CN104388579B - A kind of Arm-PCR detection methods of genetically engineered soybean and its derived varieties - Google Patents
A kind of Arm-PCR detection methods of genetically engineered soybean and its derived varieties Download PDFInfo
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- CN104388579B CN104388579B CN201410778521.XA CN201410778521A CN104388579B CN 104388579 B CN104388579 B CN 104388579B CN 201410778521 A CN201410778521 A CN 201410778521A CN 104388579 B CN104388579 B CN 104388579B
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Abstract
The invention discloses a kind of genetically engineered soybean and its Arm-PCR detection methods of derived varieties,Genetically engineered soybean and its derived varieties are genetically engineered soybean GTS40-3-2,Mon89788,A5547-127 and its derived varieties,Its Arm-PCR detection primer group includes internal reference Lectin primer sets,The sleeve type PCR primer of specificity,Super sense primer Fs and super downstream primer Rs,The first step of the sleeve type PCR primer of specificity for Arm-PCR expands,Second steps of the super sense primer Fs and super downstream primer Rs for Arm-PCR expands,Detection kit includes primer liquid,Reaction solution,Archaeal dna polymerase and control,Using six pairs of nested primers and a pair of super primer,Under the action of archaeal dna polymerase,DNA profiling is expanded,With capillary electrophoresis detection amplification.The present invention has the advantages such as low cost, quick, high-throughput and positive rate height, and with compatibility, specificity, sensibility, semi-quantitative, flexibility, repeatability and high efficiency.
Description
Technical field
The present invention relates to biology techniques field, the Arm-PCR of specifically a kind of genetically engineered soybean and its derived varieties is examined
Survey method.
Background technology
In the 1980s, herbicide production company clones from petunia and obtains resistant gene-EPSPS genes, i.e. agriculture
The 5- ketenes pyruvic acid shikimic acid -3- phosphatase genes of bacillus.Transfer DNA technology is mediated using Ti-plasmids, by petunia Ti matter
In the EPSPS Gene Into Soybean genomes that 35S promoter controls in grain, and then the transgenosis for being bred as antiweed glyphosate is big
Beans kind, abbreviation RR soybean.This genetically engineered soybean obtained U.S. FDA approval in 1994, and became carry out business earlier
Change one of the genetically modified crops of large-scale promotion production.RR soybean is to nonselective herbicide --- and agriculture is up to there is high tolerance, i.e.,
Make to apply glyphosate herbicidal in big Tanaka, nor affect on soybean yields, is suitable for large area planting with machinery.According to agricultural biotechnologies
It is shown using international service mechanism data, 72,000,000 hectares of world's plantation soybean gross area in 2001, and genetically engineered soybean has
33300000 hectares occupy global genetically modified crops 63%, and be Roundup Ready soybean.Certainly in addition to this, more also change
The new varieties soybean of benign shape is also come out one after another, soybean of the oleic acid content cultivated such as E.I.Du Pont Company up to 70% or more；In addition,
In the U.S., low linolenic soybean, low brown eleostearic acid soybean, high stearic acid soybean, high palm fibre put the transformed varieties such as sour soybean and have also cultivated
Success.The high oleic acid content turned out using gene engineering method, while content of polyunsaturated fatty acid is reduced, and not
There are trans-fatty acids, therefore are a kind of ideal vegetable oil.Nutritionist then claims 21 century to be " soybean century ".Thus may be used
See, genetically engineered soybean occupies an important position in genetically modified crops and the following food.
In order to make overall merit to genetically modified crops and products thereof, in addition to needing national governments and international body's system
Determine outside the Safety Assessment System and stringent laws and regulations on the management of science, establishes effective method system and genetically modified crops are examined
Survey is also critically important, this is to carry out safety evaluatio to genetically modified crops and implement basis and the International Agricultural Trade of supervision
The important leverage of sound development.The testing requirements method of transgenic product is quick, accurately, sensitive, and must consider to adapt to
The features such as large sample amount, target gene type is more.The detection method of genetically modified crops and products thereof is broadly divided into the world at present
Two kinds：Enzyme-linked immunization and PCR method, and the round pcr based on molecular biology principle is that detection turns in recent years
The common method of gene biological body, including qualitative PCR method, meet PCR method, nested PCR method, competitive quantitative PCR side
Method and fluorescence quantifying PCR method etc..But often detection efficiency is low, the consuming time is long for above-mentioned PCR method, and routine is multiple
Round pcr is related to multiple targets and primer pair, and with the increase of reaction system inner primer quantity, mistake causes amplification and primer phase
The probability mutually interfered greatly increases, and the problems such as incompatible, amplification background is excessively high, repeatable difference, limit are expanded so as to cause target spot
Practical ranges of the technology in the synchronous detection of more targets are made.
Amplicon saves multiple PCR technique, hereinafter referred to as Arm-PCR technologies, and principle is in multiplex PCR system
Each target sequence separately designs two pairs of nested primers so that it can form 4 kinds of amplimer combinations；Due to each target sequence
There are 4 kinds of possible amplification patterns.So that finding the best general amplification condition of different target sequences becomes simple possible, finally
Realize the high specific to a variety of target genes, highly sensitive synchronous amplification.It is quick there has been no Arm-PCR methods to be applied at present
Detect the kit of genetically engineered soybean and its derived varieties.
Invention content
It is high, the strong genetically engineered soybean and its derived varieties of sensibility that the purpose of the present invention is to provide a kind of compatibility
Arm-PCR detection methods, to solve the problems mentioned in the above background technology.
To achieve the above object, the present invention provides the following technical solutions：
The Arm-PCR detection methods of a kind of genetically engineered soybean and its derived varieties, the genetically engineered soybean and its spin-off
Kind is genetically engineered soybean GTS40-3-2, Mon89788, A5547-127 and its derived varieties, the genetically engineered soybean GTS40-3-
2, the Arm-PCR detection primer groups of Mon89788, A5547-127 and its derived varieties include internal reference Lectin primer sets, it is special
Property sleeve type PCR primer, super sense primer Fs and super downstream primer Rs, the sleeve type PCR primer of the specificity includes just
Outside primers F o, reversed outer primer Ro, positive inner primer Fi, reversed inner primer Ri, the sleeve type PCR primer of the specificity are used for
The first step of Arm-PCR expands, and the second step of the super sense primer Fs and super downstream primer Rs for Arm-PCR expands
Increase；The internal reference Lectin primer sets, the sleeve type PCR primer of specificity, super sense primer Fs and super downstream primer Rs
Nucleotide sequence difference is as follows：
GTS40-3-2 forward direction outer primers Fo：CTTCCCGTTACCTTGCGCGG；
The reversed outer primer Ro of GTS40-3-2：CGGTGAGCTTGCCGCGGCCT；
GTS40-3-2 forward direction inner primers Fi：
GCAGTCGTGCAGCAAGTTTTACGTCCGCCGTGCTGCTCGCCG；
The reversed inner primer Ri of GTS40-3-2：
CGAGTCCTGCGGTCTCAAATGTCAGGCGGATGGTGCGCACGC；
Mon89788 forward direction outer primers Fo：GAGACTGATGCTGACGGTGT；
The reversed outer primer Ro of Mon89788：CACTTCGATGTCGGCACCCA；
Mon89788 forward direction inner primers Fi：
GCAGTCGTGCAGCAAGTTTTACTGCTTTCCCATTGGTTGCT；
The reversed inner primer Ri of Mon89788：
CGAGTCCTGCGGTCTCAAATGTATGAGACCAGTACGGGTTGG；
A5547-127 forward direction outer primers Fo：GGACAGGGTACCCGGGGATC；
The reversed outer primer Ro of A5547-127：TAGCCTTCCAGGGCCCAGCG；
A5547-127 forward direction inner primers Fi：
GCAGTCGTGCAGCAAGTTTTACGCTGATATGGCCGCGGTTTG；
The reversed inner primer Ri of A5547-127：
CGAGTCCTGCGGTCTCAAATGTGTGGTGTTTGTGGCTCTGTC；
Lectin forward direction outer primers Fo：CCAAGTCATCGACGTGCCGG；
The reversed outer primer Ro of Lectin：GCCACGTCTTCGCCGCCGGC；
Lectin forward direction inner primers Fi：
GCAGTCGTGCAGCAAGTTTTACGCCTTCCCGCTGGTTGCGGC；
The reversed inner primer Ri of Lectin：
CGAGTCCTGCGGTCTCAAATGTGATGACTTCGATGTCGGCGC；
Super sense primer Fs：GCAGTCGTGCAGCAAGTTTTAC；
Super downstream primer Rs：CGAGTCCTGCGGTCTCAAATGT.
As a further solution of the present invention：The Arm-PCR detection kits include primer liquid, reaction solution, DNA polymerizations
Enzyme and reference material, the primer liquid include sleeve type PCR primer, super sense primer Fs and the super downstream primer Rs of specificity,
A concentration of 1-3 μM of wherein positive outer primer Fo, a concentration of 1-3 μM of reversed outer primer Ro, positive inner primer Fi's is a concentration of
1-3 μM, a concentration of 1-3 μM of reversed inner primer Ri, a concentration of 8-12 μM of super sense primer Fs, super downstream primer Rs's
A concentration of 8-12 μM；MgCl comprising Arm-PCR reaction buffers, a concentration of 6mM in the reaction solution2With a concentration of 10mM's
dNTP；A concentration of 7-9U/ μ l of the archaeal dna polymerase；Positive control is that soybean standard internal reference Lectin draws in the reference material
Object group, negative control are the reaction mixture without target gene.
As a further solution of the present invention：A concentration of 2 μM of positive outer primer Fo, reversed outer primer in the primer liquid
A concentration of 2 μM of Ro, a concentration of 2 μM of positive inner primer Fi, a concentration of 2 μM of reversed inner primer Ri, super sense primer Fs
A concentration of 10 μM, a concentration of 10 μM of super downstream primer Rs.
As a further solution of the present invention：The archaeal dna polymerase is thermal starting archaeal dna polymerase, archaeal dna polymerase
A concentration of 8U/ μ l.
As a further solution of the present invention：The Arm-PCR detection methods of the genetically engineered soybean and its derived varieties,
It is as follows：
（1）The extraction of measuring samples DNA：Using plant DNA extraction kit extraction genetically engineered soybean GTS40-3-2,
The purification of samples DNA of Mon89788, A5547-127；
（2）Setting control reaction：When positive control reaction is set, replaced with soybean standard internal reference Lectin primer sets to be checked
When setting negative control reacts, DNA to be checked is substituted with the reaction mixture without target gene by DNA；
（3）Arm-PCR amplified reactions：
1）The first step expands：Configure reaction system：2.5 μ l of primer liquid, reaction solution mixing are added into the PCR pipe of 200 μ l
Object 12.5 μ l and DNA2-5 μ l to be checked, are used in combination sterile deionized water by solution polishing in PCR pipe to 25 μ l；By prepared PCR
It is centrifuged after pipe mixing, 15min is reacted at 95 DEG C, 30s is reacted at 94 DEG C, 90s is reacted at 60 DEG C, 60s is reacted at 72 DEG C, recycled
Reaction 15 times；15s is reacted at 94 DEG C, and 90s, circular response 6 times are reacted at 70 DEG C；The extension 30min at 60 DEG C again,
Finally preserved under 4 DEG C of environment；
2）Second step expands：Configure reaction system：1 μ l of primer liquid, reaction liquid mixture are added into the PCR pipe of 200 μ l
The PCR reaction product 1-2 μ l that 12.5 μ l and the first step expand, are used in combination sterile deionized water to arrive solution polishing in PCR pipe
25μl；It will be centrifuged after prepared PCR pipe mixing, react 15min at 95 DEG C；30s is reacted at 94 DEG C, reacts 90s at 60 DEG C,
React 60s at 72 DEG C, after circular response 30 times at 60 DEG C extension 30min, finally preserved under 4 DEG C of environment；
（4）As a result judge：Pass through capillary electrophoresis detection Arm-PCR amplifications.
As further scheme of the invention：The step（1）Middle plant DNA extraction kit extracts genetically engineered soybean
The method of the DNA of GTS40-3-2, Mon89788, A5547-127 is：
A, genetically engineered soybean GTS40-3-2, Mon89788 and the A5547-127 for taking 95-105mg, are put into mortar, are added
Liquid nitrogen is ground rapidly, and liquid nitrogen is added 3-4 times repeatedly, until genetically engineered soybean is clayed into power；
B, the RNaseAl of 400 μ l buffer solutions FP1 and 6 a concentration of 10mg/m of μ l is added, vortex vibrates 1min, is placed at room temperature for
10min；
C, 130 μ l buffer solution FP2 are added, mix well, vortex oscillation 1min；
D, 5min is centrifuged at rotating speed 12000rpm, supernatant is transferred in new centrifuge tube；
E, the isopropanol of 0.7 times of volume is added into supernatant, mixes well, cotton-shaped genomic DNA occurs, in rotating speed
2min is centrifuged under 12000rpm, abandons supernatant, retains precipitation；
F, the ethyl alcohol of 600 μ l/70% is added into precipitation, vortex oscillation 5s centrifuges 2min at rotating speed 12000rpm, abandons
Supernatant；
G, step F is repeated；
H, it uncaps inversion, is placed at room temperature for 5-10min, thoroughly dry remaining ethyl alcohol；
I, it is added appropriate elution buffer TE, water-bath 10min dissolving DNAs at 65 DEG C overturn mixing hydrotropy for several times, most therebetween
DNA solution is obtained eventually.
Compared with prior art, the beneficial effects of the invention are as follows：
The present invention has compatible, specific, quick with the advantages such as inexpensive, quick, high-throughput and positive rate height
Perception, semi-quantitative, flexibility, repeatability and high efficiency：
（1）Compatibility：In Arm-PCR reactions, each target sequence to be checked has 2 pairs of nested primers, forms 4 kinds of amplimers
Combination.Each combination may respectively have its own best amplification condition, but since each target sequence has 4 kinds of possible amplification patterns,
It is likely to search out the best general conditions for different target sequences；
（2）Specificity：Because the concentration of target sequence specificity nested primer is extremely low, even if being deposited simultaneously in multipair nested primer
Under the conditions, it can also be specifically bound with its complementary target sequence, ensure that the specificity of amplification；
（3）Sensibility：The amount of gene-specific primer is extremely low, it is not easy to form primer dimer, be only used for enriched target sequence
It arranges and adds super primer label sequence to initial p CR.In enrichment target sequence and after adding super primer label, only drawn with a pair
Object is expanded, and only there are one primer marks, all reduce the background noise of reaction；
（4）Sxemiquantitative：Super primer is used only in Arm-PCR reactions as the primer that can carry out geometric progression amplification, because
And the target sequence amplification efficiency having the same of coamplification, the product amount for expanding terminal being capable of reaction template to a certain extent
Starting copies amount；
（5）Flexibility：Different nested primer combinations can be designed, according to the difference of sequence to be detected to be directed to difference
The target sequence of monoid combines.Can existing combination be deleted or be added nested primer, prepare new combination；Furthermore, it is possible to
New amplification target is added in the combination of former target sequence, the sensibility without reducing original target sequence detection；
（6）Repeatability：The cleaning procedure after PCR is not needed, simplifies operating process, while only there are one universal primer quilts
Label also strengthens the stability and repeatability of product；
（7）High efficiency：Up to 20 kinds of target sequences can be expanded and be marked simultaneously in primary first-order equation, a reaction tube.
Description of the drawings
Fig. 1 is the individual line agarose gel electrophoresis testing result schematic diagram of the embodiment of the present invention 1.
Fig. 2 is the Arm-PCR capillary electrophoresis detection result schematic diagrams that part primer sets carry out in the embodiment of the present invention 1.
Fig. 3 is the hair that the embodiment of the present invention 1 is free of genetically engineered soybean GTS40-3-2, Mon89788, A5547-127 ingredient
Cons electrophoresis testing result schematic diagram.
Fig. 4 is the capillary electrophoresis detection result schematic diagram of the embodiment of the present invention 2.
Specific implementation mode
The technical solution of this patent is described in more detail With reference to embodiment.
Embodiment 1
Please refer to Fig.1-3, in the embodiment of the present invention, the detection sides Arm-PCR of a kind of genetically engineered soybean and its derived varieties
Method, the Arm-PCR detection kits include primer liquid, reaction solution, archaeal dna polymerase and reference material, and the primer liquid includes spy
Anisotropic sleeve type PCR primer, super sense primer Fs and super downstream primer Rs, wherein a concentration of 2 μM of positive outer primer Fo,
A concentration of 2 μM of reversed outer primer Ro, it is a concentration of 2 μM of positive inner primer Fi, a concentration of 2 μM of reversed inner primer Ri, super
A concentration of 10 μM of sense primer Fs, a concentration of 10 μM of super downstream primer Rs；It is reacted comprising Arm-PCR in the reaction solution
The MgCl of buffer solution, a concentration of 6mM2With the dNTP of a concentration of 10mM；A concentration of 8U/ μ l of the archaeal dna polymerase；The control
Positive control is soybean standard internal reference Lectin primer sets in object, and negative control is the reaction mixture without target gene.
Sleeve type PCR primer, super sense primer Fs and the super downstream primer of the internal reference Lectin primer sets, specificity
The nucleotide sequence difference of Rs is as follows：
GTS40-3-2 forward direction outer primers Fo：CTTCCCGTTACCTTGCGCGG；
The reversed outer primer Ro of GTS40-3-2：CGGTGAGCTTGCCGCGGCCT；
GTS40-3-2 forward direction inner primers Fi：
GCAGTCGTGCAGCAAGTTTTACGTCCGCCGTGCTGCTCGCCG；
The reversed inner primer Ri of GTS40-3-2：
CGAGTCCTGCGGTCTCAAATGTCAGGCGGATGGTGCGCACGC；
Mon89788 forward direction outer primers Fo：GAGACTGATGCTGACGGTGT；
The reversed outer primer Ro of Mon89788：CACTTCGATGTCGGCACCCA；
Mon89788 forward direction inner primers Fi：
GCAGTCGTGCAGCAAGTTTTACTGCTTTCCCATTGGTTGCT；
The reversed inner primer Ri of Mon89788：
CGAGTCCTGCGGTCTCAAATGTATGAGACCAGTACGGGTTGG；
A5547-127 forward direction outer primers Fo：GGACAGGGTACCCGGGGATC；
The reversed outer primer Ro of A5547-127：TAGCCTTCCAGGGCCCAGCG；
A5547-127 forward direction inner primers Fi：
GCAGTCGTGCAGCAAGTTTTACGCTGATATGGCCGCGGTTTG；
The reversed inner primer Ri of A5547-127：
CGAGTCCTGCGGTCTCAAATGTGTGGTGTTTGTGGCTCTGTC；
Lectin forward direction outer primers Fo：CCAAGTCATCGACGTGCCGG；
The reversed outer primer Ro of Lectin：GCCACGTCTTCGCCGCCGGC；
Lectin forward direction inner primers Fi：
GCAGTCGTGCAGCAAGTTTTACGCCTTCCCGCTGGTTGCGGC；
The reversed inner primer Ri of Lectin：
CGAGTCCTGCGGTCTCAAATGTGATGACTTCGATGTCGGCGC；
Super sense primer Fs：GCAGTCGTGCAGCAAGTTTTAC；
Super downstream primer Rs：CGAGTCCTGCGGTCTCAAATGT.
As a further solution of the present invention：A concentration of 2 μM of positive outer primer Fo, reversed outer primer in the primer liquid
A concentration of 2 μM of Ro, a concentration of 2 μM of positive inner primer Fi, a concentration of 2 μM of reversed inner primer Ri, super sense primer Fs
A concentration of 10 μM, a concentration of 10 μM of super downstream primer Rs.
The Arm-PCR detection methods of the genetically engineered soybean and its derived varieties, are as follows：
（1）The extraction of measuring samples DNA：Using plant DNA extraction kit extraction genetically engineered soybean GTS40-3-2,
The purification of samples DNA of Mon89788, A5547-127：
A, genetically engineered soybean GTS40-3-2, Mon89788 and the A5547-127 for taking 100mg, are put into mortar, and liquid is added
Nitrogen is ground rapidly, and liquid nitrogen is added 3 times repeatedly, until genetically engineered soybean is clayed into power；
B, the RNaseAl of 400 μ l buffer solutions FP1 and 6 a concentration of 10mg/m of μ l is added, vortex vibrates 1min, is placed at room temperature for
10min；
C, 130 μ l buffer solution FP2 are added, mix well, vortex oscillation 1min；
D, 5min is centrifuged at rotating speed 12000rpm, supernatant is transferred in new centrifuge tube；
E, the isopropanol of 0.7 times of volume is added into supernatant, mixes well, cotton-shaped genomic DNA occurs, in rotating speed
2min is centrifuged under 12000rpm, abandons supernatant, retains precipitation；
F, the ethyl alcohol of 600 μ l/70% is added into precipitation, vortex oscillation 5s centrifuges 2min at rotating speed 12000rpm, abandons
Supernatant；
G, step F is repeated；
H, it uncaps inversion, is placed at room temperature for 8min, thoroughly dry remaining ethyl alcohol；
I, it is added appropriate elution buffer TE, water-bath 10min dissolving DNAs at 65 DEG C overturn mixing hydrotropy for several times, most therebetween
DNA solution is obtained eventually.
（2）Setting control reaction：When positive control reaction is set, replaced with soybean standard internal reference Lectin primer sets to be checked
When setting negative control reacts, DNA to be checked is substituted with the reaction mixture without target gene by DNA；
（3）Arm-PCR amplified reactions：
1）The first step expands：Configure reaction system：2.5 μ l of primer liquid, reaction solution mixing are added into the PCR pipe of 200 μ l
Object 12.5 μ l and DNA2 μ l to be checked, are used in combination sterile deionized water by solution polishing in PCR pipe to 25 μ l；By prepared PCR pipe
It is centrifuged after mixing, 15min is reacted at 95 DEG C, 30s is reacted at 94 DEG C, 90s is reacted at 60 DEG C, 60s is reacted at 72 DEG C, cycle is anti-
It answers 15 times；15s is reacted at 94 DEG C, and 90s, circular response 6 times are reacted at 70 DEG C；The extension 30min at 60 DEG C again, most
It is preserved under 4 DEG C of environment afterwards；
2）Second step expands：Configure reaction system：1 μ l of primer liquid, reaction liquid mixture are added into the PCR pipe of 200 μ l
The 1 μ l of PCR reaction products that 12.5 μ l and the first step expand, are used in combination sterile deionized water by solution polishing in PCR pipe to 25 μ
l；It will be centrifuged after prepared PCR pipe mixing, react 15min at 95 DEG C；30s is reacted at 94 DEG C, reacts 90s at 60 DEG C, 72
React 60s at DEG C, after circular response 30 times at 60 DEG C extension 30min, finally preserved under 4 DEG C of environment；
（4）As a result judge：Pass through capillary electrophoresis detection Arm-PCR amplifications.
Referring to Fig. 1, each strain and positive control separately design 2 groups of primers and are expanded and use fine jade in the present embodiment
Sepharose electrophoresis is detected：1 and 2 be amplification of the two groups of primers of positive control Lectin in 3 strains；3 and 4 are
Amplification of the two groups of primers of GST40-3-2 in the strain；5 and 6 be amplification of the two groups of primers of MON89788 in the strain；7 and 8
For amplification of the two groups of primers of A5547-127 in the strain.
Referring to Fig. 2, selecting preferable primer sets according to the result of Fig. 1 carries out Arm-PCR capillary electrophoresis detection results
Negative control is shown as without band, the band of positive control Lectin is 173bp, and the PCR product size of measuring samples is
198bp, 134bp and 129bp, show to contain in soybean seed representative to be checked or all genetically engineered soybean GTS40-3-2,
Mon89788, A5547-127 and its derived varieties, containing genetically engineered soybean GTS40-3-2, Mon89788, A5547-127 at
Point.
Referring to Fig. 3, sample to be tested occurs through capillary electrophoresis detection without peak figure, show that measuring samples are big without transgenosis
Beans GTS40-3-2, Mon89788, A5547-127 ingredient.
Embodiment 2
Specificity experiments
With the identification method of embodiment 1 respectively to isolate and purify transgenic corns BT176, Event98140,59122,
MON810, MON88017, MON89034, MON863, GA21, MIR604, genetically engineered soybean MON89788, genetically engineered soybean
GTS40-3-2, genetically engineered soybean A5547-127, transgene rape T45, transgene cotton GHB614, non-transgenic corn, water
Rice, wheat, rape, cotton carry out Arm-PCR identifications.
Referring to Fig. 4, qualification result is shown：Transgenic corns BT176, Event98140,59122, MON810,
MON88017, MON89034, MON863, GA21, MIR604, transgenic paddy rice BT63, transgene rape T45, transgene cotton
GHB614, Non-transgenic soybean, rice, wheat, rape, corn, cotton reaction tube capillary electrophoresis detection are without peak, i.e., without expansion
Increase；Genetically engineered soybean MON89788, genetically engineered soybean GTS40-3-2, genetically engineered soybean A5547-127 and soybean endogenous gene
Lectin capillary electrophoresis detections have peak, that is, have amplification, this experimental result to show good specificity.
The present invention is based on amplicons to save multiplexed PCR amplification technology, according to each target sequence in multiplex PCR system, divides
Not She Ji two pairs of nested primers so that it can form the combination of 4 kinds of amplimers；Since each target sequence has 4 kinds of possible moulds
Formula has low cost, quick, high pass so that finding the best general amplification condition of different target sequences becomes simple possible
Amount and the advantages such as positive rate height have compatibility, specificity, sensibility, semi-quantitative, flexibility, repeatability and efficient
Property：
（1）Compatibility：In Arm-PCR reactions, each target sequence to be checked has 2 pairs of nested primers, forms 4 kinds of amplimers
Combination.Each combination may respectively have its own best amplification condition, but since each target sequence has 4 kinds of possible amplification patterns,
It is likely to search out the best general conditions for different target sequences；
（2）Specificity：Because the concentration of target sequence specificity nested primer is extremely low, even if being deposited simultaneously in multipair nested primer
Under the conditions, it can also be specifically bound with its complementary target sequence, ensure that the specificity of amplification；
（3）Sensibility：The amount of gene-specific primer is extremely low, it is not easy to form primer dimer, be only used for enriched target sequence
It arranges and adds super primer label sequence to initial p CR.In enrichment target sequence and after adding super primer label, only drawn with a pair
Object is expanded, and only there are one primer marks, all reduce the background noise of reaction；
（4）Sxemiquantitative：Super primer is used only in Arm-PCR reactions as the primer that can carry out geometric progression amplification, because
And the target sequence amplification efficiency having the same of coamplification, the product amount for expanding terminal being capable of reaction template to a certain extent
Starting copies amount；
（5）Flexibility：Different nested primer combinations can be designed, according to the difference of sequence to be detected to be directed to difference
The target sequence of monoid combines.Can existing combination be deleted or be added nested primer, prepare new combination；Furthermore, it is possible to
New amplification target is added in the combination of former target sequence, the sensibility without reducing original target sequence detection；
（6）Repeatability：The cleaning procedure after PCR is not needed, simplifies operating process, while only there are one universal primer quilts
Label also strengthens the stability and repeatability of product；
（7）High efficiency：Up to 20 kinds of target sequences can be expanded and be marked simultaneously in primary first-order equation, a reaction tube.
The better embodiment of this patent is explained in detail above, but this patent is not limited to above-mentioned embodiment party
Formula, one skilled in the relevant art within the scope of knowledge, can also be under the premise of not departing from this patent objective
Various changes can be made.
Claims (6)
1. the Arm-PCR detection methods of a kind of genetically engineered soybean and its derived varieties, which is characterized in that the genetically engineered soybean and
Its derived varieties is genetically engineered soybean GTS40-3-2, Mon89788, A5547-127 and its derived varieties, the genetically engineered soybean
The Arm-PCR detection primer groups of GTS40-3-2, Mon89788, A5547-127 and its derived varieties include internal reference Lectin primers
Sleeve type PCR primer, super sense primer Fs and the super downstream primer Rs of group, specificity, the sleeve type PCR primer of the specificity
Including positive outer primer Fo, reversed outer primer Ro, positive inner primer Fi, reversed inner primer Ri, the sleeve type PCR of the specificity draws
The first step of the object for Arm-PCR expands, and the super sense primer Fs and super downstream primer Rs are used for the second of Arm-PCR
Step amplification；Sleeve type PCR primer, super sense primer Fs and the super downstream primer of the internal reference Lectin primer sets, specificity
The nucleotide sequence difference of Rs is as follows：
GTS40-3-2 forward direction outer primers Fo：CTTCCCGTTACCTTGCGCGG；
The reversed outer primer Ro of GTS40-3-2：CGGTGAGCTTGCCGCGGCCT；
GTS40-3-2 forward direction inner primers Fi：
GCAGTCGTGCAGCAAGTTTTACGTCCGCCGTGCTGCTCGCCG；
The reversed inner primer Ri of GTS40-3-2：
CGAGTCCTGCGGTCTCAAATGTCAGGCGGATGGTGCGCACGC；
Mon89788 forward direction outer primers Fo：GAGACTGATGCTGACGGTGT；
The reversed outer primer Ro of Mon89788：CACTTCGATGTCGGCACCCA；
Mon89788 forward direction inner primers Fi：
GCAGTCGTGCAGCAAGTTTTACTGCTTTCCCATTGGTTGCT；
The reversed inner primer Ri of Mon89788：
CGAGTCCTGCGGTCTCAAATGTATGAGACCAGTACGGGTTGG；
A5547-127 forward direction outer primers Fo：GGACAGGGTACCCGGGGATC；
The reversed outer primer Ro of A5547-127：TAGCCTTCCAGGGCCCAGCG；
A5547-127 forward direction inner primers Fi：
GCAGTCGTGCAGCAAGTTTTACGCTGATATGGCCGCGGTTTG；
The reversed inner primer Ri of A5547-127：
CGAGTCCTGCGGTCTCAAATGTGTGGTGTTTGTGGCTCTGTC；
Lectin forward direction outer primers Fo：CCAAGTCATCGACGTGCCGG；
The reversed outer primer Ro of Lectin：GCCACGTCTTCGCCGCCGGC；
Lectin forward direction inner primers Fi：
GCAGTCGTGCAGCAAGTTTTACGCCTTCCCGCTGGTTGCGGC；
The reversed inner primer Ri of Lectin：
CGAGTCCTGCGGTCTCAAATGTGATGACTTCGATGTCGGCGC；
Super sense primer Fs：GCAGTCGTGCAGCAAGTTTTAC；
Super downstream primer Rs：CGAGTCCTGCGGTCTCAAATGT.
2. the Arm-PCR detection methods of genetically engineered soybean according to claim 1 and its derived varieties, which is characterized in that
The Arm-PCR detection kits include primer liquid, reaction solution, archaeal dna polymerase and reference material, and the primer liquid includes specificity
Sleeve type PCR primer, super sense primer Fs and super downstream primer Rs, wherein a concentration of 1-3 μM of positive outer primer Fo, instead
To a concentration of 1-3 μM of outer primer Ro, a concentration of 1-3 μM of positive inner primer Fi, a concentration of 1-3 μM of reversed inner primer Ri,
A concentration of 8-12 μM of super sense primer Fs, a concentration of 8-12 μM of super downstream primer Rs；Include in the reaction solution
The MgCl of Arm-PCR reaction buffers, a concentration of 6mM2With the dNTP of a concentration of 10mM；A concentration of 7- of the archaeal dna polymerase
9U/μl；Positive control is soybean standard internal reference Lectin primer sets in the reference material, and negative control is without target gene
Reaction mixture.
3. the Arm-PCR detection methods of genetically engineered soybean according to claim 2 and its derived varieties, which is characterized in that
A concentration of 2 μM of positive outer primer Fo in the primer liquid, a concentration of 2 μM of reversed outer primer Ro, positive inner primer Fi's is dense
Degree is 2 μM, and a concentration of 2 μM of reversed inner primer Ri, a concentration of 10 μM of super sense primer Fs, super downstream primer Rs's is dense
Degree is 10 μM.
4. the Arm-PCR detection methods of genetically engineered soybean according to claim 2 and its derived varieties, which is characterized in that
The archaeal dna polymerase is thermal starting archaeal dna polymerase, a concentration of 8U/ μ l of archaeal dna polymerase.
5. according to the Arm-PCR detection methods of any genetically engineered soybeans and its derived varieties of claim 1-4, feature
It is, is as follows：
(1) extraction of measuring samples DNA：Using plant DNA extraction kit extraction genetically engineered soybean GTS40-3-2,
The purification of samples DNA of Mon89788, A5547-127；
(2) setting control reaction：When positive control reaction is set, DNA to be checked is replaced with soybean standard internal reference Lectin primer sets,
When negative control reaction is set, DNA to be checked is substituted with the reaction mixture without target gene；
(3) Arm-PCR amplified reactions：
The A first steps expand：Configure reaction system：2.5 μ l of primer liquid, reaction liquid mixture 12.5 are added into the PCR pipe of 200 μ l
μ l and DNA2-5 μ l to be checked are used in combination sterile deionized water by solution polishing in PCR pipe to 25 μ l；By prepared PCR pipe mixing
After centrifuge, 15min is reacted at 95 DEG C, 30s is reacted at 94 DEG C, 90s is reacted at 60 DEG C, 60s, circular response 15 are reacted at 72 DEG C
It is secondary；15s is reacted at 94 DEG C, and 90s, circular response 6 times are reacted at 70 DEG C；The extension 30min at 60 DEG C again, finally exists
It is preserved under 4 DEG C of environment；
B second steps expand：Configure reaction system：1 μ l of primer liquid, 12.5 μ l of reaction liquid mixture are added into the PCR pipe of 200 μ l
The PCR reaction product 1-2 μ l expanded with the first step, are used in combination sterile deionized water by solution polishing in PCR pipe to 25 μ l；It will
It is centrifuged after prepared PCR pipe mixing, reacts 15min at 95 DEG C；30s is reacted at 94 DEG C, reacts 90s at 60 DEG C, at 72 DEG C
React 60s, after circular response 30 times at 60 DEG C extension 30min, finally preserved under 4 DEG C of environment；
(4) result judges：Pass through capillary electrophoresis detection Arm-PCR amplifications.
6. the Arm-PCR detection methods of genetically engineered soybean according to claim 5 and its derived varieties, which is characterized in that
The DNA of plant DNA extraction kit extraction genetically engineered soybean GTS40-3-2, Mon89788, A5547-127 in the step (1)
Method be：
A, genetically engineered soybean GTS40-3-2, Mon89788 and the A5547-127 for taking 95-105mg, are put into mortar, and liquid nitrogen is added
It grinds rapidly, liquid nitrogen is added 3-4 times repeatedly, until genetically engineered soybean is clayed into power；
B, the RNaseAl of 400 μ l buffer solutions FP1 and 6 a concentration of 10mg/m of μ l is added, vortex vibrates 1min, is placed at room temperature for
10min；
C, 130 μ l buffer solution FP2 are added, mix well, vortex oscillation 1min；
D, 5min is centrifuged at rotating speed 12000rpm, supernatant is transferred in new centrifuge tube；
E, the isopropanol of 0.7 times of volume is added into supernatant, mixes well, cotton-shaped genomic DNA occurs, in rotating speed
2min is centrifuged under 12000rpm, abandons supernatant, retains precipitation；
F, the ethyl alcohol of 600 μ l/70% is added into precipitation, vortex oscillation 5s centrifuges 2min at rotating speed 12000rpm, abandons
Clearly；
G, step F is repeated；
H, it uncaps inversion, is placed at room temperature for 5-10min, thoroughly dry remaining ethyl alcohol；
I, it is added appropriate elution buffer TE, water-bath 10min dissolving DNAs at 65 DEG C overturn mixing hydrotropy for several times therebetween, final
To DNA solution.
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