CN103740855B - Based on the eel species discrimination method of DNA bar code - Google Patents

Based on the eel species discrimination method of DNA bar code Download PDF

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CN103740855B
CN103740855B CN201410045586.3A CN201410045586A CN103740855B CN 103740855 B CN103740855 B CN 103740855B CN 201410045586 A CN201410045586 A CN 201410045586A CN 103740855 B CN103740855 B CN 103740855B
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eel
bar code
dna
dna bar
species
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CN103740855A (en
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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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    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

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Abstract

The present invention relates to a kind of eel species discrimination method based on DNA bar code, more specifically relate to the species discrimination method of a kind of Cuscuta japonicoa based on DNA bar code, eel and European eel.The method is by eel DNA extraction, then DNA bar code primer is utilized to carry out pcr amplification, amplify 244? the product of bp, does is described DNA bar code primer EEL? 244-F:CCTGTTCC? TCGTGGGGGC? TTTT;? EEL? 244-R:ATGGCATAACGAGGGTTTAACTG, finally carries out species discriminating according to species nucleotide sequence Feature change site.The method is consuming time short, easy and simple to handle, high specificity, can differentiate through processing, the Pu that cannot be identified 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.

Description

Based on the eel species discrimination method of DNA bar code
Technical field
The present invention relates to a kind of eel species discrimination method based on DNA bar code, more specifically relate to a kind of Cuscuta japonicoa based on DNA bar code ( anguillajaponica), eel ( anguillarostrata) and European eel ( anguillaanguilla) 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, namely Cuscuta japonicoa ( anguillajaponica), eel ( anguillarostrata), European eel ( anguillaanguilla), annual export amount reaches billions of unit.Because eel processing is as barbecue or after making other shape prepared food, cannot distinguish its kind from form, different types of eel price is different.In order to prevent adulterating, protection enterprise and consumer's interests, answer enterprise and law enforcement agency's requirement, set up the biological assay method of eel kind.The present invention answers foreign trade and inspection and quarantine requirements of one's work, significant to the foreign trade as the large Fujian Province of economizing of eel outlet.
DNA bar code (DNAbarcode) to refer in organism and can represent these species, standard, have enough variations, easily amplification and relatively short DNA fragmentation.DNA bar code has become the important tool of ecological study, not only for species identification, also helps biologist to understand the interaction occurred in the ecosystem further simultaneously.But there is no the report utilizing DNA bar code 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 bar code.
The present invention provide firstly the DNA bar code primer of a kind of Cuscuta japonicoa, eel and European eel, and the PCR primer base number of DNA bar code object fragment is 244bp, and the nucleotides sequence of described DNA bar code primer is classified as:
EEL244-F：CCTGTTCCTCGTGGGGGCTTTT；
EEL244-R：ATGGCATAACGAGGGTTTAACTG。
The present invention is by comparing to the PCR primer base sequence for the Cuscuta japonicoa of examination, the DNA bar code object fragment of eel and European eel, 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 bar code of the present invention, the method comprises the following steps:
(1) eel DNA extraction;
(2) utilize DNA bar code primer to carry out pcr amplification, amplify the product of 244bp, described DNA bar code primer is EEL244-F:CCTGTTCCTCGTGGGGGCTTTT, EEL244-R:ATGGCATAACGAGGGTTTAACTG;
(3) carrying out the sequence signature comparison of species discriminating, wherein, is that species discriminating is carried out in the nucleotide sequence Feature change site shown according to the form below:
。
The condition of step (2) described pcr amplification is: 94 DEG C of denaturation 3min, 94 DEG C of sex change 30s, 45 DEG C of annealing 30s, and 72 DEG C extend 60s, 5 circulations; 94 DEG C of sex change 30s, 51 DEG C of annealing 60s, 72 DEG C extend 60s, 35 circulations; 72 DEG C extend 10min; Reaction system is: 10 × taqbuffer2.5 μ L, taqenzyme 1.0U, template DNA 200ng, MgCl 23.5mmol/L, dNTPs300 μm of ol/L, upstream primer 0.5 μm of ol/L, downstream primer 0.5 μm of ol/L, uses ddH 2cumulative volume is mended to 25 μ L by O.
Method of the present invention be used for specifically identify three kinds of eel Cuscuta japonicoa ( anguillajaponica), eel ( anguillarostrata), European eel ( anguillaanguilla) kind, the method is consuming time short, easy and simple to handle, high specificity, can differentiate through processing, the Pu that cannot be identified 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:DNAMarker; 1. Japanese eel ( anguillajaponica); 2. American eel ( anguillarostrata); 3. European eel ( anguillaanguilla).
Fig. 2 is 3 kinds of eel 16SrDNA Partial Fragment specific base sequence comparison charts, wherein, underscore part is primer sequence, adding boldface type in frame is 3 eel species specificity base sequences, namely from forward primer first base 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 the synthesis of Shanghai Sheng Gong company limited, and DNA sequence dna entrusts Guangzhou English Wei to create bio tech ltd's test.
primer synthesizes
According to the Cuscuta japonicoa of GenBank inquiry anguillajaponica(AJ244813.1), eel anguillarostrata(AJ244829.1), European eel anguillaanguilla(AJ244826.1) three kinds of eels
16SrRNA coding gene sequence (16SrDNA), apply PrimerPremier5.0 Automated Design 3 pairs of PCR primer, the PCR primer amplified checks order, and select and can amplify 1 pair of primer that 3 kinds of eels have special base sequence separately, sequence is as table 1.
Table 1PCR primer and annealing temperature thereof
The pcr amplification product sequencing result of 3 kinds of eel samples is at Genbank(gene library) middle comparison, the sequence corresponding to respective species conforms to completely, sequence is following, and (underscore part is primer, 5 ' end is forward primer, 3 ' end is the reverse complementary sequence of reverse primer base sequence, is eel species specificity base composition in frame):
Cuscuta japonicoa ( anguillajaponica) 5 '- cCTGTTCCT tCGTGGGGGCTTTT tTCTCCCCCATGGTCGCCCCAACCGAAGACATTTGGATCAGTTTACGTCGGGTTTC GTGGCCTTTGTGTTCCTTTTGGTT aACtTGGTTTCTTTACATGTTTGATCTTTTGTCTAAAGCTCCATAGGGTCTTCTCGTCT TATGTATTTATGTCCGCTTCTGCACGGGCAGATCAATTTCATTGACTAGGGGAAGG AGA cAGTTAAACCCTCGTTATGCCAT-3 ' (244bp),
Eel ( anguillarostrata)
5’- CCTGTTCCTTCGTGGGGGCTTTTCTTTCCCCCATGGTCGCCCCAACCGAAGACATTTAGATCAGTTTACGTTGGGTTTTGTGGCCTTCGTGTTCCTTTTGGTT GTTTTGGTTTCTTTACATGTTTGATCTTTTGTCTAAAGCTCCATAGGGTCTTCTCGTCTTATGTATTTATGTCCGCTTCTGCACGGGCAGATCAATTTCATTGACTGGGGGAAGGAGA CAGTTAAACCCTCGTTATGCCAT-3’(244bp)，
Europe eel ( anguillaanguilla)
5’- CCTGTTCCTTCGTGGGGGCTTTTCTTTTCCCCCATGGTCGCCCCAACCGAAGACATTTAGATCAGTTTACGTTGGGTTTTGTGGCCTTCGTGTTCCTTTTGGTT GGTTGGTTTCTTTACATGTTTGATCTTTTGTCTAAAGCTCCATAGGGTCTTCTCGTCTTATGTGTTTATGTCCGCTTCTGCACGGGCAGATCAATTTCATTGACTAGGGGAAGGAGA CAGTTAAACCCTCGTTATGCCAT-3’(244bp)。
eel DNA extraction
Add about 50mg sample powder in 1.5mL centrifuge tube, add 200 μ LTE 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, the centrifugal 10min of 12000g; Get supernatant liquor, add equal-volume chloroform: primary isoamyl alcohol (24:1), thermal agitation, the centrifugal 10min of 12000g; Get supernatant liquor, add 0.8 times of volume isopropanol, the centrifugal 10min of 12000g; Get precipitation, add 400 μ LNaCl(1mol/L) dissolve in 65 DEG C of temperature baths, then add 5 μ L Proteinase Ks (20mg/mL), 5 μ LRnaseA(10 μ g/mL)], thermal agitation more than 30 seconds, 37 DEG C of temperature bath 1h, period regularly rocks.Add isopyknic water-saturated phenol: chloroform: primary isoamyl alcohol (25:24:1), vibration, the centrifugal 15min of 12,000r/min, repeats this step 1-2 time, until protein removal is clean.Get supernatant, add the dehydrated alcohol of 2 times of volumes, weak vibrations, the centrifugal 10min of 12,000r/min.Get precipitation, 70% ethanol purge 2 times, dry, add 100 μ LTE(pH8.0) dissolve.
2 repetitions established by each sample.The test kit being applied to animal DNA extraction that city also can be used to buy to resell.
concentration and purity testing
Sample DNA nucleic acid-protein analysis-e/or determining 260nm and 280nm place absorption value, calculate DNA purity and concentration respectively, calculation formula is as follows: DNA purity=OD 260/ OD 280, ratio is ideal between 1.7-2.0; Double stranded DNA concentration (μ g/mL)=50 × OD 260value.
reaction system and condition
Reaction system is: 10 × taqbuffer2.5 μ L, taqenzyme 1.0U, template DNA 200ng, MgCl 23.5mmol/L, dNTPs300 μm of ol/L, upstream primer 0.5 μm of ol/L, downstream primer 0.5 μm of ol/L, uses ddH 2cumulative volume is mended to 25 μ L by O.
The condition of pcr amplification is: 94 DEG C of denaturation 3min.94 DEG C of sex change 30s, 45 DEG C of annealing 30s, 72 DEG C extend 60s, 5 circulations; 94 DEG C of sex change 30s, 51 DEG C of annealing 60s, 72 DEG C extend 60s, 35 circulations.72 DEG C extend 10min.Preserve at 4 DEG C.
interpretation of result
PCR primer electrophoresis as Fig. 1,1,2, No. 3 be respectively Cuscuta japonicoa ( anguillajaponica), eel ( anguillarostrata), European eel ( anguillaanguilla), detecting PCR primer size is 244bp, and 3 kinds of Pus burn eel sample (Hua Sheng Food Co., Ltd provides by Sanming City, Fujian Province), all amplify PCR primer.Fig. 2 is DNA bar code (16SrDNA Partial Fragment, 244bp) the base pairing figure of Cuscuta japonicoa, eel, European eel 3 kinds.Visible, the method has specificity to 3 kinds of eels.
<110> Inspection & Quarantine Technology Center of Fujian Entry-Exit Inspection & Quar
<120> is based on the eel species discrimination method of DNA bar code
<160>5
<170>PatentInversion3.3
<210>1
<211>22
<212>DNA
<213> synthetic
<400>1
cctgttcctcgtgggggctttt22
<210>2
<211>23
<212>DNA
<213> synthetic
<400>2
atggcataacgagggtttaactg23
<210>3
<211>244
<212>DNA
<213> Cuscuta japonicoa (Anguillajaponica)
<400>3
cctgttccttcgtgggggcttttttctcccccatggtcgccccaaccgaagacatttgga60
tcagtttacgtcgggtttcgtggcctttgtgttccttttggttaacttggtttctttaca120
tgtttgatcttttgtctaaagctccatagggtcttctcgtcttatgtatttatgtccgct180
tctgcacgggcagatcaatttcattgactaggggaaggagacagttaaaccctcgttatg240
ccat244
<210>4
<211>244
<212>DNA
<213> eel (Anguillarostrata)
<400>4
cctgttccttcgtgggggcttttctttcccccatggtcgccccaaccgaagacatttaga60
tcagtttacgttgggttttgtggccttcgtgttccttttggttgttttggtttctttaca120
tgtttgatcttttgtctaaagctccatagggtcttctcgtcttatgtatttatgtccgct180
tctgcacgggcagatcaatttcattgactgggggaaggagacagttaaaccctcgttatg240
ccat244
<210>5
<211>244
<212>DNA
<213> Europe eel (Anguillaanguilla)
<400>5
cctgttccttcgtgggggcttttcttttcccccatggtcgccccaaccgaagacatttag60
atcagtttacgttgggttttgtggccttcgtgttccttttggttggttggtttctttaca120
tgtttgatcttttgtctaaagctccatagggtcttctcgtcttatgtgtttatgtccgct180
tctgcacgggcagatcaatttcattgactaggggaaggagacagttaaaccctcgttatg240
ccat244

Claims (2)

1., based on an eel species discrimination method for DNA bar code, it is characterized in that: the method comprises the following steps:
(1) eel DNA extraction;
(2) utilize DNA bar code primer to carry out pcr amplification, amplify the product of 244bp, described DNA bar code primer is EEL244-F:CCTGTTCCTCGTGGGGGCTTTT, EEL244-R:ATGGCATAACGAGGGTTTAACTG;
(3) carrying out the sequence signature comparison of species discriminating, wherein, is that species discriminating is carried out in the nucleotide sequence Feature change site shown according to the form below:
。
2. the eel species discrimination method based on DNA bar code according to claim 1, is characterized in that: the condition of step (2) described pcr amplification is: 94 DEG C of denaturation 3min, 94 DEG C of sex change 30s, 45 DEG C of annealing 30s, and 72 DEG C extend 60s, 5 circulations; 94 DEG C of sex change 30s, 51 DEG C of annealing 60s, 72 DEG C extend 60s, 35 circulations; 72 DEG C extend 10min; Reaction system is: 10 × taqbuffer2.5 μ L, taqenzyme 1.0U, template DNA 200ng, MgCl 23.5mmol/L, dNTPs300 μm of ol/L, upstream primer 0.5 μm of ol/L, downstream primer 0.5 μm of ol/L, uses ddH 2cumulative volume is mended to 25 μ L by O.
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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
CN104450883A (en) * 2014-10-22 2015-03-25 甘肃农业大学 DNA Barcoding-based identification kit for 9 normal fishes
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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