CN103436595A - LAMP detection primer group of NOS terminator, kit and detection method - Google Patents
LAMP detection primer group of NOS terminator, kit and detection method Download PDFInfo
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Abstract
The invention relates to an LAMP detection primer group of an NOS terminator, a kit and a detection method, wherein the LAMP detection primer group includes the following primers of an outer primer F3: CTCGATCTATCTATTGGCA, an outer primer B3: GCGCTTATTATTGATTACTTACG, an inner primer FIP: TCTAGCTTATCGTATTACTTCAGTTGATTGATTCCAGTTGCCG and an inner primer BIP: AGCTTGACGTTTTATTTGTGTAGGCGTTATTATTGATTATTAGCGGGT. The LAMP detection primer group provided by the invention has good specificity for the NOS terminator.
Description
[technical field]
The invention belongs to the food safety field, relate to loop-mediated isothermal amplification technique, the LAMP of the genetically modified crops that are specifically related to contain the NOS terminator detects primer sets, test kit and detection method.
[background technology]
Genetically modified crops refer to utilize recombinant DNA technology by exogenous origin gene integrator in the recipient plant genome, change the plant and the offspring thereof that produce after its genetic composition, also referred to as genetically modified organism, GMO (Genetically modified organisms, GMOs).Since nineteen eighty-three, the first transgenic plant were come out, cultivated area and the sales revenue of global genetically modified crops all increase with multiple.But from serious scientific meaning, say, the biological safety of transgene agricultural product there is no definite conclusion at present.Although China's regulation was from 20 days March in 2002, all transgenic crops and byproduct thereof all should be indicated, under the situation of being on the increase at transgenic crop, still seldom see the agricultural-food of filling transgenosis printed words.Therefore develop easy genetically engineered soybean detection technique and related products is detected imperative.
The detection technique of generally applying based on DNA is at present detected genetically modified crops.Such technology mainly comprises traditional qualitative PCR and quantitative PCR.These detection techniques all need special DNA cloning instrument (PCR instrument) to complete.And isothermal gene amplification (the Loop-Media tedLsothcrmal AmpliFication of ring mediation, LAMP) you rely in the primer that can identify 6 special zones on target sequence and a kind of archaeal dna polymerase with strand displacement characteristic, utilize the strand displacement archaeal dna polymerase to be incubated dozens of minutes at constant temperature, complete nucleic acid amplification reaction; Can the target gene DNA fragmentation be increased to 109-1010 doubly within an hour, as long as and the white casse precipitation that detects by an unaided eye, just can identify whether increase, do not need the electrophoresis detection process.
Consult genetically engineered soybean strain MON89788 pertinent literature, understand NOS terminator sequence information, partial sequence information is specially:
TCTCGTTCAAACATTTGGCAATAAAGTTTCTTAAGATTGAATCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAGCATGTA ATAATTAACATGTAATGCATGACGTTATTTATGAGATGGGTTTTTATGATTAGAGTCCCGCAATTATACATTTAATACGCGATAGAAAACAAAATATAGCGCGCAAACTAGGATAAATTATCGCGCGCGGTGTCATCTGTCGAGGGGGGGCCCGGTAC。
[summary of the invention]
The LAMP that first technical problem that the present invention will solve is to provide a kind of NOS terminator detects primer sets, and it has good specificity to the NOS terminator.
Second technical problem that the present invention will solve is to provide a kind of LAMP detection kit of NOS terminator, and it is convenient to the NOS terminator is carried out to the LAMP detection.
The 3rd technical problem that the present invention will solve is to provide a kind of LAMP detection method of NOS terminator, its have sensitive higher, specificity good, detect characteristics fast and reliable, simple to operate.
Above-mentioned first technical problem is achieved through the following technical solutions:
A kind of LAMP of NOS terminator detects primer sets, it is characterized in that, comprises following primer:
Outer primer F3:CTCGATCTATCTATTGGCA;
Outer primer B3:GCGCTTATTATTGATTACTTACG;
Inner primer FIP:
TCTAGCTTATCGTATTACTTCAGTTGATTGATTCCAGTTGCCG；
Inner primer BIP:
AGCTTGACGTTTTATTTGTGTAGGCGTTATTATTGATTATTAGCGGGT。
Proof by experiment, LAMP provided by the invention detect primer sets to Semen Phaseoli Vulgaris, corn, peanut, pea, oat, string bean, soybean, wheat, in the DNA of French beans, tomato, walnut, jordan almond, mung bean, Radix Et Rhizoma Fagopyri Tatarici, buckwheat, potato, grass shrimp, celery, paddy rice, pig, grapefruit, Radix Dauci Sativae, goose, ox, the GTS40-3-2 that contains the NOS terminator, MON89788, EVENT98140, BT176, MON810, A5547-127 all without non-specific amplification.Therefore, LAMP detection primer sets provided by the invention has good specificity to the NOS terminator.
Above-mentioned second technical problem is achieved through the following technical solutions:
The LAMP detection kit of NOS terminator, is characterized in that, comprises following composition:
; Wherein, the LAMP that each primer pair in primer liquid should be described NOS terminator detects each primer in primer sets.
Also comprise developer; Described developer is fluorescence dye SYBR Green I.
Also comprise contrast: the DNA solution that positive control is the genetically modified corn MON 863 that contains the NOS terminator that purity is 5%, concentration is 100ng/ul, negative control is ddH
2o.
LAMP detection kit provided by the invention set above-mentioned LAMP detect the relevant materials that primer and LAMP detect, be convenient to the user NOS terminator carried out to the LAMP detection.
Above-mentioned the 3rd technical problem is achieved through the following technical solutions:
The LAMP detection method of NOS terminator, is characterized in that, comprises the following steps:
(101) treat the sample product and extract template DNA to be checked;
(102) carry out the ring mediated isothermal gene amplification reaction:
Configuration LAMP reaction system, the LAMP reaction system of every 25 μ L volumes is specially:
, the LAMP reaction system is placed in to the LAMP turbidimeter and carries out 63 ℃ of isothermal reaction 60min, then under 80 ℃, be incubated 5min; Wherein, the LAMP that each primer pair in primer liquid should be described NOS terminator detects each primer in primer sets.
Proof by experiment, LAMP detection method provided by the invention, in conjunction with the good specificity of above-mentioned primer and the advantage of LAMP method, has following characteristics: sensitivity is higher, fast and reliable, have good stability, simple to operate, identify simple.
[accompanying drawing explanation]
Fig. 1 carries out the result schematic diagram of LAMP reaction in embodiment mono-;
Fig. 2 carries out the result schematic diagram of sensitivity experiment in embodiment bis-;
Fig. 3-Fig. 6 carries out the result schematic diagram of specificity experiment in embodiment tri-;
Fig. 7-Fig. 9 is the result schematic diagram that embodiment tetra-carries out stability experiment.
[embodiment]
Embodiment mono-
The LAMP of the NOS terminator that the present embodiment one provides detects primer sets, and it comprises following primer:
Outer primer F3:CTCGATCTATCTATTGGCA;
Outer primer B3:GCGCTTATTATTGATTACTTACG;
Inner primer FIP:
TCTAGCTTATCGTATTACTTCAGTTGATTGATTCCAGTTGCCG；
Inner primer BIP:
AGCTTGACGTTTTATTTGTGTAGGCGTTATTATTGATTATTAGCGGGT。
The test kit of the NOS terminator that the present embodiment one provides, it comprises following composition:
; Wherein, the LAMP that each primer pair in primer liquid should be described NOS terminator detects each primer in primer sets.
The present embodiment one provides treats the method that the sample product are detected, and specifically comprises the following steps:
(101) treat the sample product and extract template DNA to be checked;
Extracting method can adopt CTAB method or commercialization that the method provided on the DNA test kit is provided;
(102) carry out the ring mediated isothermal gene amplification reaction:
The LAMP reaction system that the configuration cumulative volume is 25uL is specially:
, wherein, the LAMP that each primer pair in primer liquid should be described NOS terminator detects each primer in primer sets; The LAMP reaction system is placed in to the LAMP turbidimeter and carries out 63 ℃ of isothermal reaction 60min, then under 80 ℃, be incubated 5min;
(103) result judgement, can be by a kind of carrying out in following two kinds of modes:
First kind of way: change to judge amplification by the turbidity precipitated in the observing response pipe, be precipitated as the positive if occur, if feminine gender do not occur being precipitated as;
The second way is: add respectively 1-2 μ L developer (consumption is 2 μ L usually) in above-mentioned reaction tubes, mix, if be green positive, be orange negative.
Select above-mentioned primer to carry out the LAMP detection, have appearance time early, characteristics that signal is strong, as shown in Figure 1, with the DNA solution of the vegetable seed cypress that contains the NOS terminator, replace template DNA to be checked, carry out in addition above-mentioned steps (102) twice; With ddH
2o is as negative control, specifically with ddH
2o replaces template DNA to be checked, carries out in addition above-mentioned steps (102) twice; Above-mentioned four LAMP reaction results are shown in Fig. 1, in Fig. 1, and CH1 line, the corresponding ddH of CH2 line
2o, the DNA solution of the vegetable seed cypress that CH3 line, CH4 line correspondence contain the NOS terminator.
The parameter of the DNA solution of the vegetable seed cypress that in this article, contains the NOS terminator is: purity is that 5%, DNA concentration is 100ng/ul.
In this article, being determined as of DNA concentration and purity:
Get 5 μ L DNA solutions and add ddH
2the O gradient dilution, to 1mL, is used nucleic acid-protein analyser or ultraviolet spectrophotometer to survey the optical density value at 260nm and 280nm place; The concentration of DNA is calculated and is obtained according to following formula:
C＝A×N×50/1000；
In formula, C---DNA concentration (μ g/ μ L), A---the light absorption value at 260nm place, N---nucleic acid extension rate, 1OD
260nm=50 μ g/mL double-stranded DNAs; Work as OD
260/ OD
280ratio between 1.7～1.9 the time, is suitable for LAMP and detects.
Embodiment bis-
In this example, the detection method mainly embodiment mono-provided is carried out sensitivity experiment, specifically:
(purity is 10% will to contain the DNA solution of the genetically modified corn MON 863 of NOS terminator, concentration is 100ng/ μ l) be diluted to respectively the mixing solutions of six different purity with the DNA solution of non-transgenic paddy rice, in five mixing solutionss, the purity of the DNA of the genetically modified corn MON 863 that contains the NOS terminator is respectively 5%, 1%, 0.5%, 0.1%, 0.05%, 0.01%; Each mixing solutions is got respectively 2 μ L, all replaces template DNA to be checked and operates in addition the step described in embodiment mono-(102); With ddH
2o is as negative control, (purity is 10% to take the DNA solution of the genetically modified corn MON 863 that contains the NOS terminator, concentration is 100ng/ μ l) as positive control, replace respectively template DNA to be checked and operate in addition the step described in embodiment mono-(102).
Experimental result as shown in Figure 2, in Fig. 2, DNA solution (purity is 10%, and concentration is 100ng/ μ l), the ddH of the genetically modified corn MON 863 that CH21 line-CH28 line corresponds respectively to contain the NOS terminator
2the mixing solutions that the mixing solutions that the mixing solutions that the mixing solutions that the mixing solutions that the mixing solutions that O, purity are 5%, purity are 1%, purity are 0.5%, purity are 0.1%, purity are 0.05%, purity are 0.01%.
As shown in Figure 2, the DNA solution (containing the NOS terminator) of the detectability of the detection method of embodiment mono-genetically modified corn MON 863 that can to reach in purity be 0.5% detects the NOS terminator; During minimum detectability, about 30min goes out peak, and false positive does not occur, the reaction times can be defined as to 60min.Therefore, present method has higher sensitivity.
Embodiment tri-
In this example, the primer mainly embodiment mono-provided carries out the specificity experiment, specifically:
With Semen Phaseoli Vulgaris, corn, peanut, pea, oat, string bean, soybean, wheat, replace respectively template DNA to be checked in the DNA of French beans, tomato, walnut, jordan almond, mung bean, Radix Et Rhizoma Fagopyri Tatarici, buckwheat, potato, grass shrimp, celery, paddy rice, pig, grapefruit, Radix Dauci Sativae, goose, ox, the GTS40-3-2 that contains the NOS terminator, MON89788, EVENT98140, BT176, MON810, A5547-127 and operate in addition the step described in embodiment mono-(102); With ddH
2o is as negative control, (purity is 10% to take the DNA solution of the genetically modified corn MON 863 that contains the NOS terminator, concentration is 100ng/ μ l) as positive control, replace respectively template DNA to be checked and operate in addition the step described in embodiment mono-(102).
As shown in Figure 3, in Fig. 3, CH31 line-CH38 line corresponds respectively to Semen Phaseoli Vulgaris, corn, peanut, pea, oat, string bean, soybean, wheat to part of test results.
As shown in Figure 4, in Fig. 4, CH41 line-CH48 line corresponds respectively to French beans, tomato, walnut, jordan almond, mung bean, Radix Et Rhizoma Fagopyri Tatarici, buckwheat, potato to part of test results.
As shown in Figure 5, in Fig. 5, CH51 line-CH58 corresponds respectively to grass shrimp, celery, paddy rice, pig, grapefruit, Radix Dauci Sativae, goose, ox to part of test results.
As shown in Figure 6, in Fig. 6, CH61 line-CH68 line corresponds respectively to DNA solution (purity is 10%, and concentration is 100ng/ μ l), the ddH of the genetically modified corn MON 863 that contains the NOS terminator to part of test results
2o, the GTS40-3-2 that contains the NOS terminator, MON89788, EVENT98140, BT176, MON810, A5547-127.
The shown result from Fig. 3 to Fig. 6, above-mentioned primer only has specificity to the NOS terminator, to other containing the transgenic strain of NOS terminator and non-transgenic species all without increasing, therefore, the specificity of above-mentioned primer is good, therefore, putting before this, detection method provided by the invention is very reliable.
Embodiment tetra-
In this example, the detection method mainly embodiment mono-provided is carried out stability experiment, specifically comprises:
(purity is 10% will to contain the DNA solution of the genetically modified corn MON 863 of NOS terminator, concentration is 100ng/ μ l) to be diluted to purity with the non-transgenic maize dna be 5%, getting respectively 20 deals is the DNA solution after the above-mentioned dilution of 2 μ L, all replaces template DNA to be checked and operates in addition step described in embodiment mono-(102); With ddH
2o is as negative control, (purity is 10% to the DNA solution of the genetically modified corn MON 863 that contains the NOS terminator, concentration is 100ng/ μ l) as positive control, replace respectively template DNA to be checked and operate in addition the step described in embodiment mono-(102).
The results are shown in Figure 7-Fig. 9,8 curves in the CH73-CH78 curve of Fig. 7, Fig. 8 and 6 curves in Fig. 9 are the DNA solutions after corresponding above-mentioned 20 parts of dilutions, DNA solution (purity is 10%, and concentration is 100ng/ μ l), the ddH of CH71 line, the CH72 line genetically modified corn MON 863 that correspondence contains the NOS terminator respectively in Fig. 7
2o; From figure, the appearance position of 20 curves is all very approaching, therefore, and the good stability of this detection method as seen.
The present invention is not limited to above-described embodiment, based on above-described embodiment, simple replacement that do not make creative work, should belong to the scope that the present invention discloses.
Sequence table
<110>
<120 > LAMP of the genetically modified crops of NOS terminator detects primer sets, test kit and detection method
<130 > Feng Jiawang; Kuang Xiaoshan; Hu Songnan
<160>4
<170>PatentIn version3.5
<210>1
<211>19
<212>DNA
<213 > artificial sequence
<400>1
CTCGATCTATCTATTGGCA 19
<210>2
<211>23
<212>DNA
<213 > artificial sequence
<400>2
GCGCTTATTATTGATTACTTACG 23
<210>3
<211>43
<212>DNA
<213 > artificial sequence
<400>3
TCTAGCTTATCGTATTACTTCAGTTGATTGATTCCAGTTGCCG 43
<210>4
<211>48
<212>DNA
<213 > artificial sequence
<400>4
AGCTTGACGTTTTATTTGTGTAGGCGTTATTATTGATTATTAGCGGGT 48 。
Claims (6)
1.NOS the LAMP of terminator detects primer sets, it is characterized in that, comprises following primer:
Outer primer F3:CTCGATCTATCTATTGGCA;
Outer primer B3:GCGCTTATTATTGATTACTTACG;
Inner primer FIP:
TCTAGCTTATCGTATTACTTCAGTTGATTGATTCCAGTTGCCG；
Inner primer BIP:
AGCTTGACGTTTTATTTGTGTAGGCGTTATTATTGATTATTAGCGGGT。
2.NOS the LAMP detection kit of terminator, is characterized in that, comprises following composition:
; Wherein,
Outer primer F3:CTCGATCTATCTATTGGCA;
Outer primer B3:GCGCTTATTATTGATTACTTACG;
Inner primer FIP:
TCTAGCTTATCGTATTACTTCAGTTGATTGATTCCAGTTGCCG；
Inner primer BIP:
AGCTTGACGTTTTATTTGTGTAGGCGTTATTATTGATTATTAGCGGGT。
3. the LAMP detection kit of NOS terminator according to claim 2, is characterized in that, also comprises developer.
4. the LAMP detection kit of NOS terminator according to claim 3, is characterized in that, described developer is fluorescence dye SYBR Green I.
5. the LAMP detection kit of NOS terminator according to claim 2, it is characterized in that, also comprise contrast: the DNA solution that positive control is the genetically modified corn MON 863 that contains the NOS terminator that purity is 5%, concentration is 100ng/ul, negative control is ddH
2o.
6.NOS the LAMP detection method of terminator, is characterized in that, comprises the following steps:
(101) treat the sample product and extract template DNA to be checked;
(102) carry out the ring mediated isothermal gene amplification reaction:
Configuration LAMP reaction system, the LAMP reaction system of every 25LLL volume is specially:
, the LAMP reaction system is placed in to the LAMP turbidimeter and carries out 63 ℃ of isothermal reaction 60min, then under 80 ℃, be incubated 5min; Wherein,
Outer primer F3:CTCGATCTATCTATTGGCA;
Outer primer B3:GCGCTTATTATTGATTACTTACG;
Inner primer FIP:
TCTAGCTTATCGTATTACTTCAGTTGATTGATTCCAGTTGCCG；
Inner primer BIP:
AGCTTGACGTTTTATTTGTGTAGGCGTTATTATTGATTATTAGCGGGT。
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| CN104726583A (en) * | 2015-03-17 | 2015-06-24 | 苏州华麦生物科技有限公司 | Constant temperature detection primers, detection kit and detection method of genetically modified ingredient nos terminator gene |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104726583A (en) * | 2015-03-17 | 2015-06-24 | 苏州华麦生物科技有限公司 | Constant temperature detection primers, detection kit and detection method of genetically modified ingredient nos terminator gene |
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