CN104404145B - A kind of test kit quickly detecting meat adulteration based on LAMP method - Google Patents
A kind of test kit quickly detecting meat adulteration based on LAMP method Download PDFInfo
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Abstract
The invention discloses a kind of test kit quickly detecting meat adulteration based on LAMP method.Containing well-designed multiple meat primer in test kit, specific can amplify corresponding product, and then be easy to quickly detect.Compared with traditional PCR, test kit of the present invention has high specificity, highly sensitive: can detect bottom line respectively: Carnis Sus domestica 10‑4Ng, fox meat 10‑4Ng, mouse meat 10‑ 5Ng, Carnis Gallus domesticus 10‑3Ng, duck meat 10‑5ng；In 1 h, just can complete detection, quickly save time；Simple to operate, and the requirement to operator is relatively low；Being prone to detection: end reaction result only need to the naked eye be observed identifying, positive findings be green, and negative findings is orange, without use gel electrophoresis, it is to avoid the toxic pharmaceuticals such as use ethidium bromide, it is to avoid potential safety hazard；The test kit of the present invention, can disposably detect multiple meat, substantially increase detection efficiency.
Description
Technical field
The present invention relates to a kind of molecular detecting method, particularly to a kind of meat adulteration that quickly detects based on LAMP method
Test kit.
Background technology
Meat and its products is one of Main Foods of mankind's daily life consumption.Due to price, religion, healthy etc. former
Cause, the food labelling of many national requirements meat and its products classes must mark meat sources to true, unambiguous, forbid adulterated row
For, to protect the interests of consumer.
In recent years, the meat products consumption figure of China is the most always in soaring situation, but food safety annoyings consumption always
Person, makes the problems such as tripolycyanamide that compatriots shock, clenbuterol hydrochloride, false beef event emerge in an endless stream.More seriously, city in 2013
Occur in that on face that the malignant event of Carnis caprae seu ovis pretended to be by the fox meat utilizing source not clear, mouse meat, mink meat, cause various circles of society
Extensive concern.And the illegal retailer of part also to mix the price such as Carnis Sus domestica, Carnis Gallus domesticus, duck meat in Carnis caprae seu ovis, mutton roll, mutton cubes roasted on a skewer relatively low
Honest and clean, invade consumer legitimate right.Because adulterated meat product the most seriously compromises consumer rights, even jeopardize consumer
Life and health, be therefore badly in need of a kind of easy and simple to handle, with low cost, the detection method of rapid sensitive.
It is adulterated to meat products that traditional meat morphology relying on sense organ and experience differentiates that means can not meet market
The needs that phenomenon is controlled and supervises.At present the authentication method of meat products is mainly carried out at protein and nucleic acid level.
At protein level, the technology such as ELISA, liquid chromatograph, high performance liquid chromatography of commonly using detect, and common being limited in that is right
Instrument, reagent and sample treatment require height, and during detection processed sample, specificity and accuracy are poor, waste time and energy.Relatively come
Saying, the mode with inspection nucleic acid is more convenient accurately.In nucleic acid level, conventional Accounting Level detection technique has PCR with in real time
PCR.Round pcr is the method for the most the most frequently used detection meat derived component, because its response time is short, has higher sensitive
Degree, specificity and operability, but because its late detection needs to use gel electrophoresis, and the EB in gel electrophoresis has strong cause
Carcinous, there is certain potential safety hazard；And compare and normal PCR, real-time PCR has higher specificity and sensitivity, but its
Need expensive real-time machine, and operator are had higher requirements, it is difficult to promoted in basic unit.
Summary of the invention
It is an object of the invention to provide a kind of test kit for meat detection.
The technical solution used in the present invention is:
A kind of LAMP kit for meat detection, including reaction buffer, archaeal dna polymerase and specific primer, its
It is characterised by: specific primer sequence is as follows:
Pork detection specific sequence:
P-F3:5 '-CCTCACCCTAGTAGAACGAA-3 ' (SEQ ID NO:1)
P-B3:5 '-GCTTGACATGGCTAGCAT-3 ' (SEQ ID NO:2)
P-FIP:5 '-GGTGAATAGTTTTAGGGCATCGGCTTTTACTACGAAAAGGACCCAACG-3 ' (SEQ ID
NO:3)
P-BIP:5 '-TTGCACCAATCCTAGCCTTATCCCTTTTGGGTAGGGTATTGGTAGTGGA-3 ' (SEQ ID
NO:4)
Fox meat detection specific sequence:
F-F3:5 '-TCTGAGGGGCAACCGTAAT-3 ' (SEQ ID NO:5)
F-B3:5 '-GTCGGATGTGATTCCTGAAGG-3 ' (SEQ ID NO:6)
F-FIP:5 '-CTTTGTCTACTGAGAAGCCCCCTCA-CTGCTATCCCCTATATCGGAACC-3 ' (SEQ ID
NO:7)
F-BIP:5 '-CCATTCATCATCGCAGCATTAGCG-GTTGTTGGATCCTGTTTCGTGG-3 ' (SEQ ID
NO:8)
Mouse meat detection specific sequence:
M-F3:5 '-CTCTATTTCTACCATCCTCCG-3 ' (SEQ ID NO:9)
M-B3:5 '-GATTAGACCCGTTACCATCG-3 ' (SEQ ID NO:10)
M-FIP:5 '-TTCAGTATAGTCACCCCCAGGACTTTTTGAAATCAACAACCCGCC-3 ' (SEQ ID NO:
11)
M-BIP:5 '-ACCAGGCATCTGGTTCTTACTTTTTAAGGGGAACGTATGGACG-3 ' (SEQ ID NO:
12)
Carnis Gallus domesticus detection specific sequence:
C-F3:5 '-CTCACGAGAGATCAGCAACC-3 ' (SEQ ID NO:13)
C-B3:5 '-GAAGAGAGAAGATGCCGCG-3 ' (SEQ ID NO:14)
C-FIP:5 '-GTACGGTGGAAGGCAAGTAGGGTTTTGTACTTCATGACCAGTCTCAGG-3 ' (SEQ ID
NO:15)
C-BIP:5 '-GGCACATCCCATGCATAACTCCTTTTTATCACGGACTAAAGAGGGGA-3 ' (SEQ ID
NO:16)
Duck meat detection specific sequence:
D-F3:5 '-GATTCTACTTCACCGCCCTA-3 ' (SEQ ID NO:17)
D-B3:5 '-AATCCGAAGTGGTGGTCTG-3 ' (SEQ ID NO:18)
D-FIP:5 '-CCGGTGGCAACAAAGAAAGTGGTTTTTAGAGTACCATGAAGCCCCA-3 ' (SEQ ID
NO:19)
D-BIP:5 '-CCACGGACTCCACGTGATCATCTTTTTTGATTAGTCGGAGGAGGCA-3 ' (SEQ ID
NO:20).
As a further improvement on the present invention, mentioned reagent box is possibly together with DNA extraction liquid.
As a further improvement on the present invention, mentioned reagent box for the reaction system of meat detection is: 1 ×
ThermoPol reaction buffer, MgSO4, dNTPs, Betaine, Calcein, MnCl2, Bst archaeal dna polymerase, outer primer,
Inner primer, template DNA and sterilizing ddH2O。
As a further improvement on the present invention, mentioned reagent box concentration of each component in the reaction system of meat detection
For MgSO41-10 mM, dNTPs0.1-5 mM, Betain0.1-1 M, Calcein 10-50 μM, MnCl20.1-5 mM,
1-40U Bst archaeal dna polymerase, 0.1-2 μ l outer primer, 1-10 μ l inner primer, 0.1-5 μ l template DNA, supply sterilizing ddH2O
To 25 μ l；The concentration of primer is 1-20 μm ol/L, and the concentration of template DNA is 10-100ng/ μ l.
Particularly, the specific primer of mentioned reagent box reacts in same reaction system, and this reaction system is:
1 × ThermoPol reaction buffer, MgSO4 7 mM, dNTPs 2.0 mM, Betaine 0.4 M, Calcein 25 μ
M, MnCl20.5 mM, 8U Bst archaeal dna polymerase, each 0.5 μ l of outer primer F3, B3 of 10 μm ol/L, 10 μm ol/L
Inner primer FIP, BIP each 4 μ l and template DNA 1 μ l, supply sterilizing ddH2O to 25 μ l；The concentration of primer is 10 μm ol/L,
The concentration of template DNA is 50 ng/ μ l.
The invention has the beneficial effects as follows:
The LAMP specific primer that the test kit of the present invention is used can detect Carnis Sus domestica, Vulpes quick, special, delicately
Carnis Felis bengalensis, mouse meat, Carnis Gallus domesticus, duck meat composition, compared with traditional PCR, test kit of the present invention has the advantage that
1) high specificity: primer used in the present invention, is according to 4 designed by corresponding species mitochondrion specific sequence
Bar primer, it is possible to 6 sites of specific recognition, specificity has exceeded normal PCR, has the strongest specificity；
2) highly sensitive: test kit of the present invention, bottom line can be detected respectively: Carnis Sus domestica 10-4Ng, fox meat 10- 4Ng, mouse meat 10-5Ng, Carnis Gallus domesticus 10-3Ng, duck meat 10-5ng；
3) quickly save time: in 1h, just can complete detection, at least need more than 2h compared to normal PCR, saved inspection
The survey time；
4) simple to operate: to have only to utilize water-bath to carry out isothermal reaction, without expensive PCR instrument, and to operation
The requirement of personnel is relatively low；
5) being prone to detection: end reaction result only need to the naked eye be observed identifying, positive findings is green, negative
Result is orange, without using gel electrophoresis, it is to avoid toxic pharmaceuticals such as use ethidium bromides, it is to avoid potential safety hazard
6) test kit of the present invention, can disposably detect multiple meat, substantially increase detection efficiency.
Accompanying drawing explanation
Fig. 1 is that naked eyes detect Carnis Sus domestica LAMP specific reaction amplification
In figure, 1-10 template is respectively as follows: 1. Carnis Sus domesticas；2. beef；3. Carnis caprae seu ovis；4. fox meat；5. Carnis Leporis；6. mouse meat；7.
Carnis Gallus domesticus；8. duck meat；9. Cyprinus carpio meat；10. water
Fig. 2 is that naked eyes detect fox meat LAMP specific reaction amplification
In figure, 1-10 template is respectively as follows: 1. Carnis Sus domesticas；2. beef；3. Carnis caprae seu ovis；4. fox meat；5. Carnis Leporis；6. mouse meat；7.
Carnis Gallus domesticus；8. duck meat；9. Cyprinus carpio meat；10. water
Fig. 3 is that naked eyes detect mouse meat LAMP specific reaction amplification
In figure, 1-10 template is respectively as follows: 1. Carnis Sus domesticas；2. beef；3. Carnis caprae seu ovis；4. fox meat；5. Carnis Leporis；6. mouse meat；7.
Carnis Gallus domesticus；8. duck meat；9. Cyprinus carpio meat；10. water
Fig. 4 is that naked eyes detect Carnis Gallus domesticus LAMP specific reaction amplification
In figure, 1-10 template is respectively as follows: 1. Carnis Sus domesticas；2. beef；3. Carnis caprae seu ovis；4. fox meat；5. Carnis Leporis；6. mouse meat；7.
Carnis Gallus domesticus；8. duck meat；9. Cyprinus carpio meat；10. water
Fig. 5 is that naked eyes detect duck meat LAMP specific reaction amplification
In figure, 1-10 template is respectively as follows: 1. Carnis Sus domesticas；2. beef；3. Carnis caprae seu ovis；4. fox meat；5. Carnis Leporis；6. mouse meat；7.
Carnis Gallus domesticus；8. duck meat；9. Cyprinus carpio meat；10. water
Fig. 6 is that naked eyes detect multiple meat LAMP specific reaction amplification simultaneously
In figure, 1-10 template is respectively as follows:. Carnis Sus domestica；2. beef；3. Carnis caprae seu ovis；4. fox meat；5. Carnis Leporis；6. mouse meat；7.
Carnis Gallus domesticus；8. duck meat；9. Cyprinus carpio meat；10. water
Fig. 7 is that naked eyes detect Carnis Sus domestica LAMP sensitivity response amplification
In figure, 1-10 template concentrations is respectively as follows: 1. 50 ng；2. 5 ng；3. 500 pg；4. 50 pg；5. 5 pg；
6. 500 fg；7. 50 fg；8. 5 fg；9. 500 ag；10. water
Fig. 8 is that naked eyes detect fox meat LAMP sensitivity response amplification
In figure, 1-10 template concentrations is respectively as follows: 1. 50 ng；2. 5 ng；3. 500 pg；4. 50 pg；5. 5 pg；
6. 500 fg；7. 50 fg；8. 5 fg；9. 500 ag；10. water
Fig. 9 is that naked eyes detect mouse meat LAMP sensitivity response amplification
In figure, 1-10 template concentrations is respectively as follows: 1. 50 ng；2. 5 ng；3. 500 pg；4. 50 pg；5. 5 pg；
6. 500 fg；7. 50 fg；8. 5 fg；9. 500 ag；10. water
Figure 10 is that naked eyes detect Carnis Gallus domesticus LAMP sensitivity response amplification
In figure, 1-10 template concentrations is respectively as follows: 1. 50 ng；2. 5 ng；3. 500 pg；4. 50 pg；5. 5 pg；
6. 500 fg；7. 50 fg；8. 5 fg；9. 500 ag；10. water
Figure 11 is that naked eyes detect duck meat LAMP sensitivity response amplification
In figure, 1-10 template concentrations is respectively as follows: 1. 50 ng；2. 5 ng；3. 500 pg；4. 50 pg；5. 5 pg；
6. 500 fg；7. 50 fg；8. 5 fg；9. 500 ag；10. water.
Detailed description of the invention
Below in conjunction with embodiment, further illustrate technical scheme.
If no special instructions, all technical terms in file all have logical with one skilled in the art of the present invention
Understand identical implication.
(1) LAMP reaction reagent and consumptive material:
Glycine betaine (Betaine) is purchased from Sigma company；
Bst DNA Large fragment polymerase (Bst DNA polymerase large fragment), 10 × ThermoPol
Reaction buffer is purchased from New England Biolabs company；
Triphosphate deoxy-nucleotide (dNTPs) is purchased from green skies biotechnology research institute；
Calcein (Calcein) is purchased from Dojindo company；
Magnesium sulfate (MgSO4), manganese chloride (MnCl2) purchased from Beijing Chemical Plant；
Loopamp reaction tube is purchased from Rong Yan company of Japan.
(2) LAMP primer (forward outer primer F3, reverse outer primer B3, forward inner primer FIP, reverse inner primer BIP), south
Jing Jinsirui bio tech ltd.
(3) all meat products sample standard deviations are provided by animal science institute of Jilin University.
Synthesis the LAMP primer sterilizing redistilled water obtained be diluted to 10 μm ol/L respectively, be placed in-20 DEG C frozen
Standby.
Full-length genome extracts: utilize blood/cell/tissue genome DNA extracting reagent kit (TIANamp Genomic
DNA Kit, TIANGEN) extract each animal species muscular tissue genome, measure genome concentration, be diluted to 50 ng/ μ subsequently
L, be placed in-20 DEG C frozen standby.
LAMP visual method inspection primer specificity
(1) visualization inspection Carnis Sus domestica primer specificity:
It is 25 μ l by pork detection reaction system, including: 1 × ThermoPol reaction buffer； MgSO4 7 mM；
dNTPs 2.0 mM； Betaine 0.2 M；Calcein 25 μM； MnCl20.5 mM；8U Bst archaeal dna polymerase；10
The each 0.5 μ l of outer primer P-F3, P-B3 of μm ol/L；Each 4 μ l and the template DNAs of inner primer P-FIP, P-BIP of 10 μm ol/L
1 μl；Supply sterilizing ddH2O to 25 μ l adds in 200 μ l centrifuge tubes, uses the pig that described in embodiment 2, method is extracted respectively
Meat, beef, Carnis caprae seu ovis, fox meat, Carnis Leporis, mouse meat, Carnis Gallus domesticus, duck meat, Cyprinus carpio meat genomic DNA and sterilizing redistilled water are made
For template.Mixing centrifuge tube after centrifugal is placed in thermostat water bath and reacts 60 min, be placed in 80 DEG C of water-baths the most again and go out
Live 5 min, changes judgment experiment result by naked-eye observation color
In Fig. 1 No. 1-10 respectively represent template be Carnis Sus domestica, beef, Carnis caprae seu ovis, fox meat, Carnis Leporis, mouse meat, Carnis Gallus domesticus, duck meat,
Cyprinus carpio meat and water.Result represents, only Carnis Sus domestica is become green as the centrifuge tube reactant liquor of template from orange, and result is positive；
And beef, Carnis caprae seu ovis, fox meat, Carnis Leporis, mouse meat, Carnis Gallus domesticus, duck meat, Cyprinus carpio meat and the reaction tube reactant liquor that water is template are appointed for orange
Color, result is negative.Accordingly, it is determined that use LAMP visual method can detect pork content with specificity.
(2) visualization inspection fox meat primer specificity:
It is 25 μ l by fox meat detection reaction system, including: 1 × ThermoPol reaction buffer； MgSO4 6 mM；
dNTPs 1.6 mM； Betaine 0.4 M；Calcein 25 μM； MnCl20.5 mM；8U Bst archaeal dna polymerase；10
The each 0.5 μ l of outer primer F-F3, F-B3 of μm ol/L；Each 4 μ l and the template DNAs of inner primer F-FIP, F-BIP of 10 μm ol/L
1 μl；Supply sterilizing ddH2O to 25 μ l adds in 200 μ l centrifuge tubes, uses the pig that described in embodiment 2, method is extracted respectively
Meat, beef, Carnis caprae seu ovis, fox meat, Carnis Leporis, mouse meat, Carnis Gallus domesticus, duck meat, Cyprinus carpio meat genomic DNA and sterilizing redistilled water are made
For template.Mixing centrifuge tube after centrifugal is placed in thermostat water bath and reacts 60 min, be placed in 80 DEG C of water-baths the most again and go out
Live 5 min, changes judgment experiment result by naked-eye observation color
In Fig. 2 No. 1-10 respectively represent template be Carnis Sus domestica, beef, Carnis caprae seu ovis, fox meat, Carnis Leporis, mouse meat, Carnis Gallus domesticus, duck meat,
Cyprinus carpio meat and water.Result represents, only fox meat is become green as the centrifuge tube reactant liquor of template from orange, and result is sun
Property；And Carnis Sus domestica, beef, Carnis caprae seu ovis, Carnis Leporis, mouse meat, Carnis Gallus domesticus, duck meat, Cyprinus carpio meat and the reaction tube reactant liquor that water is template are appointed and are
Orange, result is negative.Accordingly, it is determined that use LAMP visual method can detect fox meat composition with specificity.
(3) visualization inspection mouse meat primer specificity:
It is 25 μ l by mouse meat detection reaction system, including: 1 × ThermoPol reaction buffer； MgSO4 7 mM；
dNTPs 1.2 mM； Betaine 0.4 M；Calcein 25 μM； MnCl20.5 mM；8U Bst archaeal dna polymerase；10
The each 0.5 μ l of outer primer M-F3, M-B3 of μm ol/L；Each 4 μ l and the template DNAs of inner primer M-FIP, M-BIP of 10 μm ol/L
1μl；Supply sterilizing ddH2O to 25 μ l adds in 200 μ l centrifuge tubes, uses the pig that described in embodiment 2, method is extracted respectively
Meat, beef, Carnis caprae seu ovis, fox meat, Carnis Leporis, mouse meat, Carnis Gallus domesticus, duck meat, Cyprinus carpio meat genomic DNA and sterilizing redistilled water are made
For template.Mixing centrifuge tube after centrifugal is placed in thermostat water bath and reacts 60 min, be placed in 80 DEG C of water-baths the most again and go out
Live 5 min, changes judgment experiment result by naked-eye observation color
In Fig. 3 No. 1-10 respectively represent template be Carnis Sus domestica, beef, Carnis caprae seu ovis, fox meat, Carnis Leporis, mouse meat, Carnis Gallus domesticus, duck meat,
Cyprinus carpio meat and water.Result represents, only mouse meat is become green as the centrifuge tube reactant liquor of template from orange, and result is sun
Property；And Carnis Sus domestica, beef, Carnis caprae seu ovis, fox meat, Carnis Leporis, Carnis Gallus domesticus, duck meat, Cyprinus carpio meat and the reaction tube reactant liquor that water is template are appointed and are
Orange, result is negative.Accordingly, it is determined that use LAMP visual method can detect mouse meat composition with specificity.
(4) visualization inspection Carnis Gallus domesticus primer specificity:
It is 25 μ l by Carnis Gallus domesticus detection reaction system, including: 1 × ThermoPol reaction buffer； MgSO4 8 mM；
dNTPs 2.0 mM； Betaine 0.2 M；Calcein 25 μM； MnCl20.5 mM；8U Bst archaeal dna polymerase；10
The each 0.5 μ l of outer primer C-F3, C-B3 of μm ol/L；Each 4 μ l and the template DNAs of inner primer C-FIP, C-BIP of 10 μm ol/L
1 μl；Supply sterilizing ddH2O to 25 μ l adds in 200 μ l centrifuge tubes, uses the pig that described in embodiment 2, method is extracted respectively
Meat, beef, Carnis caprae seu ovis, fox meat, Carnis Leporis, mouse meat, Carnis Gallus domesticus, duck meat, Cyprinus carpio meat genomic DNA and sterilizing redistilled water are made
For template.Mixing centrifuge tube after centrifugal is placed in thermostat water bath and reacts 90 min, be placed in 80 DEG C of water-baths the most again and go out
Live 5 min, changes judgment experiment result by naked-eye observation color
In Fig. 4 No. 1-10 respectively represent template be Carnis Sus domestica, beef, Carnis caprae seu ovis, fox meat, Carnis Leporis, mouse meat, Carnis Gallus domesticus, duck meat,
Cyprinus carpio meat and water.Result represents, only Carnis Gallus domesticus is become green as the centrifuge tube reactant liquor of template from orange, and result is positive；
And Carnis Sus domestica, beef, Carnis caprae seu ovis, fox meat, Carnis Leporis, mouse meat, duck meat, Cyprinus carpio meat and the reaction tube reactant liquor that water is template are appointed for orange
Color, result is negative.Accordingly, it is determined that use LAMP visual method can detect Carnis Gallus domesticus composition with specificity.
(5) visualization inspection duck meat primer specificity:
It is 25 μ l by duck meat detection reaction system, including: 1 × ThermoPol reaction buffer； MgSO4 7 mM；
dNTPs 2.0 mM； Betaine 0.4 M；Calcein 25 μM； MnCl20.5 mM；8U Bst archaeal dna polymerase；10
The each 0.5 μ l of outer primer D-F3, D-B3 of μm ol/L；Each 4 μ l and the template DNAs of inner primer D-FIP, D-BIP of 10 μm ol/L
1 μl；Supply sterilizing ddH2O to 25 μ l adds in 200 μ l centrifuge tubes, uses the pig that described in embodiment 2, method is extracted respectively
Meat, beef, Carnis caprae seu ovis, fox meat, Carnis Leporis, mouse meat, Carnis Gallus domesticus, duck meat, Cyprinus carpio meat genomic DNA and sterilizing redistilled water are made
For template.Mixing centrifuge tube after centrifugal is placed in thermostat water bath and reacts 60 min, be placed in 80 DEG C of water-baths the most again and go out
Live 5 min, changes judgment experiment result by naked-eye observation color
In Fig. 5 No. 1-10 respectively represent template be Carnis Sus domestica, beef, Carnis caprae seu ovis, fox meat, Carnis Leporis, mouse meat, Carnis Gallus domesticus, duck meat,
Cyprinus carpio meat and water.Result represents, only duck meat is become green as the centrifuge tube reactant liquor of template from orange, and result is positive；
And Carnis Sus domestica, beef, Carnis caprae seu ovis, fox meat, Carnis Leporis, mouse meat, Carnis Gallus domesticus, Cyprinus carpio meat and the reaction tube reactant liquor that water is template are appointed for orange
Color, result is negative.Accordingly, it is determined that use LAMP visual method can detect duck meat composition with specificity.
(6) pig, fox, mouse, chicken, duck meat primer specificity are checked in visualization simultaneously:
Detect reaction system by multiple meat be 25 μ l simultaneously, including: 1 × ThermoPol reaction buffer, MgSO4 7
MM, dNTPs 2.0 mM, Betaine 0.4 M, Calcein 25 μMs, MnCl20.5 mM, 8U Bst DNA is polymerized
Enzyme, primer premixed liquid 9 μ l (wherein comprises P-F3, P-B3, F-F3, F-B3, M-F3, M-B3, C-F3, C-B3, D-
Each 5 pmol of F3, D-B3; P-FIP, P-BIP, F-FIP, F-BIP, M-FIP, M-BIP, C-FIP, C-BIP,
Each 40 pmol of D-FIP, D-BIP) (primer join method for be added in the ratio of 1/5th by 5 kinds of primers, but because move
Liquid device minimum range is unsatisfactory for, it is possible to above-mentioned 5 kinds of primers F 3, and B3 respectively takes 0.5 μ l, each 4 μ l of FIP, BIP, fully mixes
After take 9 μ l and use, the concentration herein diluting primer is 50 μm ol/L), and template DNA 2.5 μ l；Supply sterilizing ddH2O to 25
μ l adds in 200 μ l centrifuge tubes, uses the Carnis Sus domestica that described in embodiment 2, method is extracted, beef, Carnis caprae seu ovis, fox meat, rabbit respectively
Meat, mouse meat, Carnis Gallus domesticus, duck meat, Cyprinus carpio meat genomic DNA and sterilizing redistilled water are as template.After centrifugal for mixing
Centrifuge tube is placed in thermostat water bath and reacts 60 min, is placed in 80 DEG C of water-baths the most again and inactivates 5 min, by naked-eye observation face
Complexion changed judgment experiment result
In Fig. 6 No. 1-10 respectively represent template be Carnis Sus domestica, beef, Carnis caprae seu ovis, fox meat, Carnis Leporis, mouse meat, Carnis Gallus domesticus, duck meat,
Cyprinus carpio meat and water.Result represents, Carnis Sus domestica, fox meat, mouse meat, Carnis Gallus domesticus, duck meat as the centrifuge tube reactant liquor of template by orange
Becoming green, result is positive；And beef, Carnis caprae seu ovis, Carnis Leporis, Cyprinus carpio meat and the reaction tube reactant liquor that water is template are appointed for orange,
Result is negative.Accordingly, it is determined that use LAMP visual method to detect multiple meat derived component simultaneously.
The inspection primer sensitivity of LAMP visual method
(1) visualization inspection Carnis Sus domestica primer sensitivity:
It is 25 μ l by pork detection reaction system, including: 1 × ThermoPol reaction buffer； MgSO4 7 mM；
dNTPs 2.0 mM； Betaine 0.2 M；Calcein 25 μM； MnCl20.5 mM；8U Bst archaeal dna polymerase；10
The each 0.5 μ l of outer primer P-F3, P-B3 of μm ol/L；Each 4 μ l and the template DNAs of inner primer P-FIP, P-BIP of 10 μm ol/L
1 μl；Supply sterilizing ddH2O to 25 μ l adds in 200 μ l centrifuge tubes, uses the Carnis Sus domestica base that described in embodiment 2, method is extracted
Because group does 10 times of gradient dilutions to 10-8Ng is as template, using sterilizing two distilled water as negative control.By mixing being centrifuged after centrifugal
Pipe is placed in thermostat water bath and reacts 60 min, is placed in 80 DEG C of water-baths the most again and inactivates 5 min, is become by naked-eye observation color
Change judgment experiment result.
In Fig. 7 No. 1-9 respectively represent Carnis Sus domestica template concentrations be 50 ng, 5 ng, 500 pg, 50 pg, 5 pg, 500 fg,
50 fg, 5 fg, 500 ag, No. 10 templates are two distilled waters.Result represents, the limit that Carnis Sus domestica primer can be detected by out is 500 fg.
(2) visualization inspection fox meat cited thing sensitivity:
It is 25 μ l by fox meat detection reaction system, including: 1 × ThermoPol reaction buffer； MgSO4 6 mM；
dNTPs 1.6 mM； Betaine 0.4 M；Calcein 25 μM； MnCl20.5 mM；8U Bst archaeal dna polymerase；10
The each 0.5 μ l of outer primer F-F3, F-B3 of μm ol/L；Each 4 μ l and the template DNAs of inner primer F-FIP, F-BIP of 10 μm ol/L
1 μl；Supply sterilizing ddH2O to 25 μ l adds in 200 μ l centrifuge tubes, uses the fox meat that described in embodiment 2, method is extracted
Genome does 10 times of gradient dilutions to 10-8Ng is as template, using sterilizing two distilled water as negative control.By after centrifugal for mixing from
Heart pipe is placed in thermostat water bath and reacts 60 min, is placed in 80 DEG C of water-baths the most again and inactivates 5 min, by naked-eye observation color
Change judgment experiment result
In Fig. 8 No. 1-9 respectively represent fox meat template concentrations be 50 ng, 5 ng, 500 pg, 50 pg, 5 pg, 500
Fg, 50 fg, 5 fg, 500 ag, No. 10 templates are two distilled waters.Result represents, the limit that fox meat cited thing can be detected by out is
500 fg。
(3) visualization inspection mouse meat cited thing sensitivity:
It is 25 μ l by mouse meat detection reaction system, including: 1 × ThermoPol reaction buffer； MgSO4 7 mM；
dNTPs 1.2 mM； Betaine 0.4 M；Calcein 25 μM； MnCl20.5 mM；8U Bst archaeal dna polymerase；10
The each 0.5 μ l of outer primer M-F3, M-B3 of μm ol/L；Each 4 μ l and the template DNAs of inner primer M-FIP, M-BIP of 10 μm ol/L
1 μl；Supply sterilizing ddH2O to 25 μ l adds in 200 μ l centrifuge tubes, uses the mouse meat that described in embodiment 2, method is extracted
Genome does 10 times of gradient dilutions to 10-8Ng is as template, using sterilizing two distilled water as negative control.By after centrifugal for mixing from
Heart pipe is placed in thermostat water bath and reacts 60 min, is placed in 80 DEG C of water-baths the most again and inactivates 5 min, by naked-eye observation color
Change judgment experiment result
In Fig. 9 No. 1-9 respectively represent mouse meat template concentrations be 50 ng, 5 ng, 500 pg, 50 pg, 5 pg, 500
Fg, 50 fg, 5 fg, 500 ag, No. 10 templates are two distilled waters.Result represents, the limit that mouse meat cited thing can be detected by out is 50
fg。
(4) visualization inspection Carnis Gallus domesticus primer sensitivity:
It is 25 μ l by Carnis Gallus domesticus detection reaction system, including: 1 × ThermoPol reaction buffer； MgSO4 8 mM；
dNTPs 2.0 mM； Betaine 0.2 M；Calcein 25 μM； MnCl20.5 mM；8U Bst archaeal dna polymerase；10
The each 0.5 μ l of outer primer C-F3, C-B3 of μm ol/L；Each 4 μ l and the template DNAs of inner primer C-FIP, C-BIP of 10 μm ol/L
1 μl；Supply sterilizing ddH2O to 25 μ l adds in 200 μ l centrifuge tubes, uses the Carnis Gallus domesticus base that described in embodiment 2, method is extracted
Because group does 10 times of gradient dilutions to 10-8Ng is as template, using sterilizing two distilled water as negative control.By mixing being centrifuged after centrifugal
Pipe is placed in thermostat water bath and reacts 90 min, is placed in 80 DEG C of water-baths the most again and inactivates 5 min, is become by naked-eye observation color
Change judgment experiment result
In Figure 10 No. 1-9 respectively represent Carnis Gallus domesticus template concentrations be 50 ng, 5 ng, 500 pg, 50 pg, 5 pg, 500 fg,
50 fg, 5 fg, 500 ag, No. 10 templates are two distilled waters.Result represents, the limit that Carnis Gallus domesticus primer can be detected by out is 5 pg.
(5) visualization inspection duck meat primer sensitivity:
It is 25 μ l by duck meat detection reaction system, including: 1 × ThermoPol reaction buffer； MgSO4 7 mM；
dNTPs 2.0 mM； Betaine 0.4 M；Calcein 25 μM； MnCl20.5 mM；8U Bst archaeal dna polymerase；10
The each 0.5 μ l of outer primer D-F3, D-B3 of μm ol/L；Each 4 μ l and the template DNAs of inner primer D-FIP, D-BIP of 10 μm ol/L
1 μl；Supply sterilizing ddH2O to 25 μ l adds in 200 μ l centrifuge tubes, uses the duck meat base that described in embodiment 2, method is extracted
Because group does 10 times of gradient dilutions to 10-8Ng is as template, using sterilizing two distilled water as negative control.By mixing being centrifuged after centrifugal
Pipe is placed in thermostat water bath and reacts 60 min, is placed in 80 DEG C of water-baths the most again and inactivates 5 min, is become by naked-eye observation color
Change judgment experiment result
In Figure 11 No. 1-9 respectively represent duck meat template concentrations be 50 ng, 5 ng, 500 pg, 50 pg, 5 pg, 500 fg,
50 fg, 5 fg, 500 ag, No. 10 templates are two distilled waters.Result represents, the limit that duck meat primer can be detected by out is 50 fg.
<110>Guangzhou Oasis Biochemistry Technology Co., Ltd.
<120>a kind of test kit quickly detecting meat adulteration based on LAMP method
<130>
<160> 20
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213>artificial primer
<400> 1
cctcacccta gtagaacgaa 20
<210> 2
<211> 18
<212> DNA
<213>artificial primer
<400> 2
gcttgacatg gctagcat 18
<210> 3
<211> 48
<212> DNA
<213>artificial primer
<400> 3
ggtgaatagt tttagggcat cggcttttac tacgaaaagg acccaacg 48
<210> 4
<211> 49
<212> DNA
<213>artificial primer
<400> 4
ttgcaccaat cctagcctta tcccttttgg gtagggtatt ggtagtgga 49
<210> 5
<211> 19
<212> DNA
<213>artificial primer
<400> 5
tctgaggggc aaccgtaat 19
<210> 6
<211> 21
<212> DNA
<213>artificial primer
<400> 6
gtcggatgtg attcctgaag g 21
<210> 7
<211> 48
<212> DNA
<213>artificial primer
<400> 7
ctttgtctac tgagaagccc cctcactgct atcccctata tcggaacc 48
<210> 8
<211> 46
<212> DNA
<213>artificial primer
<400> 8
ccattcatca tcgcagcatt agcggttgtt ggatcctgtt tcgtgg 46
<210> 9
<211> 21
<212> DNA
<213>artificial primer
<400> 9
ctctatttct accatcctcc g 21
<210> 10
<211> 20
<212> DNA
<213>artificial primer
<400> 10
gattagaccc gttaccatcg 20
<210> 11
<211> 45
<212> DNA
<213>artificial primer
<400> 11
ttcagtatag tcacccccag gactttttga aatcaacaac ccgcc 45
<210> 12
<211> 43
<212> DNA
<213>artificial primer
<400> 12
accaggcatc tggttcttac tttttaaggg gaacgtatgg acg 43
<210> 13
<211> 20
<212> DNA
<213>artificial primer
<400> 13
ctcacgagag atcagcaacc 20
<210> 14
<211> 19
<212> DNA
<213>artificial primer
<400> 14
gaagagagaa gatgccgcg 19
<210> 15
<211> 48
<212> DNA
<213>artificial primer
<400> 15
gtacggtgga aggcaagtag ggttttgtac ttcatgacca gtctcagg 48
<210> 16
<211> 47
<212> DNA
<213>artificial primer
<400> 16
ggcacatccc atgcataact cctttttatc acggactaaa gagggga 47
<210> 17
<211> 20
<212> DNA
<213>artificial primer
<400> 17
gattctactt caccgcccta 20
<210> 18
<211> 19
<212> DNA
<213>artificial primer
<400> 18
aatccgaagt ggtggtctg 19
<210> 19
<211> 46
<212> DNA
<213>artificial primer
<400> 19
ccggtggcaa caaagaaagt ggtttttaga gtaccatgaa gcccca 46
<210> 20
<211> 46
<212> DNA
<213>artificial primer
<400> 20
ccacggactc cacgtgatca tcttttttga ttagtcggag gaggca 46
Claims (2)
1., for a LAMP kit for meat detection, including reaction buffer, archaeal dna polymerase and specific primer, it is special
Levy and be: specific primer sequence is as follows:
Pork detection specific sequence:
P-F3:5 '-CCTCACCCTAGTAGAACGAA-3 '
P-B3:5 '-GCTTGACATGGCTAGCAT-3 '
P-FIP:5 '-GGTGAATAGTTTTAGGGCATCGGCTTTTACTACGAAAAGGACCCAACG-3 '
P-BIP:5 '-TTGCACCAATCCTAGCCTTATCCCTTTTGGGTAGGGTATTGGTAGTGGA-3 '
Fox meat detection specific sequence:
F-F3:5 '-TCTGAGGGGCAACCGTAAT-3 '
F-B3:5 '-GTCGGATGTGATTCCTGAAGG-3 '
F-FIP:5 '-CTTTGTCTACTGAGAAGCCCCCTCA-CTGCTATCCCCTATATCGGAACC-3 '
F-BIP:5 '-CCATTCATCATCGCAGCATTAGCG-GTTGTTGGATCCTGTTTCGTGG-3 '
Mouse meat detection specific sequence:
M-F3: 5’-CTCTATTTCTACCATCCTCCG-3’
M-B3: 5’-GATTAGACCCGTTACCATCG-3’
M-FIP: 5’-TTCAGTATAGTCACCCCCAGGACTTTTTGAAATCAACAACCCGCC-3’
M-BIP: 5’-ACCAGGCATCTGGTTCTTACTTTTTAAGGGGAACGTATGGACG-3’
Carnis Gallus domesticus detection specific sequence:
C-F3: 5’-CTCACGAGAGATCAGCAACC-3’
C-B3: 5’-GAAGAGAGAAGATGCCGCG-3’
C-FIP: 5’-GTACGGTGGAAGGCAAGTAGGGTTTTGTACTTCATGACCAGTCTCAGG-3’
C-BIP: 5’-GGCACATCCCATGCATAACTCCTTTTTATCACGGACTAAAGAGGGGA-3’
Duck meat detection specific sequence:
D-F3: 5’-GATTCTACTTCACCGCCCTA-3’
D-B3: 5’-AATCCGAAGTGGTGGTCTG-3’
D-FIP: 5’-CCGGTGGCAACAAAGAAAGTGGTTTTTAGAGTACCATGAAGCCCCA-3’
D-BIP: 5’-CCACGGACTCCACGTGATCATCTTTTTTGATTAGTCGGAGGAGGCA-3’ ；
Wherein, test kit for the reaction system of meat detection is: 1 × ThermoPol reaction buffer, MgSO4, dNTPs,
Betaine, Calcein, MnCl2, Bst archaeal dna polymerase, outer primer, inner primer, template DNA and sterilizing ddH2O；Reactant
In system, the concentration of each component is MgSO41-10 mM, dNTPs0.1-5 mM, Betain0.1-1 M, Calcein 10-50 μ
M, MnCl20.1-5 mM, 1-40U Bst archaeal dna polymerase, 0.1-2 μ l outer primer, 1-10 μ l inner primer, 0.1-5 μ l template
DNA, supplies sterilizing ddH2O to 25 μ l；The concentration of primer is 1-20 μm ol/L, and the concentration of template DNA is 10-100ng/ μ l.
LAMP kit the most according to claim 1, it is characterised in that: described test kit is possibly together with DNA extraction liquid.
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| CN105132569A (en) * | 2015-09-18 | 2015-12-09 | 李欣南 | Whole set of LAMP (loop-mediated isothermal amplification) primers for identifying or assisting in identifying murine components as well as applications of whole set of primers |
| CN108396067A (en) * | 2017-02-06 | 2018-08-14 | 上海市农业科学院 | A kind of the LAMP primer group and detection method of duck derived component |
| CN107937562A (en) * | 2017-12-05 | 2018-04-20 | 厦门出入境检验检疫局检验检疫技术中心 | Detect the primer and its design method of the LAMP method of duck derived component |
| CN110791568A (en) * | 2018-08-03 | 2020-02-14 | 上海市质量监督检验技术研究院 | LAMP primer group for rapidly detecting chicken-derived components in beef and mutton, detection kit, detection method and application |
| CN109182471A (en) * | 2018-10-12 | 2019-01-11 | 上海市农业科学院 | A kind of the ddPCR detection method and primer of duck derived component |
| CN109504785A (en) * | 2018-12-29 | 2019-03-22 | 博奥生物集团有限公司 | Primer combination and its application in Species estimation and/or the chicken identification of chicken |
| CN109517907A (en) * | 2018-12-29 | 2019-03-26 | 博奥生物集团有限公司 | Primer combines the application in Species estimation and/or the duck identification of duck |
| CN109706250A (en) * | 2018-12-29 | 2019-05-03 | 博奥生物集团有限公司 | Primer combination and its application in Species estimation and/or the pork identification of pig |
| CN110195112A (en) * | 2019-04-11 | 2019-09-03 | 中国农业大学 | Pig derived components rapid detection method and kit in a kind of food |
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| Title |
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| 环介导恒温扩增法鉴定牛羊肉中的搀杂肉;侯东军等;《食品工业科技》;20121231;第33卷(第22期);60-62页 * |
| 环介导等温扩增法检测牛羊肉中的猪肉成分;杨丽霞等;《食品与机械》;20130930;第29卷(第5期);63-65页 * |
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Address after: 510530 Guangdong province high tech Industrial Development Zone, Science City, Kaiyuan Avenue, building, floor 11, A2, three Patentee after: GUANGDONG DAYUAN OASIS FOOD SAFETY TECHNOLOGY CO., LTD. Address before: 901 room 7, No. 510663, 1st floor, science Avenue, hi tech Industrial Development Zone, Guangdong, Guangzhou Patentee before: Guangzhou Oasis Biochemistry Technology Co.,Ltd. |
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Effective date of registration: 20180919 Address after: 510663 Guangdong Guangzhou high tech Industrial Development Zone, Science City, open source road 11, A2 third. Co-patentee after: Guangdong Zhong Da Da Detection Technology Co., Ltd. Patentee after: GUANGDONG DAYUAN OASIS FOOD SAFETY TECHNOLOGY CO., LTD. Address before: 510530 Guangdong Guangzhou high tech Industrial Development Zone, Science City, open source road 11, A2 three. Patentee before: GUANGDONG DAYUAN OASIS FOOD SAFETY TECHNOLOGY CO., LTD. |