CN103757127B - Rape transgenosis detecting kit - Google Patents
Rape transgenosis detecting kit Download PDFInfo
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- CN103757127B CN103757127B CN201410044476.5A CN201410044476A CN103757127B CN 103757127 B CN103757127 B CN 103757127B CN 201410044476 A CN201410044476 A CN 201410044476A CN 103757127 B CN103757127 B CN 103757127B
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Abstract
The invention relates to a rape transgenosis detecting kit in the field of detecting articles. The rapeseed transgenosis detecting kit comprises a primer combination and a membrane chip, and is characterized in that the primer combination is a 11-double-starting oligonucleotide primer combination, the membrane chip refers to 11 groups of probe sequences and one group of positive control probe sequences, which are fixed on the surface of a supporting film, and the 5' end of each probe is provided with an amino marker; asymmetric PCR (polymerase chain reaction) amplification primers, which are Tag primers with biotin marks at 5'ends, are included in the primer combination. The rapeseed transgenosis detecting kit provided by the invention overcomes the defects of time and energy waste and high cost in the prior art, the rape transgenosis detecting kit provided is simple to operate, is quick and sensitive, is high in detection flux, has visual detection results and is low in cost.
Description
Affiliated technical field
The present invention relates to detect articles for use field, be specially a kind of rape transgenosis detection kit.
Background technology
Rape is Cruciferae brassica plant, be in genetically modified crops research research the earliest, maximum class farm crop.1985, Ooms etc. utilized agrobacterium-mediated transformation to cultivate successful first case transgenic brassica napus.After this Ge great transnational company has been bred as multiple transgene rape strain, and on North America, Japan and other places, by Biosafety, assesses successively after 1995, approval commercialization plantation.End 2010, global transgene rape cultivated area has surpassed 7,000,000 hm
2, account for 23% of the total cultivated area of global rape, rank after genetically engineered soybean, corn, cotton, be the genetically modified crops in the 4th, the world.Rape is important oil crops, the rape that derives from over half of the current edible oil of China.Annual China need to be from north America region import more than 3,000,000 tons Semen Brassicae campestris, more than 70% be wherein transgenic rapeseed.Due to transgenic product emerging in multitude in world market, its environment and food-safety problem also cause international showing great attention to.Various countries formulate and have put into effect corresponding laws and regulations in succession, and environment release and the production marketing of genetically modified crops are taked to strict control measures.China has issued the 304th command < < agricultural genetically modified organism security control regulations > > of State Council May 23 calendar year 2001, realized the standardized administration to genetically modified organism, and in 20 days March in 2002, implemented < < agricultural genetically modified organism identity management way > >, require 5 class genetically modified organisms that comprise transgene rape and products thereof to identify.Therefore, transgenic product detection technique quick, accurate, sensitive, cheap and corresponding flux is very crucial.The detection technique of transgenic product mainly comprises detection method and the detection method based on nucleic acid based on albumen.Enzyme-linked immunosorbent assay (ELISA), the methods such as immunity test strip and Western hybridization are the current main detection methods based on albumen, its common feature is from sample, to discharge target protein (antigen) by certain treatment step, then utilize the conjugated antigen albumen of antibodies specific, finally by enzyme mark or metal mark coupling antibody, be combined with immune complex, thereby produce detectable signal, the shortcoming of these class methods is that detection sensitivity is relatively low, and need to prepare antibody with high specificity, detect the impact that is easily subject to many factors simultaneously, there is false positive.And detection method based on nucleic acid be at present the most generally, transgenosis detection technique the most accurately, detection method mainly comprises PCR(polymerase chain reaction) technology and the biochip technology based on molecular hybridization.Still with round pcr, be most widely used at present, its conventional technological method comprises the methods such as common qualitative PCR, nest-type PRC, multiplex PCR, quantitative fluorescent PCR.Common qualitative PCR method detection efficiency is low, while especially detecting polygene object, more wastes time and energy; Although nest-type PRC has improved the sensitivity detecting, there is equally the problem of detection efficiency; Multiple PCR method can detect a plurality of genes in a reaction simultaneously, but conventional electrophoresis detection result is difficult to judgement, easily occurs false positive and false negative result; Fluorescence quantifying PCR method is highly sensitive, but its cost is high, and needs special detection instrument.Gene chip is also conventional transgenic product detection technique, and the general sheet glass that adopts is as solid support at present, and experimentation needs corresponding point sample instrument and chip identification reading instrument, and same cost is higher.Therefore researching and developing a kind of quick, convenient, sensitive, cheap, visual and high-throughout detection method will have extraordinary application prospect.
Summary of the invention
The object of the invention is, in order to overcome the prior art high defect of cost that wastes time and energy, provides a kind of simple to operate, and rapid sensitive, detects flux large, and detected result is visual, rape transgenosis detection kit with low cost.
The object of the invention is to be achieved through the following technical solutions:
Rape transgenosis detection kit of the present invention is comprised of combination of primers and film chip, it is characterized in that combination of primers is 11 heavy double startup Oligonucleolide primers (DPO) combinations, each base sequence 5 '-3 ' to primer is as follows, and wherein base I is inosinic acid:
EPSPS: forward primer: CTTTCTGGAACCGTCCGTATIIIIIGTGACAA
Reverse primer: CCAAGTATCACCTTCCTTACIIIIICTGGCAC;
GOX: forward primer: CTGCTGCTCCTAACTGGAAGIIIIITCACGTT
Reverse primer: GGTCATTGGAGCACCAGTCAIIIIIAGGTGAC;
PAT: forward primer: TGAGACGTCTACAGTGAACTIIIIIACAGAGC
Reverse primer: CTTCCAGGGCCCAGCGTAAGIIIIICCAGCCA;
NPTII: forward primer: CGAAGTGCCGGGGCAGGATCIIIIITCATCTC
Reverse primer: CGCGAGCCCCTGATGCTCTTIIIIIAGATCAT;
BAR: forward primer: GACGACCTCGTCCGTCTGCGIIIIIGCTATCC
Reverse primer: CAGCAGGTGGGTGTAGAGCGIIIIICCCAGTC;
BARNASE: forward primer: ATTATCGTGGATTTCCGTTTIIIIICTGGTGC
Reverse primer: GCTTATGATATGTCTGAAGAIIIIICGCAACC;
BARSTAR: forward primer: CCTGGACGCTTTATGGGATTIIIIIACCGGAT
Reverse primer: TTCCGCTTTCGCTTCACGGAIIIIITGAAGCA;
FMV35S: forward primer: GGAAGAATTCTCAGTCCAAAIIIIIAACAAGG
Reverse primer: CAAAGATGCCCACTAACTTTIIIIITTCGGTG;
CaMV35S: forward primer: GATAAAGGAAAGGCCATCGTIIIIIATGCCTC
Reverse primer: TGGGATTGTGCGTCATCCCTIIIIICAGTGGA;
NOS 3 ': forward primer: TTAAGATTGAATCCTGTTGCIIIIITTGCGAT
Reverse primer: CGCGCGCGATAATTTATCCTIIIIIGCGCGCT;
PEP: forward primer: ATACGCTACAACTACCAATAIIIIICGAGTGG
Reverse primer: GGAAAAAAGGTCGAGAAGTTIIIIICACGAGG;
Film chip is fixed on 11 groups of probe sequences and one group of positive control (Positive) probe sequence on supporting film surface for order, and the base sequence 5 '-3 ' of 11 groups of probe sequences and positive control probe sequence is as follows, and 5 ' end of each probe is the amino (NH of sign
2):
EPSPS probe: NH
2-5 '-TATCACCGGTCTTTTGGAAGGTGAAGATGTTATCAACA
CTGGTAAGGCTATGCAAGCTA-3’；
GOX probe: NH
2-5 '-CCCTTCCAGTGATTGGTCGTGCTACCCGTACTCCAGACG
TTATCTACGCTTTCGGTCAC-3’；
PAT probe: NH
2-5 '-GGATTGATGATCTAGAGAGGTTGCAAGATAGATACCCTT
GGTTGGTTGCTGAGGTTGAG-3’；
NPTII probe: NH
2-5 '-CACCAAGCGAAACATCGCATCGAGCGAGCACGTACTC
GGATGGAAGCCGGTCTTGTCGA-3’；
BAR probe: NH
2-5 '-CGCAACGCCTACGACTGGACGGCCGAGTCGACCGTGTA
CGTCTCCCCCCGCCACCAGCG-3’；
BARNASE probe: NH
2-5 '-AACGGAAACATCTTCTCACAAGGCACACACAGAA
GCACAGGTTATCAACACGTTTGACG-3’；
BARSTAR probe: NH
2-5 '-CGTTTTGGAATGGAGGCAGTTTGAACAAAGCAAG
CAGCTGACTGAAAATGGCGCCGAGA-3’；
FMV35S probe: NH
2-5 '-CAAAGTAAACTACTGTTCCAGCACATGCATCATGGT
CAGTAAGTTTCAGAAAAAGACAT-3’；
CaMV35S probe: NH
2-5 '-AGGAGCATCGTGGAAAAAGAAGACGTTCCAACC
ACGTCTTCAAAGCAAGTGGATTGATG-3’；
NOS 3 ' probe: NH
2-5 '-ATGAGATGGGTTTTTATGATTAGAGTCCCGCAATTATA
CATTTAATACGCGATAGAAAA-3’；
PEP probe: NH
2-5 '-GAGATTTTTCCAAGGCTTCAAACTCTTTCATCACATGTC
TCTTCTCTTTCCATTGTCTT-3’；
Positive probe: NH
2-5 '-TAGCAAGGATGAGTATTCGAACAGTTAGAGGCAGT
CAGGGAGAATCACTACGTACAAGC-3’；
In such scheme, having asymmetric PCR primer in described combination of primers, is the Tag primer of 5 ' end band vitamin H sign (biotin), and its base sequence is as follows:
Tag primer: biotin-5 '-GACGGACACCTACCCATATCACCCTTGACG-3 '.
In such scheme, described rape transgenosis detection kit is comprised of combination of primers, film chip and auxiliary material, auxiliary material is deoxyribonucleoside triphosphate (dNTP), EX-Taq polysaccharase (Polymerase) and 5 ' the positive oligonucleotide single stranded DNA of holding with vitamin H sign, and 5 ' end is biotin-5'-GCTTGTACGTAGTGATTCTCCCTGACTGCCTCTAACTGTTCGAATACTCATCCTTG CTA-3 ' with the base sequence of the positive oligonucleotide single stranded DNA of vitamin H sign.
In such scheme, described rape transgenosis detection kit is comprised of combination of primers, film chip, auxiliary material and dosing, and dosing is 10 * PCR damping fluid, prehybridization solution, hybridization solution, washing lotion, confining liquid, streptavidin mark horseradish peroxidase and tetramethyl benzidine (TMB) nitrite ion.
In such scheme, described prehybridization solution is 5 * SSC(0.75M sodium-chlor, 0.075M Trisodium Citrate), 0.1% weight percent SDS(sodium laurylsulfonate) and 10 * Denhardt ' s liquid (1% Ficoll ficoll, 1% polyvinylpyrrolidone polyvinylpyrrolidone, l% BSA bovine serum albumin); Hybridization solution is 5 * SSC, 0.1% weight percent SDS, 5 * Denhardt ' s, 50% weight percent deionized formamide and 100ug/ml yeast tRNA; Washing lotion is respectively washing lotion 1:2 * SSC and 0.1% weight percent SDS, washing lotion 2:0.5 * SSC and 0.1% weight percent SDS, washing lotion 3:100 mM (mM/l) Tris-HCl, pH7.5,150 mM NaCl, washing lotion 4:100 mM Tris-HCl, pH9.5,100 mM NaCl and 100 mM MgCl
2; Confining liquid is 3% weight percent BSA(bovine serum albumin), 100 mM Tris-HCl, pH7.5,150 mM NaCl; Tetramethyl benzidine nitrite ion is respectively tetramethyl benzidine nitrite ion A liquid: 200mM Trisodium Citrate pH5.4,0.2mg/ml tetramethyl benzidine, tetramethyl benzidine nitrite ion B liquid: the H of 3% weight percent
2o
2with the distilled water diluent of 800 volumes, before using, A liquid B liquid balanced mix is made into tetramethyl benzidine nitrite ion.
In such scheme, described supporting film is nitrocellulose filter or nylon membrane.
Rape transgenosis detection kit of the present invention adopts the method amplification target sequence of multiplex PCR amplification.Multiplex PCR amplification technique principle is that multipair primer is put in same reaction tubes and carries out pcr amplification, reaches the object of simultaneously increase 2 kinds or two or more target dna sequences.The present invention has adopted 11 heavy pcr amplification system amplification target sequences, comprise seven kinds of common transgenic sequence EPSPS, GOX, PAT, NPTII, BAR, BARNASE, BARSTAR, two exogenous promoter sequence FMV35S and CaMV35S, external source terminator sequence NOS 3 ' and rape native gene sequence PEP.
The present invention designs and adopts DPO primer (Dual Priming Oligonucleotide, double startup Oligonucleolide primers) to carry out target sequence amplification.The base sequence of DPO primer of the present invention, through studying intensively with great many of experiments, obtain, be characterized in the difficult secondary structure that forms between primer, the high specificity of multiplex amplification, little with the mispairing probability of template, as long as there are 3 mispairing more than base just can successfully not increase, both can significantly improve the specificity of multiplex PCR amplification, can improve again the amplification efficiency of target sequence.
The present invention adopts the amino (NH of 5 ' end band
2) probe modified carries out target sequence hybridization check.Amido modified probe is combined more firm with film, and the combination of probe and film has directivity, is more conducive to the hybridization of probe and target area, thereby improves the sensitivity detecting.
The present invention adopts asymmetric PCR technology (asymmetric PCR) in order to improve detection sensitivity.Asymmetric PCR know-why is the forward primer and reverse primer (or different forward primer and the reverse primers of amplification extension condition) that adopts inequality in pcr amplification process, after pcr amplification, produce a large amount of single stranded DNAs, this single stranded DNA can effectively and be fixed on the probe hybridization on supporting film, thereby improves detection sensitivity.
The present invention carries out asymmetric PCR in multiplex PCR amplification simultaneously, the technical scheme adopting is the Tag sequence of holding with vitamin H sign in 5 ' end interpolation 5 ' of multiplex PCR reverse primer, in PCR reaction system, add 5 ' excessive end with the Tag primer of vitamin H sign simultaneously, the base sequence of Tag primer is identical with the base sequence of above-mentioned Tag sequence, this Tag primer Tm value (melting temperature(Tm)) is higher 10 ~ 15 ℃ than multiplex PCR forward primer Tm value, for asymmetric pcr stage amplification single stranded DNA.In whole pcr amplification process, first adopt low temperature thermal oxidation (55 ~ 60 ℃) to carry out multi-PRC reaction, its amplified production is mainly double-stranded DNA, improve afterwards annealing temperature (65 ~ 68 ℃), make forward primer not can be incorporated in template, only have Tag primer and reverse primer can be incorporated in template and extend amplification, thereby produce a large amount of 5 ' ends with the single stranded DNA of vitamin H sign, this single stranded DNA detects for ensuing film chip hybridization.
The present invention adopts the visual film chip technology based on reverse dot blot hybridization to carry out genetically modified molecular hybridization detection, its principle of work is first the single-stranded probe for each transgenic sequence to be arranged to the specific region of being fixed on supporting film surface in order, again multiple PCR products to be measured is hybrid with it, like this transgenic sequence single stranded DNA in PCR product will with supporting film on probe hybridization, through washing, remove unconjugated DNA sample, because the DNA of PCR product to be measured is upper with vitamin H sign, the probe points that combines DNA to be measured with regard in coupling vitamin H mark, through corresponding color reaction, just can read corresponding hybridization signal again, on such film chip, just can detect a plurality of transgenosis target sequences simultaneously.The detection probes of rape transgenosis detection kit of the present invention is the oligonucleotide probe of 59 bases, and it is sequentially arranged in the Special Areas on supporting film surface, forms low density probe array.Film chip can form the visual detected result of naked eyes with sample to be tested hybridization by substrate colour developing.
The invention provides a kind of many indexs and detect the genetically modified visual film chip agent box of rape, wherein target sequence to be detected comprises common rape transgenic sequence EPSPS, GOX, PAT, NPTII, BAR, BARNASE, BARSTAR, exogenous promoter sequence FMV35S, CaMV35S, external source terminator sequence NOS 3 ' and rape native gene PEP.By the detection of this test kit, can judge fast and accurately in rape product to be checked whether contain above-mentioned transgenic sequence, be suitable for the extensive examination of transgene rape product.
The invention has the beneficial effects as follows: 1) simple to operate, through a Sample pretreatment, single tube pcr amplification, single-chip hybridization just can synchronous detection sample in multiple transgenic sequence, there is the feature of parallel analysis and multiple judgement; 2) checked object is complete, has comprised current common transgenic sequence, and can add very easily new detection sequence; 3) system has been carried out asymmetric PCR at multiplex PCR amplification procedure simultaneously, has improved sensitivity and the accuracy of system; 4) test kit has adopted the detection mode of visual film chip, has improved the detection flux of system, and detected result can directly with the naked eye judge, convenient, quick; 5) test kit is simple to operate, and economy, without special expensive instrument, is applicable to the extensive detection of transgene rape.
In sum, the present invention has overcome the prior art high defect of cost that wastes time and energy, and the rape transgenosis detection kit providing is simple to operate, and rapid sensitive detects flux large, and detected result is visual, with low cost.
Accompanying drawing explanation
Fig. 1 be the present invention for detection of the results of hybridization figure of transgene rape strain GT73/RT73, wherein Figure 1A is chip results mode chart, the detected result figure that Figure 1B is genetically modified rape GT 73/RT73.
Fig. 2 be the present invention for detection of the results of hybridization figure of transgene rape strain HCN92, wherein Fig. 2 A is chip results mode chart, Fig. 2 B is the detected result figure of transgene rape HCN92.
Fig. 3 be the present invention for detection of the results of hybridization figure of transgene rape strain MS1 * RF1, wherein Fig. 3 A is chip results mode chart, Fig. 3 B is the detected result figure of transgene rape MS1 * RF1.
Fig. 4 be the present invention for detection of the results of hybridization figure of the wild strain 3529 of non-transgenic rape, wherein Fig. 4 A is chip results mode chart, Fig. 4 B is the detected result figure of the wild strain 3529 of non-transgenic rape.
In accompanying drawing, EPSPS:(5-shikimic acid-3-phosphate synthase gene), GOX:(glyphosate oxidoreductase gene), PAT:(grass fourth phosphinothricin acetyl CoA transferase gene), NPTII:(Liu Suanyan NEOMYCIN SULPHATE-3 '-phosphoric acid transferase gene), BAR:(grass fourth phosphinothricin acetyl transferase gene), BARNASE:(derives from bacillus
bacillus amylolique faciensribonuclease gene), BARSTAR:(derives from bacillus
bacillus amylolique faciensthe special suppressor gene of barnase gene), FMV35S:(radix scrophulariae mosaic virus 35 S promoter), CaMV35S:(cauliflower mosaic virus 35 S promoter), NOS 3 ': (rouge alkali synthetase gene terminator), PEP:(phosphoric acid enol pyruvic acid carboxylase), Positive:(positive control), Barcode:(film chip bar code).
Specific embodiment
Below in conjunction with drawings and Examples, be described in further detail the present invention, but the present invention is not limited only to described embodiment.
Embodiment mono-
This routine rape transgenosis detection kit is comprised of combination of primers and film chip, combination of primers is 11 heavy DPO primer (Dual Priming Oligonucleotide, double startup Oligonucleolide primers) combination, each base sequence 5 '-3 ' to primer is as follows, and wherein base I is inosinic acid:
EPSPS: forward primer: CTTTCTGGAACCGTCCGTATIIIIIGTGACAA
Reverse primer: CCAAGTATCACCTTCCTTACIIIIICTGGCAC;
GOX: forward primer: CTGCTGCTCCTAACTGGAAGIIIIITCACGTT
Reverse primer: GGTCATTGGAGCACCAGTCAIIIIIAGGTGAC;
PAT: forward primer: TGAGACGTCTACAGTGAACTIIIIIACAGAGC
Reverse primer: CTTCCAGGGCCCAGCGTAAGIIIIICCAGCCA;
NPTII: forward primer: CGAAGTGCCGGGGCAGGATCIIIIITCATCTC
Reverse primer: CGCGAGCCCCTGATGCTCTTIIIIIAGATCAT;
BAR: forward primer: GACGACCTCGTCCGTCTGCGIIIIIGCTATCC
Reverse primer: CAGCAGGTGGGTGTAGAGCGIIIIICCCAGTC;
BARNASE: forward primer: ATTATCGTGGATTTCCGTTTIIIIICTGGTGC
Reverse primer: GCTTATGATATGTCTGAAGAIIIIICGCAACC;
BARSTAR: forward primer: CCTGGACGCTTTATGGGATTIIIIIACCGGAT
Reverse primer: TTCCGCTTTCGCTTCACGGAIIIIITGAAGCA;
FMV35S: forward primer: GGAAGAATTCTCAGTCCAAAIIIIIAACAAGG
Reverse primer: CAAAGATGCCCACTAACTTTIIIIITTCGGTG;
CaMV35S: forward primer: GATAAAGGAAAGGCCATCGTIIIIIATGCCTC
Reverse primer: TGGGATTGTGCGTCATCCCTIIIIICAGTGGA;
NOS 3 ': forward primer: TTAAGATTGAATCCTGTTGCIIIIITTGCGAT
Reverse primer: CGCGCGCGATAATTTATCCTIIIIIGCGCGCT;
PEP: forward primer: ATACGCTACAACTACCAATAIIIIICGAGTGG
Reverse primer: GGAAAAAAGGTCGAGAAGTTIIIIICACGAGG.
In the combination of primers of the rape transgenosis detection kit that this is routine, comprise asymmetric PCR primer, be the Tag primer of 5 ' end band vitamin H sign (biotin) simultaneously, and its base sequence is as follows:
Tag primer: biotin-5 '-GACGGACACCTACCCATATCACCCTTGACG-3 '.
Film chip is fixed on 11 groups of probe sequences and one group of positive control (Positive) probe sequence on supporting film surface for order, and the base sequence 5 '-3 ' of 11 groups of probe sequences and positive control probe sequence is as follows, and 5 ' end of each probe is the amino (NH of sign
2):
EPSPS probe: NH
2-5 '-TATCACCGGTCTTTTGGAAGGTGAAGATGTTATCAACA
CTGGTAAGGCTATGCAAGCTA-3’；
GOX probe: NH
2-5 '-CCCTTCCAGTGATTGGTCGTGCTACCCGTACTCCAGACG
TTATCTACGCTTTCGGTCAC-3’；
PAT probe: NH
2-5 '-GGATTGATGATCTAGAGAGGTTGCAAGATAGATACCCTT
GGTTGGTTGCTGAGGTTGAG-3’；
NPTII probe: NH
2-5 '-CACCAAGCGAAACATCGCATCGAGCGAGCACGTACTC
GGATGGAAGCCGGTCTTGTCGA-3’；
BAR probe: NH
2-5 '-CGCAACGCCTACGACTGGACGGCCGAGTCGACCGTGTA
CGTCTCCCCCCGCCACCAGCG-3’；
BARNASE probe: NH
2-5 '-AACGGAAACATCTTCTCACAAGGCACACACAGAA
GCACAGGTTATCAACACGTTTGACG-3’；
BARSTAR probe: NH
2-5 '-CGTTTTGGAATGGAGGCAGTTTGAACAAAGCAAG
CAGCTGACTGAAAATGGCGCCGAGA-3’；
FMV35S probe: NH
2-5 '-CAAAGTAAACTACTGTTCCAGCACATGCATCATGGT
CAGTAAGTTTCAGAAAAAGACAT-3’；
CaMV35S probe: NH
2-5 '-AGGAGCATCGTGGAAAAAGAAGACGTTCCAACC
ACGTCTTCAAAGCAAGTGGATTGATG-3’；
NOS 3 ' probe: NH
2-5 '-ATGAGATGGGTTTTTATGATTAGAGTCCCGCAATTATA
CATTTAATACGCGATAGAAAA-3’；
PEP probe: NH
2-5 '-GAGATTTTTCCAAGGCTTCAAACTCTTTCATCACATGTC
TCTTCTCTTTCCATTGTCTT-3’；
Positive probe: NH
2-5 '-TAGCAAGGATGAGTATTCGAACAGTTAGAGGCAGT
CAGGGAGAATCACTACGTACAAGC-3’；
Supporting film is nitrocellulose filter.
By corresponding multiple PCR primer, design, adopt synthetic above-mentioned 11 heavy PCR combination primer and the Tag primers that obtain of solid phase phosphoramidite triester method.According to multiplex PCR amplified production design detection probes, adopt solid phase phosphoramidite triester method synthesising probing needle, when probe is synthetic, synthesize positive probe (positive probe) and 5 ' corresponding positive oligonucleotide (positive oligo) single stranded DNA of holding with vitamin H sign, 5 ' end with the base sequence of the positive oligonucleotide single stranded DNA of vitamin H sign is simultaneously:
biotin-5'-GCTTGTACGTAGTGATTCTCCCTGACTGCCTCTAACTGTTCGAATAC
TCATCCTTGCTA-3’。
The film bar that supporting film is cut into 1.2cm * 1.8cm, the film cutting out soaks 15min in distilled water, then uses 15 * SSC to soak 15min, take out, be placed on filter paper, 60 ℃ are dried 1.5 hours, and film bar cools to after room temperature, probe (5uM) order is put on film, each probe repeats point sample twice, and to verify the repeatability of detected result, point sample pattern is shown in Figure 1A, film bar dries latter 80 ℃ and dries 2 hours with stationary probe, the film chip room temperature preservation of handling well.
According to plant genes group DNA extraction test kit (the NuCleanPlantGen DNA Kit of Kang Wei century bio tech ltd, Beijing, CW0531) the rape genomic dna of transgene rape strain GT73/RT73 is extracted in explanation, and the initial sample of every 30mg is used 50ul ddH after extracting
2o(distilled water) dissolve genomic dna, and with ultraviolet spectrophotometer, DNA solution is diluted to same mass concentration (50ng/ul).
The reaction system of pcr amplification is as follows:
DdH
2o: add to cumulative volume 50ul
10×PCR Buffer: 5ul
dNTP (2.5mM each): 5ul
PCR primer (20 μ M): 0.5ul each
Tag primer (20μM): 3.5ul
EX-Taq Polymerase (Takara, 5U/ul): 0.5ul
The rape genomic dna (about 50ng/ul) extracting: 2ul
Above-mentioned reaction system is carried out to PCR cyclic amplification, and PCR cycling condition is as follows, and reaction process is comprised of denaturation, multiplex PCR circulation 1 and asymmetric pcr circulation 2, and condition is respectively:
Denaturation: 95 ℃ of temperature, 10 minutes time.
1:30 circulation of multiplex PCR circulation forms:
Sex change: 95 ℃ of temperature, 45 seconds time.
Annealing: 55 ℃ of temperature, 30 seconds time.
Extend: 72 ℃ of temperature, 30 seconds time.
2:25 circulation of asymmetric pcr circulation forms:
Sex change: 95 ℃ of temperature, 45 seconds time.
Annealing: 65 ℃ of temperature, 30 seconds time.
Extend: 72 ℃ of temperature, 30 seconds time.
Finally extend: 72 ℃ of temperature, 10 minutes time.
Then carrying out film chip dot hybridization detects, film bar is in 42 ℃ of prehybridization solution (5 * SSC, 0.1% SDS, 10 * Denhardt ' s) in, prehybridization is 1 hour, afterwards film bar is put in to 2ml hybridization solution (5 * SSC, 0.1% SDS, 5 * Denhardt ' s, 50% deionized formamide, 100ug/ml yeast tRNA) in 42 ℃, 1 hour.Get 40ul multiple PCR products and add the positive oligonucleotide single stranded DNA (10uM) of 1ul, 100 ℃ of water-bath sex change are placed on ice for 5 minutes, add afterwards in 2ml hybridization solution, hybridize 16 hours for 42 ℃.Washing lotion 1(2 * SSC, 0.1% SDS), washing lotion 2(0.5 * SSC, 0.1% SDS) 42 ℃ respectively wash film bar twice, each 10 minutes.Film bar is placed in confining liquid (3%BSA, 100 mMTris-HCl, pH7.5,150 mMNaCl) to 37 ℃ of sealings 30 minutes, afterwards film bar is put in enzyme connection liquid (with confining liquid 1:5000 dilution streptavidin mark horseradish peroxidase) to 37 ℃, 30 minutes.With washing lotion 3(100 mM Tris-HCl, pH7.5,150 mMNaCl), washing lotion 4(100 mM Tris-HCl, pH9.5,100 mMNaCl, 100 mMMgCl
2) each rinsing film bar 3 times, each 5 minutes, afterwards film bar is put into TMB nitrite ion (100mM Trisodium Citrate pH5.4,0.1mg/ml tetramethyl benzidine TMB, the H of 1:1600 volume ratio
2o
2(3%)), lucifuge colour developing 10 ~ 15 minutes, according to the colour developing situation result of determination of hybridization point.
In this example, auxiliary material used and dosing are outsourcing or autogamy, and per-cent is weight percentage.
The rape genomic dna extracting in this example is transgene rape strain GT73/RT73, and detected result as shown in Figure 1B.
Embodiment bis-
This routine rape transgenosis detection kit is comprised of combination of primers, film chip and auxiliary material, auxiliary material is deoxyribonucleoside triphosphate (dNTP), EX-Taq polysaccharase (Polymerase) and 5 ' the positive oligonucleotide single stranded DNA of holding with vitamin H sign, and 5 ' end is biotin-5'-GCTTGTACGTAGT with the base sequence of the positive oligonucleotide single stranded DNA of vitamin H sign
GATTCTCCCTGACTGCCTCTAACTGTTCGAATACTCATCCTTGCTA -3’。
The rape genomic dna extracting is transgene rape strain HCN92, and detected result as shown in Figure 2 B.
All the other are with embodiment mono-.
Embodiment tri-
This routine rape transgenosis detection kit is comprised of combination of primers, film chip, auxiliary material and dosing, dosing is 10 * PCR damping fluid, prehybridization solution, hybridization solution, washing lotion, confining liquid, streptavidin mark horseradish peroxidase and tetramethyl benzidine (TMB) nitrite ion, wherein, prehybridization solution is 5 * SSC, 0.1% SDS and 10 * Denhardt ' s; Hybridization solution is 5 * SSC, 0.1% SDS, 5 * Denhardt ' s, 50% deionized formamide and 100ug/ml yeast tRNA; Washing lotion is respectively washing lotion 1:2 * SSC and 0.1% SDS, washing lotion 2:0.5 * SSC and 0.1% SDS, washing lotion 3:100 mM Tris-HCl, pH7.5,150 mM NaCl, washing lotion 4:100 mM Tris-HCl, pH9.5,100 mM NaCl and 100 mM MgCl
2; Confining liquid is 3% BSA, 100 mM Tris-HCl, pH7.5,150 mM NaCl; Tetramethyl benzidine nitrite ion is respectively tetramethyl benzidine nitrite ion A liquid: 200mM Trisodium Citrate, pH5.4,0.2mg/ml tetramethyl benzidine, tetramethyl benzidine nitrite ion B liquid: 3% H
2o
2with the distilled water diluent of 800 volumes, before using, A liquid B liquid balanced mix is made into tetramethyl benzidine nitrite ion.
Supporting film is nitrocellulose filter.
The rape genomic dna extracting is transgene rape strain MS1 * RF1, and detected result as shown in Figure 3 B.
All the other are with embodiment mono-.
Embodiment tetra-
This routine rape transgenosis detection kit is comprised of combination of primers, film chip, auxiliary material and dosing, wherein, multiplex PCR amplifing reagent one pipe 600ul, every 48ul reagent can detect a DNA sample, every 48ul amplification system is constructed as follows: 10 * PCR Buffer (Takara) 5ul, dNTP (2.5mM each) 5ul, 11 pairs of multiple PCR primers (20 μ M) 0.5ul each, Tag primer (20 μ M) 3.5ul, EX-Taq Polymerase (Takara, 5U/ul) 0.5ul, ddH
2o adds to 48ul.Each pattern detection need to be drawn 48ul amplifing reagent, adds 2ul genomic dna to be detected (about 50ng/ul).
Positive oligonucleotide single stranded DNA (10uM) pipe 15ul.
One bottle of 30ml of prehybridization solution (5 * SSC, 0.1% SDS, 10 * Denhardt ' s).One bottle of 30ml of hybridization solution (5 * SSC, 0.1% SDS, 5 * Denhardt ' s, 50% deionized formamide, 100ug/ml yeast tRNA).
Washing lotion 1(2 * SSC, 0.1% SDS) 10 times of one bottle of 12ml of concentrated solution, add 108ml ddH before use
2o.Washing lotion 2(0.5 * SSC, 0.1% SDS) 10 times of one bottle of 12ml of concentrated solution, add 108ml ddH before use
2o.
One bottle of 60ml of confining liquid/enzyme connection liquid (3%BSA, 100 mM Tris-HCl, pH7.5,150 mMNaCl).Streptavidin mark horseradish peroxidase (1mg/ml) pipe 5ul, is diluted to enzyme connection liquid with confining liquid 1:5000 before using.
Washing lotion 3(100 mM Tris-HCl, pH7.5,150 mMNaCl) 10 times of one bottle of 18ml of concentrated solution, before use, add 162ml ddH
2o.Washing lotion 4(100 mM Tris-HCl, pH9.5,100 mMNaCl, 100 mM MgCl
2) 10 times of one bottle of 18ml of concentrated solution, before use, add 162ml ddH
2o.
One bottle of 15ml of TMB nitrite ion A liquid (200mM Trisodium Citrate, pH5.4,0.2mg/ml tetramethyl benzidine TMB).The TMB nitrite ion B liquid (H of 1:800 volume ratio
2o
2(3%)) one bottle of 15ml.Before using, A liquid B liquid balanced mix is made into TMB nitrite ion.
The rape genomic dna extracting is the wild strain 3529 of non-transgenic rape, and detected result as shown in Figure 4 B.
All the other are with embodiment tri-.
Claims (1)
1. a rape transgenosis detection kit, is comprised of combination of primers and film chip, it is characterized in that combination of primers is 11 heavy double startup Oligonucleolide primers combinations, and each base sequence 5 '-3 ' to primer is as follows, and wherein base I is inosinic acid:
EPSPS: forward primer: CTTTCTGGAACCGTCCGTATIIIIIGTGACAA
Reverse primer: CCAAGTATCACCTTCCTTACIIIIICTGGCAC;
GOX: forward primer: CTGCTGCTCCTAACTGGAAGIIIIITCACGTT
Reverse primer: GGTCATTGGAGCACCAGTCAIIIIIAGGTGAC;
PAT: forward primer: TGAGACGTCTACAGTGAACTIIIIIACAGAGC
Reverse primer: CTTCCAGGGCCCAGCGTAAGIIIIICCAGCCA;
NPTII: forward primer: CGAAGTGCCGGGGCAGGATCIIIIITCATCTC
Reverse primer: CGCGAGCCCCTGATGCTCTTIIIIIAGATCAT;
BAR: forward primer: GACGACCTCGTCCGTCTGCGIIIIIGCTATCC
Reverse primer: CAGCAGGTGGGTGTAGAGCGIIIIICCCAGTC;
BARNASE: forward primer: ATTATCGTGGATTTCCGTTTIIIIICTGGTGC
Reverse primer: GCTTATGATATGTCTGAAGAIIIIICGCAACC;
BARSTAR: forward primer: CCTGGACGCTTTATGGGATTIIIIIACCGGAT
Reverse primer: TTCCGCTTTCGCTTCACGGAIIIIITGAAGCA;
FMV35S: forward primer: GGAAGAATTCTCAGTCCAAAIIIIIAACAAGG
Reverse primer: CAAAGATGCCCACTAACTTTIIIIITTCGGTG;
CaMV35S: forward primer: GATAAAGGAAAGGCCATCGTIIIIIATGCCTC
Reverse primer: TGGGATTGTGCGTCATCCCTIIIIICAGTGGA;
NOS 3 ': forward primer: TTAAGATTGAATCCTGTTGCIIIIITTGCGAT
Reverse primer: CGCGCGCGATAATTTATCCTIIIIIGCGCGCT;
PEP: forward primer: ATACGCTACAACTACCAATAIIIIICGAGTGG
Reverse primer: GGAAAAAAGGTCGAGAAGTTIIIIICACGAGG;
Film chip is fixed on 11 groups of probe sequences and one group of positive control (Positive) probe sequence on supporting film surface for order, and the base sequence 5 '-3 ' of 11 groups of probe sequences and positive control probe sequence is as follows, and 5 ' end of each probe is done amino sign:
EPSPS probe: NH
2-5 '-TATCACCGGTCTTTTGGAAGGTGAAGATGTTATCAACA
CTGGTAAGGCTATGCAAGCTA-3’；
GOX probe: NH
2-5 '-CCCTTCCAGTGATTGGTCGTGCTACCCGTACTCCAGACG
TTATCTACGCTTTCGGTCAC-3’；
PAT probe: NH
2-5 '-GGATTGATGATCTAGAGAGGTTGCAAGATAGATACCCTT
GGTTGGTTGCTGAGGTTGAG-3’；
NPTII probe: NH
2-5 '-CACCAAGCGAAACATCGCATCGAGCGAGCACGTACTC
GGATGGAAGCCGGTCTTGTCGA-3’；
BAR probe: NH
2-5 '-CGCAACGCCTACGACTGGACGGCCGAGTCGACCGTGTA
CGTCTCCCCCCGCCACCAGCG-3’；
BARNASE probe: NH
2-5 '-AACGGAAACATCTTCTCACAAGGCACACACAGAA
GCACAGGTTATCAACACGTTTGACG-3’；
BARSTAR probe: NH
2-5 '-CGTTTTGGAATGGAGGCAGTTTGAACAAAGCAAG
CAGCTGACTGAAAATGGCGCCGAGA-3’；
FMV35S probe: NH
2-5 '-CAAAGTAAACTACTGTTCCAGCACATGCATCATGGT
CAGTAAGTTTCAGAAAAAGACAT-3’；
CaMV35S probe: NH
2-5 '-AGGAGCATCGTGGAAAAAGAAGACGTTCCAACC
ACGTCTTCAAAGCAAGTGGATTGATG-3’；
NOS 3 ' probe: NH
2-5 '-ATGAGATGGGTTTTTATGATTAGAGTCCCGCAATTATA
CATTTAATACGCGATAGAAAA-3’；
PEP probe: NH
2-5 '-GAGATTTTTCCAAGGCTTCAAACTCTTTCATCACATGTC
TCTTCTCTTTCCATTGTCTT-3’；
Positive probe: NH
2-5 '-TAGCAAGGATGAGTATTCGAACAGTTAGAGGCAGT
CAGGGAGAATCACTACGTACAAGC-3’；
In described combination of primers, have asymmetric PCR primer, be 5 ' end with the Tag primer of vitamin H sign, its base sequence is as follows:
Tag primer: biotin-5 '-GACGGACACCTACCCATATCACCCTTGACG-3 '.
2. rape transgenosis detection kit according to claim 1, it is characterized in that described rape transgenosis detection kit is comprised of combination of primers, film chip and auxiliary material, auxiliary material is deoxyribonucleoside triphosphate, EX-Taq polysaccharase and 5 ' the positive oligonucleotide single stranded DNA of holding with vitamin H sign, and 5 ' end is biotin-5'-GCTTGTACGTAGTGATTCTCCCTGACTGCCTCTAACTGTTCGAATACTCATCCTTG CTA-3 ' with the base sequence of the positive oligonucleotide single stranded DNA of vitamin H sign.
3. rape transgenosis detection kit according to claim 1, it is characterized in that described rape transgenosis detection kit is comprised of combination of primers, film chip, auxiliary material and dosing, dosing is 10 * PCR damping fluid, prehybridization solution, hybridization solution, washing lotion, confining liquid, streptavidin mark horseradish peroxidase and tetramethyl benzidine nitrite ion.
4. rape transgenosis detection kit according to claim 3, is characterized in that described prehybridization solution is 5 * SSC, 0.1% weight percent sodium laurylsulfonate and 10 * Denhardt ' s liquid; Hybridization solution is 5 * SSC, 0.1% weight percent SDS, 5 * Denhardt ' s, 50% weight percent deionized formamide and 100ug/ml yeast tRNA; Washing lotion is respectively washing lotion 1:2 * SSC and 0.1% weight percent SDS, washing lotion 2:0.5 * SSC and 0.1% weight percent SDS, washing lotion 3:100 mM Tris-HCl, pH7.5,150 mM NaCl, washing lotion 4:100 mM Tris-HCl, pH9.5,100 mM NaCl and 100 mM MgCl
2; Confining liquid is 3% weight percent bovine serum albumin, 100 mM Tris-HCl, pH7.5,150 mM NaCl; Tetramethyl benzidine nitrite ion is respectively tetramethyl benzidine nitrite ion A liquid: 200mM Trisodium Citrate pH5.4,0.2mg/ml tetramethyl benzidine, tetramethyl benzidine nitrite ion B liquid: the H of 3% weight percent
2o
2with the distilled water diluent of 800 volumes, before using, A liquid B liquid balanced mix is made into tetramethyl benzidine nitrite ion.
5. rape transgenosis detection kit according to claim 1, is characterized in that described supporting film is nitrocellulose filter or nylon membrane.
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|---|---|---|---|---|
| CN105154437A (en) * | 2015-07-28 | 2015-12-16 | 中国检验检疫科学研究院 | DPO (dual priming oligonucleotide) primer combination and detection method for multi-PCR detection of transgenic crops |
| CN105112409B (en) * | 2015-08-18 | 2018-07-27 | 中国检验检疫科学研究院 | Detect the combination of multiple DPO-PCR primers and the method for transgenic corns MON810 and MIR604 |
| CN105861499A (en) * | 2016-06-13 | 2016-08-17 | 湛江出入境检验检疫局检验检疫技术中心 | Multi-DPO-PCR primer composition for detecting vibrio parahaemolyticus and vibrio cholerae |
| CN108841990B (en) * | 2018-07-18 | 2022-03-01 | 四川华汉三创生物科技有限公司 | Detection kit and method for transgenic rape line |
| CN114107552B (en) * | 2022-01-04 | 2023-09-08 | 江汉大学 | Primer pair combination, kit, detection method and application for detecting rape transgenic line |
| CN114277178B (en) * | 2022-01-04 | 2023-07-07 | 江汉大学 | Primer pair combination for detecting transgenic components of rape, kit, detection method and application |
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| CN103173548A (en) * | 2013-03-13 | 2013-06-26 | 深圳出入境检验检疫局动植物检验检疫技术中心 | PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) assay primer and assay method for transgenic rapeseed RF2 strain |
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| CN1420181A (en) * | 2002-10-11 | 2003-05-28 | 覃文 | Probe sequence and kit for real time fluorescence PCR detection of transgenic rapeseed |
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