CN103740855A - DNA (deoxyribonucleic acid)-barcode-based eel species identification method - Google Patents

Abstract

The invention relates to a method for identifying eel species based on DNA barcodes, more particularly to a method for identifying species of Japanese eels, American eels and European eels based on DNA barcodes. The method extracts eel DNA, and then uses DNA barcode primers for PCR amplification to amplify a 244bp product. The DNA barcode primers are EEL244-F: CCTGTTCCTCGTGGGGGCTTTT, EEL244-R: ATGGCATAACGAGGGTTTAACTG, and finally according to the nucleotide sequence characteristics of the species Variation sites for species identification. The method is time-consuming, easy to operate, and highly specific. It can identify processed eels that cannot be identified morphologically and other common eel processed products, and provides technology for maintaining the interests of enterprises and consumers as well as normal market order. support.

Description

Identification method of eel species based on DNA barcode

technical field

The invention relates to a method for identifying eel species based on DNA barcodes, more particularly to a method for identifying species of Japanese eels ( Anguilla japonica ), American eels ( Anguilla rostrata ) and European eels ( Anguilla anguilla ) based on DNA barcodes.

Background technique

Fujian Province is a large eel breeding and processing export province. There are three main products of eel, namely Japanese eel ( Anguilla japonica ), American eel ( Anguilla rostrata ) and European eel ( Anguilla anguilla ). The annual export value reaches billions of yuan. Since eels are processed, such as grilled or cooked in other shapes, the species cannot be distinguished from the shape, and the prices of different types of eels are different. In order to prevent shoddy products and protect the interests of enterprises and consumers, a biological identification method for eel species was established at the request of enterprises and law enforcement agencies. The invention meets the needs of foreign trade and inspection and quarantine work, and is of great significance to the foreign trade of Fujian Province, which is a large eel export province.

DNA barcode (DNA barcode) refers to a standard, sufficiently variable, easy-to-amplify, and relatively short DNA fragment that can represent the species in an organism. DNA barcoding has become an important tool in ecological research, not only for species identification, but also to help biologists further understand the interactions that occur within ecosystems. However, there is no report on the identification of eel species using DNA barcodes at home and abroad.

Contents of the invention

Based on the above purpose, the present invention provides a method for identifying eel species based on DNA barcodes.

The present invention firstly provides a kind of DNA barcode primer of Japanese eel, American eel and European eel, the PCR product base number of DNA barcode target fragment is 244 bp, the nucleotide sequence of described DNA barcode primer is:

EEL 244-F: CCTGTTCC TCGTGGGGGC TTTT;

EEL 244-R: ATGGCATAACGAGGGTTTAACTG.

The present invention determines the specific DNA barcode base sequences of the three eels by comparing and analyzing the base sequences of the PCR products of the DNA barcode target fragments of the Japanese eel, American eel and European eel, and establishes the molecular structure of the eel species. methods of biological identification. The eel species identification method based on DNA barcode of the present invention, the method comprises the following steps:

(1) Eel DNA extraction;

(2) Using DNA barcode primers for PCR amplification to amplify a 244 bp product, the DNA barcode primers are EEL 244-F: CCTGTTCC TCGTGGGGGC TTTT, EEL 244-R: ATGGCATAACGAGGGTTTAACTG;

(3) Sequence feature comparison for species identification, wherein, the nucleotide sequence feature variation sites shown in the following table are used for species identification:

。

.

The PCR amplification conditions in step (2) are: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 30 s, annealing at 45°C for 30 s, extension at 72°C for 60 s, 5 cycles; denaturation at 94°C for 30 s, 51°C Anneal for 60 s, extend at 72°C for 60 s, 35 cycles; extend at 72°C for 10 min; reaction system: 10 × Taq buffer 2.5 μL, Taq enzyme 1.0 U, template DNA 200 ng, MgCl ₂ 3.5 mmol/L, dNTPs 300 μmol/L, upstream primer 0.5 μmol/L, downstream primer 0.5 μmol/L, make up the total volume to 25 μL with ddH ₂ O.

The method of the present invention is used to specifically identify three types of eels Japanese eel ( Anguilla japonica ), American eel ( Anguilla rostrata ) and European eel ( Anguilla anguilla ). The method is time-consuming, easy to operate and strong in specificity. It can identify processed eels that cannot be identified morphologically and other common eel processed products, providing technical support for maintaining the interests of enterprises and consumers as well as normal market order.

Description of drawings

Figure 1 is the PCR amplification product map of three species of eels, in which M: DNA Marker; 1. Japanese eel ( Anguilla japonica ); 2. American eel ( Anguilla rostrata ); 3. European eel ( Anguilla anguilla ).

Figure 2 is the comparison of the specific base sequences of the 16S rDNA partial fragments of three species of eels, in which the underlined part is the primer sequence, and the bold font in the frame is the specific base sequence of the three eel species, that is, from the forward primer to the first The 104th-106th base sequence starting from base C is: Japanese eel AAC, American eel GTT, European eel GGT.

Detailed ways

Reagent source: 10 × Taq buffer, Taq enzyme and dNTPs were purchased from Shanghai Sangon Co., Ltd., primers were synthesized by Shanghai Sangon Co., Ltd., and DNA sequence was tested by Guangzhou Invent Biotechnology Co., Ltd.

Primer synthesis

Anguilla japonica (AJ244813.1), American eel Anguilla rostrata (AJ244829.1), European eel Anguilla anguilla (AJ244826.1) according to GenBank query

16S rRNA coding gene sequence (16S rDNA), 3 pairs of PCR primers were automatically designed by Primer Premier 5.0, the amplified PCR products were sequenced, and one of the three eels with specific base sequences was selected from them. For the primers, the sequences are shown in Table 1.

Table 1 PCR primers and their annealing temperature

The sequencing results of the PCR amplification products of the three eel samples were compared and verified in Genbank (Gene Bank), and they were completely consistent with the corresponding sequences of their respective species. The sequences are as follows (the underlined part is the primer, the 5' end is the forward primer, and the 3' end is the reverse complementary sequence of the base sequence of the reverse primer, and the eel species-specific base sequence is in the box):

日本鳗（ Anguilla japonica ）5'- CCTGTTCCT TCGTGGGGGCTTTT TTCTCCCCCATGGTCGCCCCAACCGAAGACATTTGGATCAGTTTACGTCGGGTTTCGTGGCCTTTGTGTTCCTTTTGGTT AAC TTGGTTTCTTTACATGTTTGATCTTTTGTCTAAAGCTCCATAGGGTCTTCTCGTCTTATGTATTTATGTCCGCTTCTGCACGGGCAGATCAATTTCATTGACTAGGGGAAGGAGA CAGTTAAACCCTCGTTATGCCAT -3'(244 bp)，

American Eel ( Anguilla rostrata )

5'- CCTGTTCCTTCGTGGGGGCTTTT CTTTCCCCCATGGTCGCCCCAACCGAAGACATTTAGATCAGTTTACGTTGGGTTTTGTGGCCTTCGTGTTCCTTTTGGTT GTT TTGGTTTCTTTACATGTTTGATCTTTTGTCTAAAGCTCCATAGGGTCTTCTCGTCTTATGTATTTATGTCCGCTTCTGCACGGGCAGATCAATTTCATTGACTGGGGGAAGGAGA CAGTTAAACCCTCGTTATGCCAT -3'(244 bp)，

European eel ( Anguilla anguilla )

5'- CCTGTTCCTTCGTGGGGGCTTTT CTTTTCCCCCATGGTCGCCCCAACCGAAGACATTTAGATCAGTTTACGTTGGGTTTTGTGGCCTTCGTGTTCCTTTTGGTT GGT TGGTTTCTTTACATGTTTGATCTTTTGTCTAAAGCTCCATAGGGTCTTCTCGTCTTATGTGTTTATGTCCGCTTCTGCACGGGCAGATCAATTTCATTGACTAGGGGAAGGAGA CAGTTAAACCCTCGTTATGCCAT -3'(244 bp)。

Eel DNA Extraction

Add about 50 mg of sample powder to a 1.5 mL centrifuge tube, add 200 μL TE solution (pH8.0), vortex and mix; add 400 μL lysate, vortex mix; add 600 μL phenol: chloroform: isoamyl alcohol (25: 24:1), shake vigorously, and centrifuge at 12,000 g for 10 min; take the supernatant, add an equal volume of chloroform:isoamyl alcohol (24:1), shake vigorously, and centrifuge at 12,000 g for 10 min; take the supernatant, add 0.8 times Volume isopropanol, centrifuged at 12000 g for 10 min; take the precipitate, add 400 μL NaCl (1 mol/L) to dissolve in a warm bath at 65 °C, then add 5 μL proteinase K (20 mg/mL), 5 μL Rnase A (10 μg/mL)], shake vigorously for more than 30 seconds, incubate at 37°C for 1 hour, and shake regularly during this period. Add an equal volume of water-saturated phenol: chloroform: isoamyl alcohol (25:24:1), shake, and centrifuge at 12,000 r/min for 15 min. Repeat this step 1-2 times until the protein is completely removed. Take the supernatant, add 2 times the volume of absolute ethanol, shake slightly, and centrifuge at 12,000 r/min for 10 min. Take the precipitate, wash it twice with 70% ethanol, dry it, and add 100 μL TE (pH8.0) to dissolve it.

Two replicates were set up for each sample. Commercially available kits for animal DNA extraction can also be used.

Concentration and Purity Determination

Measure the absorbance at ₂₆₀ nm and ₂₈₀ nm of the sample DNA with a nucleic acid protein analyzer, and calculate the DNA purity and concentration respectively. Concentration (μg/mL) = 50 × OD ₂₆₀ value.

Reaction system and conditions

The reaction system was: 10 × Taq buffer 2.5 μL, Taq enzyme 1.0 U, template DNA 200 ng, MgCl ₂ 3.5 mmol/L, dNTPs 300 μmol/L, upstream primer 0.5 μmol/L, downstream primer 0.5 μmol/L, ddH ₂ O to bring the total volume to 25 μL.

The conditions for PCR amplification were: pre-denaturation at 94°C for 3 min. Denaturation at 94°C for 30 s, annealing at 45°C for 30 s, extension at 72°C for 60 s, 5 cycles; denaturation at 94°C for 30 s, annealing at 51°C for 60 s, extension at 72°C for 60 s, 35 cycles. Extend at 72°C for 10 min. Store at 4°C.

Result analysis

Electrophoresis of PCR products is shown in Figure 1. Nos. 1, 2, and 3 are Japanese eel ( Anguilla japonica ), American eel ( Anguilla rostrata ), and European eel ( Anguilla anguilla ), respectively. The size of the PCR product detected is 244 bp. The samples (all provided by Huasheng Food Co., Ltd., Sanming City, Fujian Province) amplified PCR products. Figure 2 is the base pairing map of DNA barcodes (partial 16S rDNA fragment, 244 bp) of Japanese eel, American eel, and European eel. It can be seen that the method is specific to the three eels.

<110> Inspection and Quarantine Technology Center of Fujian Entry-Exit Inspection and Quarantine Bureau

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tgtttgatct tttgtctaaa gctccatagg gtcttctcgt cttatgtatt tatgtccgct 180

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Claims (3)

1. A DNA barcode primer, characterized in that: the nucleotide sequence of the DNA barcode primer is: EEL 244-F: CCTGTTCC TCGTGGGGGC TTTT; EEL 244-R: ATGGCATAACGAGGGTTTAACTG. 2. A method for eel species identification based on DNA barcodes, characterized in that: the method may further comprise the steps: (1) Eel DNA extraction; (2) Using DNA barcode primers for PCR amplification to amplify a 244 bp product, the DNA barcode primers are EEL 244-F: CCTGTTCC TCGTGGGGGC TTTT, EEL 244-R: ATGGCATAACGAGGGTTTAACTG; (3) Sequence feature comparison for species identification, wherein, the nucleotide sequence feature variation sites shown in the following table are used for species identification:

. 3. The species identification method for Japanese eel, American eel and European eel according to claim 2, characterized in that: the conditions for PCR amplification in step (2) are: pre-denaturation at 94°C for 3 min, denaturation at 94°C 30 s, annealing at 45°C for 30 s, extension at 72°C for 60 s, 5 cycles; denaturation at 94°C for 30 s, annealing at 51°C for 60 s, extension at 72°C for 60 s, 35 cycles; extension at 72°C for 10 min; the reaction system was : 10 × Taq buffer 2.5 μL, Taq enzyme 1.0 U, template DNA 200 ng, MgCl ₂ 3.5 mmol/L, dNTPs 300 μmol/L, upstream primer 0.5 μmol/L, downstream primer 0.5 μmol/L, ddH ₂ O The total volume was made up to 25 μL.

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