CN110317896A - LAMP primer group and application thereof for detecting corn derived components - Google Patents
LAMP primer group and application thereof for detecting corn derived components Download PDFInfo
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- CN110317896A CN110317896A CN201910531982.XA CN201910531982A CN110317896A CN 110317896 A CN110317896 A CN 110317896A CN 201910531982 A CN201910531982 A CN 201910531982A CN 110317896 A CN110317896 A CN 110317896A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention belongs to biomolecule detection technical fields, and in particular to a kind of LAMP primer group and application thereof for detecting corn derived components.Of the invention includes FIP primer, F3 primer, BIP primer, B3 primer and LB primer for detecting the LAMP primer group of corn derived components, LAMP detection architecture of the invention has good specificity and sensitivity, quickly, conveniently, efficiently whether can contain corn ingredient in test sample, can be used for the quick and precisely detection of sweet potato vermicelli quality, with meet country to sweet potato vermicelli raw material authenticity detection there is an urgent need to.
Description
Technical field
The invention belongs to biomolecule detection technical fields, and in particular to a kind of LAMP for detecting corn derived components draws
Object group and application thereof.
Background technique
Corn is common cereal crops, due to cheap, have mix corn in other products with secondary in the market
The case where substituting the bad for the good.
By taking vermicelli as an example, vermicelli are one of the traditional foods that our people is fond of, and there is convenient, quick, nutrition to close
The features such as reason and abundant taste, is liked and welcome by the majority of consumers, wherein sweet potato vermicelli is always with the daily drink of people
Food, nutritive value and suitable mouthfeel are liked deeply.Sweet potato (also known as sweet potato, sweet potato, pachyrhizus, sweet potato etc.) is a kind of ancient
Tuber crops, without pollution by pesticides in planting process, low fertilizer application is the green food raw material that consumer most trusts.Sweet potato is rich
Containing protein, starch, vitamin and several mineral materials, and there is anti-cancer, defaecation, beauty treatment, lengthen one's life and other effects, is known as " natural green
Color food " and " life prolonging food ", occupy first of green vegetables.In recent years, sweet potato is considered for health food more and more,
Market comsupton demand is continuously increased, and is joyfully risen with diet Leisure Culture with the continuous improvement of people's living standards, and sweet potato is
The main quality raw materials for being risen to food industry, all enjoy high reputation in the world, this also gives the sweet potato vermicelli industrial zone of China
Huge market opportunity is carried out.But there are many problems in present vermicelli market.Due to cornstarch, the valence of tapioca
Lattice are all more much lower than starch from sweet potato, so many illegal businessman are to obtain the sudden huge profits of fake products bring to form sediment toward addition corn in vermicelli
Powder, tapioca, with cornstarch make so-called " pure sweet potato vermicelli ", for allow color likeness in form, mouthfeel chewiness, or even addition
The violations additive such as prepared Chinese ink and industrial work stone wax.It is at present to detect the harmful substance contained in sweet potato vermicelli and judge it
It is true and false, it is established there are many kinds of method, but not yet establish and whether there is sweet potato from the angle of gene detection sweet potato vermicelli
The method of specific gene, whether for identifying with other starch false making sweet potato vermicelli, gene tester has the time
It is short, the features such as high specificity, high sensitivity, great researching value and meaning.
" the PCR detections of several vegetable-derived components in food " (" Food Science ", 2006, Vol.27, No.11, Chen Wen Ping,
Shao Biying etc., P404-405), disclose the primer using PCR method detection corn ingredient；CN 101701256A is also disclosed
The PCR identification primer and identification method of corn, the PCR identify primer can specific amplification corn trnL sequence, but it is also only public
The embodiment that the DNA for using corn plant tissue extraction to obtain is expanded for template is opened；CN 106399540A discloses base
In the corn detection method of ring mediated isothermal amplification delustring technology, can specific detection corn itself intrinsic IVR gene,
The gene is not included in transgenic corns then；CN 108277294A disclose specific primer for detecting maize dna and
Probe, can specific detection corn Zein rDNA, Zein be corn embryosperm major storage protein, account for endosperm gross protein
70%, it is primarily present in corn oil.But the DNA as contained in cornstarch is relatively fewer, and extraction difficulty is big, and vermicelli
Deng needing to be heat-treated cornstarch in processed food process, it be easy to cause the damage of DNA, detects cornstarch
Whether the difficulty containing corn derived components is bigger in converted products.
" food starch vegetable-derived components discrimination method-real-time fluorescence PCR method-Part VII cornstarch " (Chinese people
Republic's inspection and quarantining for import/export professional standard, referring tohttp://www.doc88.com/p-9819120496207.html)
Disclose can specific detection corn real-time fluorescence amplification PCR primer, specificity it is good, and using corn be raw material extraction DNA into
Gone the test (25 μ L of reaction system, 5 μ L of template DNA) of absolute sensitivity, absolute sensitivity up to 0.01ng/ μ L, when from
The maize dna extracted in sample can be detected when being 50pg or more；And mix cornstarch with tapioca,
The test of relative sensitivity is carried out, relative sensitivity is up to 0.1wt% (Maize Starch Content 0.1g/100g).But the standard
The detection of amplified fluorescence PCR primer application starch from sweet potato processed food (for example, vermicelli etc.) will not implemented, cornstarch exists
It usually needs to be heat-treated in process, and then the DNA in starch can be caused to damage, be in the converted products such as detection vermicelli
The no difficulty containing corn derived components is bigger.
Summary of the invention
It is an object of the present invention to provide the LAMP primer groups for detecting corn derived components, to solve in the prior art
Gene tester cannot be used for detection cornstarch processed food the problem of, specific technical solution is as follows:
The sequence of the primer sets are as follows:
Yu-F3:5`-GCGAAAAAGAACCCACGGC-3`
Yu-B3:5`-GCTTCGGGCGCAACTTG-3`
Yu-FIP:5`-TAACCGCTGCCCTGGGAGCTTTTCACCAGTACTACCTCCTGCC-3`
Yu-BIP:5`-GCAACGGATATCTCGGCTCTCGTTTTTTCGCGGGATTCTGCAATT- 3`
Yu-LB:5`-CATCGATGAAGAACGTAGCAAAATG-3`.
2nd purpose of the invention is to provide the reagent for detecting corn derived components, specific technical solution are as follows: including
dNTPs、Mg2+, Bst enzyme, Buffer and fluorescent dye and LAMP primer group as described above.
3rd kit being designed to provide for detecting corn derived components of the invention, specific technical solution are as follows:
Including as described above for detecting the LAMP primer group of corn derived components or as described above for detecting corn derived components
Reagent.
4th method being designed to provide for detecting corn derived components of the invention, specific technical solution are as follows: packet
It includes following steps: (1) extracting sample DNA；(2) using LAMP primer group as described in claim 1 or such as claim 2 institute
The reagent stated or kit as claimed in claim 3 prepare LAMP reaction system；(3) by prepared LAMP reaction system
It is placed in real-time fluorescence PCR instrument or water-bath and carries out constant-temperature amplification；(4) face of amplification curve or reaction solution is observed after amplification 1h
Color.
Preferably, the method for extracting sample DNA is CTAB method.
Preferably, the temperature of the constant-temperature amplification is 60 DEG C.
Preferably, the molar ratio of Yu-FIP:Yu-BIP:Yu-LB:Yu-F3:Yu-B3 is 8 in the LAMP reaction system:
8:4:1:1。
The object of the invention is also to provide above-mentioned for detecting the purposes of the LAMP primer group of corn derived components, specifically
Technical solution are as follows: the application in detection corn derived components.
Preferably, LAMP primer group/reagent/kit as described above for detecting corn derived components is in detection starch
Or the application in starch processed food in corn derived components.
Preferably, LAMP primer group/reagent/kit as described above for detecting corn derived components is in detection vermicelli
Application in middle corn derived components.
The beneficial effects of the present invention are: the LAMP primer group for detecting corn derived components of the invention has good spy
Anisotropic and sensitivity, can be realized the detection to corn derived components in sample under constant temperature conditions.
The LAMP primer group of the invention specificity at 60 DEG C is more preferable.
The absolute sensitivity of LAMP primer group of the invention is up to 10pg/ μ L, and relative sensitivity is up to 5%.
LAMP primer group of the invention can be used for detecting the corn derived components in vermicelli.
When detecting the corn derived components in vermicelli using LAMP primer group of the invention, CTAB method is selected to extract sample DNA
There is better sensitivity.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
Some embodiments of invention, for those of ordinary skill in the art, without any creative labor, also
Other drawings may be obtained according to these drawings without any creative labor.
Fig. 1 is that sweet potato DNA, cassava DNA, potato DNA, H2O and jade are separately added into LAMP reaction system at 59 DEG C
The amplification curve of the progress isothermal duplication of Miho Dockyard NA；
Fig. 2 is that sweet potato DNA, cassava DNA, potato DNA, H2O and jade are separately added into LAMP reaction system at 60 DEG C
The amplification curve of the progress isothermal duplication of Miho Dockyard NA；
Fig. 3 is that sweet potato DNA, cassava DNA, potato DNA, H2O and jade are separately added into LAMP reaction system at 61 DEG C
The amplification curve of the progress isothermal duplication of Miho Dockyard NA；
Fig. 4 is that sweet potato DNA, cassava DNA, potato DNA, H2O and jade are separately added into LAMP reaction system at 62 DEG C
The amplification curve of the progress isothermal duplication of Miho Dockyard NA；Wherein, GAN DNA indicates that sweet potato DNA, MA DNA indicate Ma Ling in Fig. 1-4
Potato DNA, MU DNA indicate that cassava DNA, YU DNA indicate maize dna；
Fig. 5 is the amplification curve that the absolute sensitivity of LAMP primer group of the invention is tested, and wherein 1pg indicates DNA profiling
Concentration be 1pg/ μ L, 10pg indicate DNA profiling concentration be 10pg/ μ L, 100pg indicate DNA profiling concentration be 100pg/
μL；
Fig. 6 is the amplification curve that the relative sensitivity of LAMP primer group of the invention is tested；
Fig. 7 is the melting curve that the relative sensitivity of LAMP primer group of the invention is tested, wherein in Fig. 6 and Fig. 7
20,1,10,5 respectively indicate in cornstarch and tapioca mixing sample, the mass fraction of cornstarch is 20%, 1%,
10%, 5%；
Fig. 8 is to 18-29 vermicelli sample using LAMP primer group of the invention (using RNA isolation kit extract powder bar sample
In DNA) amplification curve that is detected；
Fig. 9 is to No. 25 vermicelli samples using LAMP primer group of the invention (using in CTAB method extract powder bar sample
DNA the amplification curve) detected；
Figure 10 is to No. 25 vermicelli samples using LAMP primer group of the invention (using in CTAB method extract powder bar sample
DNA the melting curve) detected.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts it is all its
His embodiment, shall fall within the protection scope of the present invention.
The source of agents useful for same of the present invention, is detailed in the following table 1
1 test reagent source of table
Embodiment 1
(1) design of primers: it is with corn rRNA genetic interval sequence (Internal Transcribed Spacer, ITS)
Target sequence, with the primer of PrimerExplorerV5 design LAMP, the primer sets for largely being screened to obtain following sequence are (by leading to
Synthesized with biosystem (Anhui) Co., Ltd), particular sequence see the table below 2.
(2) it the extraction of template DNA: is mentioned using the plant genome DNA purchased from TIANGEN Biotech (Beijing) Co., Ltd.
Kit is taken to extract maize dna.Concrete operation step is shown in the specification of kit.
(3) specific test of LAMP primer of the invention:
The reaction system of LAMP is as shown in table 3 below
3 LAMP reaction system of table (10 μ L)
Wherein, LAMP primer be by FIP (0.8 μM of final concentration), BIP (0.8 μM of final concentration), LB (0.4 μM of final concentration),
The mixed liquor that F3 (0.1 μM of final concentration), B3 (0.1 μM of final concentration) are mixed in the ratio of 8:8:4:1:1.
Eight union of PCR is taken, is added in 8 single tubes respectively and according to the prepared reaction system of upper table 3 (does not include in system
Template DNA, 9 μ L in each pipe), wherein 1 μ L maize dna is added in two pipes, 1 μ L sweet potato DNA, 1 are in addition separately added into six pipes
μ L sweet potato DNA, 1 μ L cassava DNA, 1 μ L cassava DNA, 1 μ L potato DNA, 1 μ L potato DNA；It is put into StepOnePlus reality
When fluorescent PCR instrument in, holding stage temperature setting is 5min, and each group holding temperature is respectively 59 DEG C, 60 DEG C, 61 DEG C, 62 DEG C,
Cycle stage temperature is also respectively set to 59 DEG C, 60 DEG C, 61 DEG C, 62 DEG C, and melting curve ratio is 1.5%, continuously recycles 110
A circulation；
Observe known to amplification curve (being detailed in Figure of description Fig. 1-4): maize dna can be big under the action of primer at 59 DEG C
Amount amplification, but sweet potato DNA has an amplification, and another does not expand, and illustrates the specificity of the primer pair maize dna at 59 DEG C not
By force；Maize dna obviously expands at 60 DEG C, and other curves do not expand trend, illustrates primer at 60 DEG C to maize dna
High specificity, and two curves essentially coincide, reproducibility might as well；Maize dna obviously expands at 61 DEG C, and other curves do not have
Amplification trend illustrates that primer is also strong to the specificity of maize dna at 61 DEG C, and reproducibility might as well；All DNA are at 62 DEG C
It does not expand, illustrates not being ideal temperature that primer combination DNA is effectively expanded at 62 DEG C.To sum up, select 60 DEG C for LAMP most
Good temperature.
The sensitivity of the LAMP primer of the invention of embodiment 2
(1) absolute sensitivity: carrying out gradient dilution to the obtained maize dna of extraction, be diluted to 1ng/ μ L, 100pg/ μ L,
10pg/ μ L and 1pg/ μ L, takes eight union of PCR, is added in 8 single tubes according to the prepared reaction system (body of upper table 4 respectively
Do not include template DNA in system, 9 μ L in each pipe), and be separately added into wherein 2 single tubes 1 μ L 1ng/ μ L maize dna,
The corn for being separately added into the maize dna of 1 μ L 100pg/ μ L in another 2 single tubes, being separately added into 10pg/ μ L in another 2 single tubes
The maize dna of 1pg/ μ L is separately added into DNA, last 2 single tubes；Negative control is set, takes 2 PCR single tubes, is separately added into
According to the prepared reaction system of upper table 4 (not including template DNA in system, 9 μ L in each pipe) and 1 μ L ddH2O；By PCR
Eight unions and negative control are put into StepOnePlus real-time fluorescence PCR instrument, and holding stage temperature setting is 5min, heat preservation temperature
Degree is respectively 60 DEG C, and cycle stage temperature is also 60 DEG C, and solubility curve ratio is 1.5%, 110 circulations of continuous circulation；Observation
Amplification curve (is detailed in Figure of description Fig. 5).
As shown in figure 5, two lines when maize dna concentration is 1ng/ μ L, 100pg/ μ L, 10pg/ μ L have obvious amplification,
But DNA concentration only has a line to have amplification trend when being 1pg/ μ L, this is because the concentration of 1pg/ μ L has exceeded the inspection of this method
Limit is surveyed, makes to expand unstable.Therefore the absolute sensitivity of this method can reach 10pg/ μ L, under the reaction system of 10 μ L, from
The maize dna extracted in sample, which reaches 10pg, to be detected.
(2) relative sensitivity: cornstarch is mixed with tapioca, wherein the mass fraction of cornstarch is respectively
20%, 10%, 5% and 1%, extract DNA (plant genome DNA extracts kit, the Tiangeng, according to saying of above-mentioned mixing sample
Step in bright book is extracted), it is spare as template DNA；Eight union of PCR is taken, is added in 8 single tubes matches according to upper table 4 respectively
The reaction system (not including template DNA in system, 9 μ L in each pipe) made and 1 μ L 20%, 10%, 5% and 1% mixing
The template DNA extracted in sample, each sample do a repetition；Negative control is set, takes 2 PCR single tubes, is separately added into
According to the prepared reaction system of upper table 4 (not including template DNA in system, 9 μ L in each pipe) and 1 μ L ddH2O；By PCR
Eight unions and negative control are put into StepOnePlus real-time fluorescence PCR instrument, and holding stage temperature setting is 5min, heat preservation temperature
Degree is respectively 60 DEG C, and cycle stage temperature is also 60 DEG C, and solubility curve ratio is 1.5%, 110 circulations of continuous circulation；Observation
Amplification curve (being detailed in Figure of description Fig. 6) and melting curve (being detailed in Figure of description Fig. 7).
There is apparent amplification trend when it will be appreciated from fig. 6 that containing 20%, 10%, 5% cornstarch in mixing sample, but
Curve when adulterated 1% does not expand, and illustrates that 1% has been more than the detection limit of the method.Therefore the relative sensitivity of this method is extremely
5% can be reached less.
LAMP primer of the invention is used for the detection of vermicelli by embodiment 3
(1) 30 parts of vermicelli samples being commercially available are detected
The extraction of vermicelli DNA: using plant genome DNA extracts kit after vermicelli are crushed, Tiangeng is said according to kit
Step in bright book is extracted to obtain, respectively number 1-30.
The preparation of LAMP reaction system: it is added in PCR pipe respectively according to the prepared reaction system of upper table 3 (in system
Do not include template DNA, 9 μ L in each pipe) and the DNA that is extracted from 1-30 vermicelli sample of 1 μ L, each sample do one
Group repeats to test；PCR pipe is put into StepOnePlus real-time fluorescence PCR instrument, holding stage temperature setting is 5min, heat preservation
Temperature is respectively 60 DEG C, and cycle stage temperature is also 60 DEG C, and solubility curve ratio is 1.5%, 110 circulations of continuous circulation；It sees
Examine amplification curve.
Testing result: No. 1-24 linear without amplification with 26-30 vermicelli sample, shows do not have corn in above-mentioned vermicelli
The adulterated situation of starch；No. 25 vermicelli samples have amplification phenomenon (being detailed in Figure of description Fig. 8), but reproducibility is bad, illustrates 25
Number sample may have a small amount of cornstarch adulterated.
(2) repeated authentication is carried out to No. 25 vermicelli samples using the DNA that CTAB method is extracted:
DNA in No. 25 vermicelli samples is extracted using CTAB method: with Universalpulverizer by No. 25 vermicelli sample comminutions, being weighed
0.1g powder in the centrifuge tube of a clean 2.0ml, be added 1.5ml CTAB lysate (1gCTAB, 4.0908gNaCl,
0.37224g disodium ethylene diamine tetraacetate, 0.6057gTris are after beaker dissolution with deionized water in 50ml volumetric flask constant volume),
In 65 DEG C of water-baths one hour and turn upside down；Take 1ml supernatant in another clean centrifuge tube after 8000rpm centrifugation 15min
In, 700 μ L chloroforms are added, mix 30s, 14500rpm is centrifuged 10min；It takes 650 μ L of supernatant into clean centrifuge tube, adds
Entering 1300 μ LCTAB precipitated liquids, (0.25gCTAB, 0.11688gNaCl are after beaker dissolution with deionized water in 50ml volumetric flask
Constant volume), 30s is mixed, is stored at room temperature one hour；14500rpm is centrifuged 10min, abandons supernatant, and 350 μ L 1.2M/L are added
NaCl (3.5064gNaCl is after beaker dissolution with deionized water in 50ml volumetric flask constant volume), acutely shakes 30s, 350 μ is added
L chloroform, mixes 30s, and 14500rpm is centrifuged 10min；320 μ L of supernatant is taken, 256 μ L isopropanols are added, -20 DEG C again after mixing
Lower to place one hour, 14500rpm is centrifuged 20min, abandons supernatant, and 500 μ L70% ethyl alcohol are added, and mixes, 14500rpm centrifugation
20min abandons supernatant；It dries in the air to air-drying, 20 μ LddH is added2O dissolution.
Being added in PCR pipe according to the prepared reaction system of upper table 3 respectively (does not include template DNA, Mei Geguan in system
In 9 μ L) and 1 μ L use the DNA that extracts from No. 25 vermicelli samples of CTAB method, do one group and repeat to test；PCR pipe is put
Enter in StepOnePlus real-time fluorescence PCR instrument, holding stage temperature setting is 5min, and holding temperature is respectively 60 DEG C, circulation
Phase temperature is also 60 DEG C, and solubility curve ratio is 1.5%, 110 circulations of continuous circulation；Observing amplification curve, (specification is attached
Fig. 9) and melting curve (Figure of description 10).
By Fig. 9 and 10 it is found that having obvious amplification using the DNA that CTAB method is extracted from No. 25 vermicelli samples, show 25
The situation for having cornstarch adulterated really in number sample.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Within mind and principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Sequence table
<110>Xuchang College
<120>for detecting LAMP primer group of corn derived components and application thereof
<130> 192100
<141> 2019-06-19
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gcgaaaaaga acccacggc 19
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gcttcgggcg caacttg 17
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taaccgctgc cctgggagct tttcaccagt actacctcct gcc 43
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Claims (10)
1. the LAMP primer group for detecting corn derived components, which is characterized in that the sequence of the primer sets are as follows:
Yu-F3:5`-GCGAAAAAGAACCCACGGC-3`
Yu-B3:5`-GCTTCGGGCGCAACTTG-3`
Yu-FIP:5`-TAACCGCTGCCCTGGGAGCTTTTCACCAGTACTACCTCCTGCC-3`
Yu-BIP:5`-GCAACGGATATCTCGGCTCTCGTTTTTTCGCGGGATTCTGCAATT- 3`
Yu-LB:5`-CATCGATGAAGAACGTAGCAAAATG-3`.
2. the reagent for detecting corn derived components, which is characterized in that including dNTPs, Mg2+, Bst enzyme, Buffer and fluorescence dye
Material and LAMP primer group as described in claim 1.
3. for detecting the kits of corn derived components, which is characterized in that including LAMP primer group as described in claim 1 or
The reagent as claimed in claim 2 for being used to detect corn derived components.
4. the method for detecting corn derived components, which comprises the steps of: (1) extract sample DNA；(2) using as weighed
Benefit require 1 described in LAMP primer group or reagent as claimed in claim 2 or kit as claimed in claim 3 prepare
LAMP reaction system；(3) prepared LAMP reaction system is placed in progress constant temperature expansion in real-time fluorescence PCR instrument or water-bath
Increase；(4) color of amplification curve or reaction solution is observed after amplification 1h.
5. the method for detection corn derived components according to claim 4, which is characterized in that the side for extracting sample DNA
Method is CTAB method.
6. the method for detection corn derived components according to claim 4, which is characterized in that the temperature of the constant-temperature amplification is
60℃。
7. the method for detection corn derived components according to claim 4, which is characterized in that in the LAMP reaction system
The molar ratio of Yu-FIP:Yu-BIP:Yu-LB:Yu-F3:Yu-B3 is 8:8:4:1:1.
8. the purposes of LAMP primer group according to claim 1, which is characterized in that answering in detection corn derived components
With.
9. the purposes of LAMP primer group according to claim 8, which is characterized in that in detection starch or starch processed food
Application in middle corn derived components.
10. the purposes of LAMP primer group according to claim 8, which is characterized in that the corn derived components in detection vermicelli
In application.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112553300A (en) * | 2020-10-15 | 2021-03-26 | 德歌生物技术(山东)有限公司 | Primer design method, primer and method for isothermal amplification of nucleic acid fragment |
| CN112626181A (en) * | 2020-12-07 | 2021-04-09 | 许昌学院 | LMCP (local mean-size distribution) primer group for detecting walnut source components, detection method and application thereof |
| CN112662797A (en) * | 2020-11-09 | 2021-04-16 | 许昌学院 | LMCP (local mean-particle size distribution) primer group and kit for detecting plant source components and application of LMCP primer group and kit |
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