for PCR amplification, the target band with the size of 494bp is displayed on the electropherogram, the target band with the size of 1025bp is identified as Huang Qiji porgy, and the target band with the size of 494bp is displayed on the electropherogram, and the target band with the size of Huang Qiji bp is identified as flat porgy.
4. The method for identifying Huang Qiji and flat bream according to claim 1, wherein: in PCR amplification, the PCR amplification system adopted comprises: 90-110 ng of template DNA, 0.15 mu L of Taq enzyme, 0.15 mu L of upstream and downstream primers respectively, and filling deionized water to 10 mu L.
5. The method for identifying Huang Qiji and flat bream according to claim 1, wherein: in PCR amplification, the procedure for PCR amplification was: pre-denaturation at 95 ℃ for 5min; denaturation at 95 ℃ for 30s, annealing at 50-60 ℃ for 30s, and extension at 72 ℃ for 1min for 30 cycles; extending at 72 ℃ for 10min; preserving at 4 ℃.
6. Use of a primer or a kit for identifying Huang Qiji and flat sea bream in identifying Huang Qiji and flat sea bream, wherein the primer is primer 1 or primer 2, the primer 1 comprises a forward primer 1F and a reverse primer 1R, and the nucleotide sequence of the forward primer 1F is shown as SEQ ID NO:1, the nucleotide sequence of the reverse primer 1R is shown in SEQ ID NO:2, the preparation method comprises the following steps of (1); the primer 2 comprises a forward primer 2F and a reverse primer 2R, wherein the nucleotide sequence of the forward primer 2F is shown as SEQ ID NO:3, the nucleotide sequence of the reverse primer 2R is shown in SEQ ID NO:4, wherein the first part is a part of the second part; the kit comprises the primer.
7. Use of the method according to any one of claims 1-5 for identifying Huang Qiji sea bream and flat sea bream.
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