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

DNA (deoxyribonucleic acid)-barcode-based eel species identification method Download PDF

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CN103740855A
CN103740855A CN201410045586.3A CN201410045586A CN103740855A CN 103740855 A CN103740855 A CN 103740855A CN 201410045586 A CN201410045586 A CN 201410045586A CN 103740855 A CN103740855 A CN 103740855A
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eel
dna
anguilla
barcode
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陈文炳
邵碧英
缪婷玉
彭娟
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Inspection and Quarantine Technology Center of Fujian Entry Exit Inspection and Quarsntine Bureau
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Abstract

The invention relates to a DNA (deoxyribonucleic acid)-barcode-based eel species identification method, in particular to a DNA-barcode-based species identification method for anguilla japonica, anguilla rostrata and auguilla anguilla. The method comprises the following steps of performing eel DNA extraction, performing PCR (polymerase chain reaction) amplification to obtain a 244bp product by utilizing DNA barcode primers, and performing species identification according to nucleotide sequence characteristic variation sites of the species, wherein the DNA barcode primers are EEL244-F:CCTGTTCC TCGTGGGGGC TTTT and EEL244-R:ATGGCATAACGAGGGTTTAACTG. The method is low in time consumption, simple and convenient to operate and high in specificity, and can be used for identifying common processed eel products such as roasted eel which cannot be morphologically identified, and technical support is provided for the maintenance of enterprises and consumer benefits and normal market order.

Description

Eel species discrimination method based on DNA barcode
Technical field
The present invention relates to a kind of eel species discrimination method based on DNA barcode, more specifically relate to a kind of Japanese eel based on DNA barcode ( anguilla japonica), eel ( anguilla rostrata) and European eel ( anguilla anguilla) species discrimination method.
Background technology
Fujian Province is the large province of eel culture and export processing, and main products has three kinds of eels, Japanese eel ( anguilla japonica), eel ( anguilla rostrata), European eel ( anguilla anguilla), annual export amount reaches billions of units.Because eel processing is as roasted or making after other shape prepared food, cannot distinguish its kind from form, different types of eel price difference.In order to prevent adulterating, protection enterprise and consumer's interests, answer enterprise and law enforcement agency's requirement, sets up the biological assay method of eel kind.The present invention answers foreign trade and inspection and quarantine requirements of one's work, to significant as the foreign trade in the large Fujian Province of economizing of eel outlet.
DNA barcode (DNA barcode) refers to can represent these species in organism, standard, that have enough variations, easily amplification and relatively short DNA fragmentation.DNA barcode has become the important tool of ecological study, not only for species, identifies, also helps biologist further to understand the interaction occurring in the ecosystem simultaneously.But there is no the report that utilizes DNA barcode to identify eel species at present both at home and abroad.
Summary of the invention
Based on above-mentioned purpose, the invention provides a kind of eel species discrimination method based on DNA barcode.
First the present invention provides the DNA barcode primer of a kind of Japanese eel, eel and European eel, and the PCR product base number of DNA barcode object fragment is 244 bp, and the nucleotides sequence of described DNA barcode primer is classified as:
EEL 244-F： CCTGTTCC TCGTGGGGGC TTTT；
EEL 244-R： ATGGCATAACGAGGGTTTAACTG。
The present invention compares by the PCR product base sequence of the DNA barcode object fragment of Japanese eel, eel and European eel to for examination, determine 3 kinds of eels specific DNA barcode base sequence separately, set up the molecular biology identification method of eel species.Eel species discrimination method based on DNA barcode of the present invention, the method comprises the following steps:
(1) eel DNA extraction;
(2) utilize DNA barcode primer to carry out pcr amplification, amplify the product of 244 bp, described DNA barcode primer is EEL 244-F:CCTGTTCC TCGTGGGGGC TTTT, EEL 244-R:ATGGCATAACGAGGGTTTAACTG;
(3) sequence signature that carries out species discriminating is compared, and wherein, is that species discriminating is carried out in the nucleotide sequence Feature change site shown according to the form below:
Figure 455986DEST_PATH_IMAGE001
。
The condition of the described pcr amplification of step (2) is: 94 ℃ of denaturation 3 min, and 94 ℃ of sex change 30 s, 45 ℃ of annealing 30 s, 72 ℃ are extended 60 s, 5 circulations; 94 ℃ of sex change 30 s, 51 ℃ of annealing 60 s, 72 ℃ are extended 60 s, 35 circulations; 72 ℃ are extended 10 min; Reaction system is: 10 × taqbuffer 2.5 μ L, taqenzyme 1.0 U, template DNA 200 ng, MgCl 23.5 mmol/L, dNTPs 300 μ mol/L, upstream primer 0.5 μ mol/L, downstream primer 0.5 μ mol/L, uses ddH 2o mends cumulative volume to 25 μ L.
Method of the present invention for identify specifically three kinds of eels Japan eels ( anguilla japonica), eel ( anguilla rostrata), European eel ( anguilla anguilla) kind, the method is consuming time short, easy and simple to handle, high specificity, can differentiate through processing, the Pu that cannot identify from morphology burns the common converted productss of eel such as eel, for maintaining enterprise and consumer's interests and normally market order technical support is provided.
Accompanying drawing explanation
Fig. 1 is 3 kinds of eel pcr amplification product collection of illustrative plates, wherein M:DNA Marker; Japanese eel ( anguilla japonica); American eel ( anguilla rostrata); European eel ( anguilla anguilla).
Fig. 2 is 3 kinds of eel 16S rDNA Partial Fragment specificity base sequence comparison charts, wherein, underscore part is primer sequence, in frame, adding boldface type is 3 eel kind specificity base sequences, from first base of forward primer C, 104-106 base sequence is: Japanese eel AAC, American eel GTT, European eel GGT.
Embodiment
reagent source:10 × taqbuffer with taqenzyme, dNTPs are purchased from Shanghai Sheng Gong company limited, and primer entrusts Shanghai Sheng Gong company limited synthetic, and DNA sequence dna entrusts Guangzhou English Wei to create bio tech ltd's test.
primer is synthetic
According to the Japanese eel of GenBank inquiry anguilla japonica(AJ244813.1), eel anguilla rostrata(AJ244829.1), European eel anguilla anguilla(AJ244826.1) three kinds of eels
16S rRNA coding gene sequence (16S rDNA), application Primer Premier 5.0 Automated Designs 3 pairs of PCR primers, the PCR product amplifying checks order, and selects 1 pair of primer that can amplify 3 kinds of eels and have separately special base sequence, and sequence is as table 1.
Table 1 PCR primer and annealing temperature thereof
Figure 2014100455863100002DEST_PATH_IMAGE002
The pcr amplification product sequencing result of 3 kinds of eel samples is at Genbank(gene library) in comparison, conform to completely with the corresponding sequence of species separately, sequence is following, and (underscore part is primer, 5 ' end is forward primer, the reverse complementary sequence that 3 ' end is reverse primer base sequence is the sequence of eel species specificity base in frame):
Japan eel ( anguilla japonica) 5 '- cCTGTTCCT tCGTGGGGGCTTTT tTCTCCCCCATGGTCGCCCCAACCGAAGACATTTGGATCAGTTTACGTCGGGTTTC GTGGCCTTTGTGTTCCTTTTGGTT aACtTGGTTTCTTTACATGTTTGATCTTTTGTCTAAAGCTCCATAGGGTCTTCTCGTCT TATGTATTTATGTCCGCTTCTGCACGGGCAGATCAATTTCATTGACTAGGGGAAGG AGA cAGTTAAACCCTCGTTATGCCAT-3 ' (244 bp),
Eel ( anguilla rostrata)
5’- CCTGTTCCTTCGTGGGGGCTTTTCTTTCCCCCATGGTCGCCCCAACCGAAGACATTTAGATCAGTTTACGTTGGGTTTTGTGGCCTTCGTGTTCCTTTTGGTT GTTTTGGTTTCTTTACATGTTTGATCTTTTGTCTAAAGCTCCATAGGGTCTTCTCGTCTTATGTATTTATGTCCGCTTCTGCACGGGCAGATCAATTTCATTGACTGGGGGAAGGAGA CAGTTAAACCCTCGTTATGCCAT-3’(244 bp)，
Europe eel ( anguilla anguilla)
5’- CCTGTTCCTTCGTGGGGGCTTTTCTTTTCCCCCATGGTCGCCCCAACCGAAGACATTTAGATCAGTTTACGTTGGGTTTTGTGGCCTTCGTGTTCCTTTTGGTT GGTTGGTTTCTTTACATGTTTGATCTTTTGTCTAAAGCTCCATAGGGTCTTCTCGTCTTATGTGTTTATGTCCGCTTCTGCACGGGCAGATCAATTTCATTGACTAGGGGAAGGAGA CAGTTAAACCCTCGTTATGCCAT-3’(244 bp)。
eel DNA extraction
In 1.5 mL centrifuge tubes, add approximately 50 mg sample powder, add 200 μ L TE solution (pH8.0), vortex mixes; Add 400 μ L lysates, vortex mixes; Add 600 μ L phenol: chloroform: primary isoamyl alcohol (25:24:1), thermal agitation, centrifugal 10 min of 12000 g; Get supernatant liquor, add equal-volume chloroform: primary isoamyl alcohol (24:1), thermal agitation, centrifugal 10 min of 12000 g; Get supernatant liquor, add 0.8 times of volume Virahol, centrifugal 10 min of 12000 g; Get precipitation, add 400 μ L NaCl(1 mol/L) in bathing, 65 ℃ of temperature dissolve, then add 5 μ L Proteinase Ks (20 mg/mL), 5 μ L Rnase A(10 μ g/mL)], thermal agitation is more than 30 seconds, and 37 ℃ of temperature are bathed 1h, regularly rock during this time.Add isopyknic water-saturated phenol: chloroform: primary isoamyl alcohol (25:24:1), vibration, centrifugal 15 min of 12,000 r/min, repeat this step 1-2 time, until protein removal is clean.Get supernatant, add the dehydrated alcohol of 2 times of volumes, slightly rock, centrifugal 10 min of 12,000 r/min.Get precipitation, 70% ethanol cleans 2 times, dry, adds 100 μ L TE(pH8.0) dissolve.
Each sample is established 2 repetitions.Also can use city dealer's the test kit that animal DNA extracts that is applied to.
concentration and purity testing
Nucleic acid-protein analysis-e/or determining 260 nm and 280 nm place absorption values for sample DNA, calculate respectively DNA purity and concentration, and calculation formula is as follows: DNA purity=OD 260/ OD 280, ratio is comparatively desirable between 1.7-2.0; Double-stranded DNA concentration (μ g/mL)=50 × OD 260value.
reaction system and condition
Reaction system is: 10 × taqbuffer 2.5 μ L, taqenzyme 1.0 U, template DNA 200 ng, MgCl 23.5 mmol/L, dNTPs 300 μ mol/L, upstream primer 0.5 μ mol/L, downstream primer 0.5 μ mol/L, uses ddH 2o mends cumulative volume to 25 μ L.
The condition of pcr amplification is: 94 ℃ of denaturation 3 min.94 ℃ of sex change 30 s, 45 ℃ of annealing 30 s, 72 ℃ are extended 60 s, 5 circulations; 94 ℃ of sex change 30 s, 51 ℃ of annealing 60 s, 72 ℃ are extended 60 s, 35 circulations.72 ℃ are extended 10 min.At 4 ℃, preserve.
interpretation of result
PCR product electrophoresis is as Fig. 1, be respectively for 1,2, No. 3 Japanese eel ( anguilla japonica), eel ( anguilla rostrata), European eel ( anguilla anguilla), detecting PCR product size is 244 bp, 3 kinds of Pus are burnt eel sample (Hua Sheng Food Co., Ltd provides by Sanming City, Fujian Province), all amplify PCR product.Fig. 2 is DNA barcode (16S rDNA Partial Fragment, 244 bp) the base pairing figure of Japanese eel, eel, 3 kinds of European eel.Visible, the method has specificity to 3 kinds of eels.
<110> Inspection & Quarantine Technology Center of Fujian Entry-Exit Inspection & Quar
The eel species discrimination method of <120> based on DNA barcode
<160> 5
<170> PatentIn version 3.3
<210> 1
<211> 22
<212> DNA
<213> synthetic
<400> 1
cctgttcctc gtgggggctt tt 22
<210> 2
<211> 23
<212> DNA
<213> synthetic
<400> 2
atggcataac gagggtttaa ctg 23
<210> 3
<211> 244
<212> DNA
<213> Japan eel (Anguilla japonica)
<400> 3
cctgttcctt cgtgggggct tttttctccc ccatggtcgc cccaaccgaa gacatttgga 60
tcagtttacg tcgggtttcg tggcctttgt gttccttttg gttaacttgg tttctttaca 120
tgtttgatct tttgtctaaa gctccatagg gtcttctcgt cttatgtatt tatgtccgct 180
tctgcacggg cagatcaatt tcattgacta ggggaaggag acagttaaac cctcgttatg 240
ccat 244
<210> 4
<211> 244
<212> DNA
<213> eel (Anguilla rostrata)
<400> 4
cctgttcctt cgtgggggct tttctttccc ccatggtcgc cccaaccgaa gacatttaga 60
tcagtttacg ttgggttttg tggccttcgt gttccttttg gttgttttgg tttctttaca 120
tgtttgatct tttgtctaaa gctccatagg gtcttctcgt cttatgtatt tatgtccgct 180
tctgcacggg cagatcaatt tcattgactg ggggaaggag acagttaaac cctcgttatg 240
ccat 244
<210> 5
<211> 244
<212> DNA
<213> Europe eel (Anguilla anguilla)
<400> 5
cctgttcctt cgtgggggct tttcttttcc cccatggtcg ccccaaccga agacatttag 60
atcagtttac gttgggtttt gtggccttcg tgttcctttt ggttggttgg tttctttaca 120
tgtttgatct tttgtctaaa gctccatagg gtcttctcgt cttatgtgtt tatgtccgct 180
tctgcacggg cagatcaatt tcattgacta ggggaaggag acagttaaac cctcgttatg 240
ccat 244

Claims (3)

1. a DNA barcode primer, is characterized in that: the nucleotides sequence of described DNA barcode primer is classified as:
EEL 244-F： CCTGTTCC TCGTGGGGGC TTTT；
EEL 244-R： ATGGCATAACGAGGGTTTAACTG。
2. the eel species discrimination method based on DNA barcode, is characterized in that: the method comprises the following steps:
(1) eel DNA extraction;
(2) utilize DNA barcode primer to carry out pcr amplification, amplify the product of 244 bp, described DNA barcode primer is EEL 244-F:CCTGTTCC TCGTGGGGGC TTTT, EEL 244-R:ATGGCATAACGAGGGTTTAACTG;
(3) sequence signature that carries out species discriminating is compared, and wherein, is that species discriminating is carried out in the nucleotide sequence Feature change site shown according to the form below:
Figure 240322DEST_PATH_IMAGE001
。
3. the species discrimination method for Japanese eel, eel and European eel according to claim 2, it is characterized in that: the condition of the described pcr amplification of step (2) is: 94 ℃ of denaturation 3 min, 94 ℃ of sex change 30 s, 45 ℃ of annealing 30 s, 72 ℃ are extended 60 s, 5 circulations; 94 ℃ of sex change 30 s, 51 ℃ of annealing 60 s, 72 ℃ are extended 60 s, 35 circulations; 72 ℃ are extended 10 min; Reaction system is: 10 × taqbuffer 2.5 μ L, taqenzyme 1.0 U, template DNA 200 ng, MgCl 23.5 mmol/L, dNTPs 300 μ mol/L, upstream primer 0.5 μ mol/L, downstream primer 0.5 μ mol/L, uses ddH 2o mends cumulative volume to 25 μ L.
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CN104450883A (en) * 2014-10-22 2015-03-25 甘肃农业大学 DNA Barcoding-based identification kit for 9 normal fishes
CN104450881A (en) * 2014-10-22 2015-03-25 甘肃农业大学 Six common fish identification kits based on DNA Barcoding
CN104450882A (en) * 2014-10-22 2015-03-25 甘肃农业大学 DNA Barcoding-based identification kit for pelteobagrus fulvidraco, ictalurus nebulosus, weever and biological products of pelteobagrus fulvidraco, ictalurus nebulosus and weever
CN107130032A (en) * 2017-05-23 2017-09-05 福建出入境检验检疫局检验检疫技术中心 6 eel species discrimination methods based on a plurality of DNA bar code
CN108330169A (en) * 2017-09-26 2018-07-27 浙江省海洋水产研究所 Sea crabs class species discrimination method based on DNA bar code
CN110157817A (en) * 2019-07-02 2019-08-23 深圳华大海洋科技有限公司 A kind of molecular specificity labeled primers for identifying Cuscuta japonicoa and application thereof and discrimination method
CN111961733A (en) * 2020-08-28 2020-11-20 海南热带海洋学院 DNA bar code for identifying goby and goby of China comb and identification method and application thereof

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104450883A (en) * 2014-10-22 2015-03-25 甘肃农业大学 DNA Barcoding-based identification kit for 9 normal fishes
CN104450881A (en) * 2014-10-22 2015-03-25 甘肃农业大学 Six common fish identification kits based on DNA Barcoding
CN104450882A (en) * 2014-10-22 2015-03-25 甘肃农业大学 DNA Barcoding-based identification kit for pelteobagrus fulvidraco, ictalurus nebulosus, weever and biological products of pelteobagrus fulvidraco, ictalurus nebulosus and weever
CN107130032A (en) * 2017-05-23 2017-09-05 福建出入境检验检疫局检验检疫技术中心 6 eel species discrimination methods based on a plurality of DNA bar code
CN107130032B (en) * 2017-05-23 2020-08-07 福建出入境检验检疫局检验检疫技术中心 6 eel species identification method based on multiple DNA barcodes
CN108330169A (en) * 2017-09-26 2018-07-27 浙江省海洋水产研究所 Sea crabs class species discrimination method based on DNA bar code
CN110157817A (en) * 2019-07-02 2019-08-23 深圳华大海洋科技有限公司 A kind of molecular specificity labeled primers for identifying Cuscuta japonicoa and application thereof and discrimination method
CN111961733A (en) * 2020-08-28 2020-11-20 海南热带海洋学院 DNA bar code for identifying goby and goby of China comb and identification method and application thereof

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