CN103290128B - Method for detecting transgenic tomato by using in-situ synthetic microfluidic chip - Google Patents
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Abstract
The invention discloses a method for detecting a transgenic tomato by using an in-situ synthetic microfluidic chip. The method comprises the following steps of: 1) designing a chip probe for detecting an exogenous gene of the transgenic tomato; 2) preparing an oligonucleotide chip; 3) extracting genome deoxyribonucleic acid (DNA) in a tomato sample to be detected; 4) marking a specific segment of the genome or the entire genome by a fluorescent agent; 5) chip hybridization; 6) judging whether the sample belongs to the transgenic tomato variety. By adopting the method, an oligonucleotide probe is designed according to the sequence information of the known tomato exogenous gene which has been publically reported; the microfluidic gene chip is prepared by utilizing an in-situ synthesis method, so as to achieve simultaneous detection of existence of the exogenous genes related to insect resistance, antiviral action, herbicide resistance, delayed fruit, softening resistance, storage resistance and the like on the same chip. Thus, an effective method is provided for rapid detection and identification of the transgenic ingredients of the tomato and highly processed products; a foundation is provided for building and perfecting a transgenosis detection system and a supervisory system.
Description
Technical field
The present invention relates to detection and the authentication method of transgene component in tomato, relate in particular to the method that adopts the synthetic micro-fluid chip of original position to detect known foreign gene in tomato.
Background technology
Tomato (Solanum lycopersicum) is one of important solanaceous crops, and its fruit rich vitamin, carotene, Lyeopene, have higher nutritive value and health-care effect, is important fruit and vegetable food in world wide.The cultivated area of nearly ten years whole world tomatoes and output have increased respectively 38% and 42%, and Commercialization Trends is obvious.Cultivate from Calgene company of the U.S. in 1994 prolong ripe fresh-keeping transgenic Fructus Lycopersici esculenti FLAVR SAVR and get permission to come into the market after, various antiviral, pest-resistant, antiweeds, freeze proof evil, the anti-strain improvement tomato variety such as saline and alkaline are reported in succession, and some gets permission commercialization plantation, transgenic Fructus Lycopersici esculenti is put on market in a large number has become irresistible trend.But, there is expert to point out that through experiment genetically modified food can increase the probability that mouse suffers from tumour, therefore cause that about the security of transgenic product society disputes on widely, and its destruction to environment, the original nutrition system of food, be also the focus that people pay close attention to.
For this kind of situation, international community constantly perfects and improves the detection system of transgene component on the one hand, has put into effect on the other hand labeling system, to guarantee human consumer to the right to know of genetically modified food and to select right.China also attaches great importance to the security control of genetically modified food, has issued " agriculture genetically modified organism security control regulations ", and Safety of GM Food is risen to law aspect.But China relatively lags behind to the detection system of transgene component in food, the research of necessary reinforcement this respect.
At present, the detection set of transgenic Fructus Lycopersici esculenti, in nucleic acid level, mainly contains qualitative PCR, quantitative fluorescent PCR, multiplex PCR and nucleic acid hybridization detection technique.Traditional transgenic detection method is usually confined to term single gene, and sensing range is narrow, and efficiency is low.In the increasing situation of foreign gene kind in current importing agricultural-food, the detection of unitem can not meet foods supervision needs, in the urgent need to developing high-throughout quick transgenosis detection technique.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of method of utilizing the synthetic micro-fluid chip of original position to detect transgenic Fructus Lycopersici esculenti.
In order to solve the problems of the technologies described above, the invention provides a kind of structure and detection method of utilizing the synthetic micro-fluid chip of original position, comprise the following steps:
1) design of the chip probe that, transgenic Fructus Lycopersici esculenti foreign gene detects;
2), prepare oligonucleotide microarray;
3), the extraction of genomic dna in tomato sample to be measured;
4), the specific fragment of genome or whole genome fluorescent mark;
5), chip hybridization;
6), judge whether sample is transgenic Fructus Lycopersici esculenti kind.
As the improvement that utilizes the synthetic micro-fluid chip of original position to detect the method for transgenic Fructus Lycopersici esculenti of the present invention, described step 1) be: 895 oligonucleotide probes as described in Table 1 have been synthesized in design.
As the further improvements in methods of utilizing the synthetic micro-fluid chip of original position to detect transgenic Fructus Lycopersici esculenti of the present invention, step 2) be:
Prepare oligonucleotide microarray: first synthesize in position on micro-fluid chip 895 of synthetic chemistry modified sequence oligonucleotide probes successively; Then synthetic chip is carried out on chip scanner fluorometric analysis, with the fluorescence background of detection chip itself; Carry out, after fluorescent mark, hybridizing with chip again analysis chip specificity, sensitivity and repeatability, assessment chip quality with Quality Control cDNA or the DNA fragmentation that comes from plasmid.
As the further improvements in methods of utilizing the synthetic micro-fluid chip of original position to detect transgenic Fructus Lycopersici esculenti of the present invention, step 3) be the extraction of genomic dna in tomato sample to be measured:
Get 0.1-0.2g tomato sample, liquid nitrogen grinding, packs in the aseptic eppendorf pipe of 1.5ml; Add 1ml CTAB extracting solution, in 65 DEG C of water-bath 60-90min; Centrifuging and taking supernatant, add phenol/chloroform/primary isoamyl alcohol (volume ratio is 25:24:1, amount to consumption be supernatant volume 2/3), fully mix centrifuging and taking supernatant; Add chloroform/primary isoamyl alcohol, centrifuging and taking supernatant; Add NaAC solution and dehydrated alcohol precipitation; The centrifugal supernatant of abandoning, the alcohol washing precipitation that volumetric concentration is 70%; The centrifugal supernatant of abandoning, vacuum-drying; Add 50-100 μ 1TE solution dissolving DNA.
As the further improvements in methods of utilizing the synthetic micro-fluid chip of original position to detect transgenic Fructus Lycopersici esculenti of the present invention,
Step 4) be the specific fragment of genome or whole genome fluorescent mark, any one in the following 3 kinds of methods of optional use:
Method A, taking tomato sample DNA to be checked as template, design and filter out the primer of promotor conventional in transgenic Fructus Lycopersici esculenti, terminator, reporter gene and tomato kind internal reference gene, carry out PCR reaction, and it carried out to CY3 fluorescent mark; In PCR reaction system, replace common dCTP with the dCTP of CY3 mark; Concrete primer information: 35s-F/R:5 '-GCTCCTACAAATGCCATCA-3 '/5 '-GATAGTGGGATTGTGCGTCA-3 '; Nos-F/R:5 '-GAATCCTGTTGCCGGTCTTG-3 '/5 '-TTATCCTAGTTTGCGCGCTA-3 '; 35s-EFE-F/R:5 '-TGGGATCTAAGCACTTGCAATTGGA-3 '/5 '-GCACAAACAGACGGGACACGAAT-3 '; NptII-F/R:5 '-GAAAAATACCGCTGCGTAAAAG-3 '/5 '-TCAGGGCTTTGTTCATCTTCAT-3 '; Acc-F/R:5 '-TTTTGCCTCGCTGATCCTGGC-3 '/5 '-GGTTTCGCCTTTCGGCTTCTGT-3 '; Cry1AC-F/R:5 '-TCATCCCTTACGTCAGTGGAG-3 '/5 '-CCATCATTGCGATAAAGGAAA-3 '; Lat52-F/R:5 '-ATGCAGGAACATCAGCACAGAGGC-3 '/5 '-TCTTTGCAGTCCTCCCTTGGGCTT-3 '; Mcpi-F/R:5 '-TGGCACAAGATGCAACTCTGACGA-3 '/5 '-ACAGGCCTGACAGAACGTACCA-3 '; Apx-F/R:5 '-CTCTTGGAGCCCATTAGGGAGCA-3 '/5 '-TTGGAAGTAAGCACTACTTAGTAGTCATCAC-3 '; Fru-F/R:5 '-GGTAGTTCAGTACCTGTGTTGGACG-3 '/5 '-TGACATCTAATCCTAAGGTAGGGCG-3 ';
Method B, insert joint Head-F (GCCTTGTTCTACCATTACGC) at 5 ' end of method A forward primer used, reverse primer 5 ' end inserts joint Head-R (TCAGCCACCTCTTTGTCCTT); Taking tomato sample DNA to be checked as template, carry out first round multiplex PCR amplification by the combination of primers of belt lacing, amplification system is after totally 11 circulations; Then taking the PCR product of the first round as template, taking the general header sequence Head-F/Head-R that adds as primer, carry out second and take turns multi-PRC reaction; In PCR reaction system, replace common dCTP with the dCTP of CY3 mark;
Method C, tomato sample DNA to be checked is first used to restriction enzyme A ccII (FnuDII) digestion, enzyme tangent condition is 37 DEG C, 60min; Enzyme cuts into and carries out random primer labelling reaction with Random Primer DNA Labeling Kit Ver.2.0 (TaKaRa) afterwards; In reaction system, replace common dCTP with the dCTP of CY3 mark.
As the further improvements in methods of utilizing the synthetic micro-fluid chip of original position to detect transgenic Fructus Lycopersici esculenti of the present invention, step 5) chip hybridization is:
CY3 marked product after purifying is added to isopyknic hybridization buffer (6 × SSPE, 25% methane amide, pH6.6-6.8), and 95 DEG C of sex change 5min are placed on precooling 3min on ice; Join again in sample feeding pipe, 30 DEG C, bidirectional circulating hybridization 16h, speed 500 μ l/min; After hybridization finishes, with 1ml elution buffer (500 μ l hybridization buffers, 500 μ lH
2o, 20 μ L10%SDS) 32 DEG C of wash cycles 20min, then use 40 DEG C of wash cycles 20min of 1ml elution buffer; Take out chip, with MicroarryScanner Genepix4000B scanning, scanning resolution is 10 μ m, and wavelength is 635nm, reads scanning result, preserves picture post analysis chip data.
As the further improvements in methods of utilizing the synthetic micro-fluid chip of original position to detect transgenic Fructus Lycopersici esculenti of the present invention, step 6) for to judge whether sample is transgenic Fructus Lycopersici esculenti kind:
Obtain fluorescence signal intensity and the standard error of each probe with chip analysis software, signal value, after background noise subduction, is normalized data to same level by the average signal value of adjusting under this scan channel; Last according to the signal value of promotor, terminator, reporter gene, tomato kind internal reference gene and chip Quality Control probe, the confidence level of analysis chip results of hybridization, judges whether this sample belongs to transgenic positive tomato sample; Judging criterion is: if reference gene is detected, foreign gene is detected (also have certain recall rate, be at least 1 foreign gene and be detected), and explanation is tomato transgenic positive sample; If reference gene is detected, foreign gene does not detect, and explanation is non-transgenic sample.
In the present invention, collect to obtain all transgenic Fructus Lycopersici esculenti proterties and the gene expression information of open report up till now, obtain transgenic Fructus Lycopersici esculenti conventional 35S promoter, NOS terminator and marker gene NPTII sequence, and proceed to pest-resistant, antiviral, prolong the goal gene sequence (as accd, pg, anti-EFE, anti-pg, CMV cp, cry1Ac, aad, acc, sam-k gene) that the proterties such as ripe, storage endurance are relevant; According to above sequence information, be template according to its positive-sense strand and antisense strand respectively, utilize the software designs such as ArrayDesigner2 (PREMIER Biosoft), Primer5.0, Primer Express and screen complementary oligonucleotide hybridization probe; Probe length is about 25nt, GC content 40～60%, and self is without secondary structure; 895 oligonucleotide probes have been synthesized in design altogether, refer to table 1; Probe entrusts Atactic company of the U.S. to adopt μ Paraflo
tMtechnology original position on the micro-fluid chip of 31 × 128 matrixes is synthetic, and every probe repeats 4 times, and probe distribution situation is shown in Fig. 4.
The present invention utilizes the high throughput testing characteristic of this chip, taking disclosed all tomato external source transgenic sequences in the world as foundation, in tomato, some carry out multiple rapid detection to pest-resistant, antiviral, antiweed, the existence and the expression that prolong the relevant foreign gene compositions such as ripe, anti-softening and resistance to storage.
In the present invention, the method of design of the chip probe that described transgenic Fructus Lycopersici esculenti foreign gene detects is: by the transgenosis database http://gmdd.shgmo.org/ of Shanghai Communications University, U.S. genetically modified organism database http://usbiotechreg.nbii.gov/, the genetically modified organism database http://www.bch.biodic.go.jp/english/lmo.html of Japan's approval, European Union's genetically modified organism environment discharges and listing database http://gmoinfo.jrc.ec.europa.eu/, Canada transgenic product storehouse http://www.agbios.com/dbase.php, the OECD of OECD transgenic product database http://webdomino1.oecd.org/ehs/bioprod.nsf information, collect to obtain all transgenic Fructus Lycopersici esculenti proterties and the gene expression information of open report up till now.Comparative analysis is found, these transgenic Fructus Lycopersici esculenti kinds mainly adopt 35S promoter and NOS terminator, marker gene is mainly taking NPTII as main, and the goal gene proceeding to comprises that accd (prolongs ripe, tomato 8338 strains), pg (prolongs ripe, tomato Flavr Savr strain), anti-EFE (storage endurance, tomato Huafan No.1 strain), anti-pg (prolongs ripe, tomato B, Da, F strain), CMV cp is (antiviral, tomato 8805R strain), cry1Ac is (pest-resistant, tomato 5345 strains), aad is (pest-resistant, tomato 5345 strains), acc (prolongs ripe, tomato 1345-4 strain), sam-k (prolongs ripe, tomato 351N strain).According to above sequence information, be template according to its positive-sense strand and antisense strand respectively, utilize the software designs such as ArrayDesigner2 (PREMIER Biosoft), Primer5.0, Primer Express and screen complementary oligonucleotide hybridization probe; Probe length is about 25nt, GC content 40～60%, and self is without secondary structure.895 oligonucleotide probes have been synthesized in design altogether.Probe entrusts Atactic company of the U.S. to adopt μ Paraflo
tMtechnology original position on the micro-fluid chip of 31 × 128 matrixes is synthetic, and every probe repeats 4 times.
The described method of preparing oligonucleotide microarray is: first synthesize in position on micro-fluid chip the oligonucleotide probe sequence of synthetic chemistry modified successively; Then synthetic chip is carried out on chip scanner fluorometric analysis, with the fluorescence background of detection chip itself; Carry out, after fluorescent mark, hybridizing with chip again analysis chip specificity, sensitivity and repeatability, assessment chip quality with Quality Control cDNA or the DNA fragmentation that comes from plasmid.
The specific fragment of described genome or whole genome fluorescence labeling method are following any one method:
Method A, taking tomato sample DNA to be checked as template, design and filter out the primer of promotor conventional in transgenic Fructus Lycopersici esculenti, terminator, reporter gene and tomato kind internal reference gene, carry out PCR reaction, and it carried out to CY3 fluorescent mark.In PCR reaction system, replace common dCTP with the dCTP of CY3 mark.
Method B, insert joint Head-F (GCCTTGTTCTACCATTACGC) at 5 ' end of method A forward primer used, reverse primer 5 ' end inserts joint Head-R (TCAGCCACCTCTTTGTCCTT).Taking tomato sample DNA to be checked as template, carry out first round multiplex PCR amplification by the combination of primers of belt lacing, amplification system is after totally 11 circulations.Then taking the PCR product of the first round as template, taking the general header sequence Head-F/Head-R that adds as primer, carry out second and take turns multi-PRC reaction.In PCR reaction system, replace common dCTP with the dCTP of CY3 mark.
Method C, tomato sample DNA to be checked is first used to restriction enzyme A ccII (FnuDII) digestion, enzyme tangent condition is 37 DEG C, 60min.Enzyme cuts into and carries out random primer labelling reaction with Random Primer DNA Labeling Kit Ver.2.0 (TaKaRa) afterwards.In reaction system, replace common dCTP with the dCTP of CY3 mark.
The result that above three kinds of methods are obtained can be carried out horizontal or longitudinal comparative analysis of Fluorescent dye incorporation rate, to compare the relative merits of the whole bag of tricks.
Described chip hybridization method is: the CY3 marked product after purifying is added to equal-volume hybridization buffer, be placed in rapidly precooling 3min on ice after 95 DEG C of sex change 5min.Join again in sample feeding pipe, 30 DEG C, bidirectional circulating hybridization 16h (spending the night), speed 500 μ L/min.After hybridization finishes, with buffer40 DEG C of wash cycles 20min of 1mL Wash, speed is 100 μ L/min.Take out chip, with Microarry Scanner Genepix4000B scanning, scanning resolution is 10 μ m, and wavelength is 635nm, reads scanning result, preserves picture post analysis chip data.
Described judge that whether sample is the fluorescence signal intensity and the standard error that obtain each probe with chip analysis software as the method for transgenic Fructus Lycopersici esculenti kind, signal value, after background noise subduction, is normalized data to same level by the average signal value of adjusting under this scan channel; Last according to the signal value of promotor, terminator, reporter gene, tomato kind internal reference gene and chip Quality Control probe, the confidence level of analysis chip results of hybridization, judges whether this sample belongs to transgenic positive tomato sample.Judging criterion is: if reference gene is detected, foreign gene also has certain recall rate, and explanation is tomato transgenic positive sample.If reference gene is detected, foreign gene does not detect, and explanation is non-transgenic sample.
In sum, compared with prior art, the present invention according to all known tomato exogenous gene sequence information design of current open report oligonucleotide probe, and prepare microfluidic gene chip with in-situ synthetic method, on a chip, detect with pest-resistant, antiviral, antiweed to realize simultaneously, prolong and ripe, anti-ly soften the foreign gene relevant with resistance to storage etc. and have situation.Thereby for rapid detection and the qualification of tomato and deep processed product transgene component thereof provide effective ways, provide foundation for establishing and improve transgenosis detection system and supervisory system.
Table 1 transgenic Fructus Lycopersici esculenti foreign gene detection chip probe
Brief description of the drawings
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is described in further detail.
Fig. 1 is the chip hybridization collection of illustrative plates after regular-PCR, multiplex PCR, three kinds of method marks of random primer labelling " Bioscein " genomic dna.
Fig. 2 is the chip hybridization collection of illustrative plates after regular-PCR, Multiplex PCR mark " beautiful spring ZN " the specific fragment of genomic dna.
Fig. 3 is the chip hybridization collection of illustrative plates after regular-PCR method mark " cooperation 903 " the specific fragment of genomic dna.
Fig. 4 is the arrangement schematic diagram of probe on chip.
Embodiment
Embodiment 1, a kind of method of utilizing the synthetic micro-fluid chip of original position to detect transgenic Fructus Lycopersici esculenti, taking " Bioscein " tomato as sample to be tested, carry out successively following steps:
1), the design of transgenic Fructus Lycopersici esculenti foreign gene detection chip probe: design 895 oligonucleotide probes as described in Table 1;
2), prepare oligonucleotide microarray: the arrangement schematic diagram of probe on chip as shown in Figure 4.Synthetic chip is carried out on chip scanner to fluorometric analysis, with the fluorescence background of detection chip itself; When chip background signal intensity is less than 20, without clear signal point, think that chip meets the requirements.
The DNA fragmentation of using again Quality Control cDNA (synthetic, with one section of oligonucleotide of chip Quality Control probe complementation) or come from plasmid is (taking plasmid PBI121 as template, 35s promotor, no terminator and nptII gene fragment that pcr amplification obtains) carry out, after fluorescent mark, hybridizing with chip.When Quality Control probe, foreign gene detection probe signal are strong, repeatability is high, illustrate that chip specificity is high, reproducible.Otherwise, a little less than Quality Control probe, foreign gene detection probe signal, repeatability low, illustrate that chip specificity is low, poor repeatability.Chip of the present invention, through above-mentioned checking, meets high, the reproducible requirement of specificity.
3), the extraction of genomic dna in tomato sample to be measured:
Get 0.1g tomato sample, liquid nitrogen grinding, packs in the aseptic eppendorf pipe of 1.5ml; Add 1ml CTAB extracting solution, in 65 DEG C of water-bath 60-90min; Centrifugal (12000rpm, 10min) gets supernatant, add phenol/chloroform/primary isoamyl alcohol (volume ratio is 25:24:1, amount to consumption be supernatant volume 2/3), fully mix centrifuging and taking supernatant; Add chloroform/primary isoamyl alcohol (volume ratio is 24:1, amount to consumption be supernatant volume 2/3), centrifuging and taking supernatant; Add the concentration NaAC solution that is 3M and the dehydrated alcohol that accounts for 1 volume of supernatant that account for supernatant 1/10 volume ,-20 DEG C of precipitation 30min; The centrifugal supernatant of abandoning, twice of the alcohol washing precipitation (each consumption is 1ml) of 70% (volumetric concentration); The centrifugal supernatant of abandoning, vacuum-drying; Add 50 μ 1TE buffered soln dissolving DNAs.
4), the specific fragment of genome or whole genome fluorescent mark:
Adopt respectively following 3 kinds of methods:
Method A (regular-PCR),
Taking tomato sample DNA to be checked as template, carry out PCR reaction, the primer is:
35s-F/R:5′-GCTCCTACAAATGCCATCA-3′/5′-GATAGTGGGATTGTGCGTCA-3′；
nos-F/R:5′-GAATCCTGTTGCCGGTCTTG-3′/5′-TTATCCTAGTTTGCGCGCTA-3′；
35s-EFE-F/R:5′-TGGGATCTAAGCACTTGCAATTGGA-3′/5′-GCACAAACAGACGGGACACGAAT-3′；
nptII-F/R:5′-GAAAAATACCGCTGCGTAAAAG-3′/5′-TCAGGGCTTTGTTCATCTTCAT-3′；
acc-F/R:5′-TTTTGCCTCGCTGATCCTGGC-3′/5′-GGTTTCGCCTTTCGGCTTCTGT-3′；
cry1AC-F/R:5′-TCATCCCTTACGTCAGTGGAG-3′/5′-CCATCATTGCGATAAAGGAAA-3′；
lat52-F/R:5′-ATGCAGGAACATCAGCACAGAGGC-3′/5′-TCTTTGCAGTCCTCCCTTGGGCTT-3′；
mcpi-F/R:5′-TGGCACAAGATGCAACTCTGACGA-3′/5′-ACAGGCCTGACAGAACGTACCA-3′；
apx-F/R:5′-CTCTTGGAGCCCATTAGGGAGCA-3′/5′-TTGGAAGTAAGCACTACTTAGTAGTCATCAC-3′；
fru-F/R:5′-GGTAGTTCAGTACCTGTGTTGGACG-3′/5′-TGACATCTAATCCTAAGGTAGGGCG-3′；
Amplification system is 2.5 μ l10 × PCR buffer, 1.25 μ l dNTP mixtures (dGTP, dATP, the each 2.0mM of dTTP), 2.5 μ l Cy3-dCTP (1mM), 1.0 μ l upstream primer FP (10 μ M), 1.0 μ l downstream primer RP (10 μ M), 0.125 μ l HS-TaqDNA polysaccharase, 1 μ 1 genomic dna, deionized water 15.625 μ 1.
Amplification program is: 94 DEG C of preheating 5min, and 94 DEG C of sex change 30s, 60 DEG C of renaturation 30s, 68 DEG C are extended 35s, circulate 35 times, and 68 DEG C are extended 7min.After reaction finishes, clean PCR product.The regular-PCR product of 10 kinds of CY3 marks is mixed, and avoid CY3 fluorescent quenching with masking foil parcel ,-80 DEG C save backup.
Method B (multiplex PCR),
Method A just used/reverse primer 5 ' end insert joint sequence, the primer of belt lacing is 35s-F/R joint primer: 5 '-GCCTTGTTCTACCATTACGCGCTCCTACAAATGCCATCA-3 '/5 '-TCAGCCACCTCTTTGTCCTTGATAGTGGGATTGTGCGTCA-3 '; Nos-F/R joint primer: 5 '-GCCTTGTTCTACCATTACGCGAATCCTGTTGCCGGTCTTG-3 '/5 '-TCAGCCACCTCTTTGTCCTTTTATCCTAGTTTGCGCGCTA-3 '; 35s-EFE-F/R joint primer: 5 '-GCCTTGTTCTACCATTACGCTGGGATCTAAGCACTTGCAATTGGA-3 '/5 '-TCAGCCACCTCTTTGTCCTTGCACAAACAGACGGGACACGAAT-3 '; NptII-F/R joint primer: 5 '-GCCTTGTTCTACCATTACGCGAAAAATACCGCTGCGTAAAAG-3 '/5 '-TCAGCCACCTCTTTGTCCTTTCAGGGCTTTGTTCATCTTCAT-3 '; Acc-F/R joint primer: 5 '-GCCTTGTTCTACCATTACGCTTTTGCCTCGCTGATCCTGGC-3 '/5 '-TCAGCCACCTCTTTGTCCTTGGTTTCGCCTTTCGGCTTCTGT-3 '; Cry1AC-F/R joint primer: 5 '-GCCTTGTTCTACCATTACGCTCATCCCTTACGTCAGTGGAG-3 '/5 '-TCAGCCACCTCTTTGTCCTTCCATCATTGCGATAAAGGAAA-3 '; Lat52-F/R joint primer: 5 '-GCCTTGTTCTACCATTACGCATGCAGGAACATCAGCACAGAGGC-3 '/5 '-TCAGCCACCTCTTTGTCCTTTCTTTGCAGTCCTCCCTTGGGCTT-3 '; Mcpi-F/R joint primer: 5 '-GCCTTGTTCTACCATTACGCTGGCACAAGATGCAACTCTGACGA-3 '/5 '-TCAGCCACCTCTTTGTCCTTACAGGCCTGACAGAACGTACCA-3 '; Apx-F/R joint primer: 5 '-GCCTTGTTCTACCATTACGCCTCTTGGAGCCCATTAGGGAGCA-3 '/5 '-TCAGCCACCTCTTTGTCCTTTTGGAAGTAAGCACTACTTAGTAGTCATCAC-3 '; Fru-F/R joint primer: 5 '-GCCTTGTTCTACCATTACGCGGTAGTTCAGTACCTGTGTTGGACG-3 '/5 '-TCAGCCACCTCTTTGTCCTT TGACATCTAATCCTAAGGTAGGGCG-3 '.
Taking tomato sample DNA to be checked as template, points 3 groups carry out two to (nos+nptII+cry1AC is first group, and 35S+35S-EFE+acc is second group, and fru+apx+mcpi+lat52 is the 3rd group) and take turns multiplex PCR amplification.
First round pcr amplification system is: 2.5 μ l10 × PCR buffer, 2.0 μ l dNTP mixtures (each 2.5mM), 2.0 μ l respectively organize upstream primer mixture (each 10 μ M), 2.0 μ l respectively organize downstream primer mixture (each 10 μ M), 0.125 μ l HS-Taq archaeal dna polymerase, 1 μ 1 genomic dna template, deionized water 15.375 μ 1;
Amplification program is: 94 DEG C of preheating 5min, and 94 DEG C of sex change 30s, 56 DEG C of renaturation 30s, 68 DEG C are extended 35s, circulate 11 times, and 68 DEG C are extended 7min.Second takes turns pcr amplification system is: 2.5 μ l10 × PCR buffer, 1.25 μ l dNTP mixtures (2.0mMdGTP, dATP, dTTP), 2.5 μ l Cy3-dCTP (1mM), 1.0 μ l Head-F (10 μ M), 1.0 μ l Head-R (10 μ M), 0.125 μ l HS-Taq archaeal dna polymerase, 1.0 μ l first round PCR products, deionized water 15.625 μ 1; Amplification program is: 94 DEG C of preheating 5min, and 94 DEG C of sex change 30s, 56 DEG C of renaturation 30s, 68 DEG C are extended 35s, circulate 35 times, and 68 DEG C are extended 7min.After reaction finishes, clean PCR product.The multiple PCR products of three groups of CY3 marks is mixed, and avoid CY3 fluorescent quenching with masking foil parcel ,-80 DEG C save backup.
Method C (random primer labelling), first use restriction enzyme A ccII (FnuDII) to be cut into the fragment differing in size tomato sample DNA to be checked.The enzyme system of cutting is: 10.0 μ l10 × M Buffer, 5.0 μ l AccII, 25.0 μ l genomic dnas, 60.0 μ 1 deionized waters; Enzyme tangent condition is 37 DEG C, 60min; Enzyme cuts into and carries out enzyme with PCR cleaning agents box to cut product clean afterwards, and is dissolved in 50 μ 1TE buffered soln.Then carry out random primer labelling reaction, reaction system is: 5.0 μ l10 × Buffer, 5.0 μ l dNTP mixtures (0.2mM dGTP, dATP, dTTP), 1.0 μ l Cy3-dCTP (1mM), 4.0 μ l9mer random primer 5 ' d (NNN NNN NNN) 3 ', 2.0 μ l Exo-free Klenow Fragment (2U/ μ L), 20.0 μ l DNA enzymes are cut product, 13.0 μ 1 deionized waters.Reaction conditions is 37 DEG C, more than 2 hours; React rear 95 DEG C of heating 3min, fast cold on ice, add 100 μ L dehydrated alcohols, the NaAC of 15 μ L3M ,-20 DEG C of precipitation 3h; 4 DEG C, 8000rpm is centrifugal, and 20min abandons filtrate, and twice (each consumption is 1ml) of 70% washing with alcohol precipitation, is dissolved in 50 μ L TE buffered soln, and after wrapping with masking foil ,-80 DEG C save backup.
5), chip hybridization:
By step 4) CY3 marked product after the purifying of 3 kinds of method gained carries out respectively following operation:
In CY3 marked product after purifying, add isopyknic hybridization buffer (6 × SSPE, 25% methane amide, pH6.6-6.8), after 95 DEG C of sex change 5min, be placed in rapidly precooling 3min on ice; Join again in sample feeding pipe, 30 DEG C, bidirectional circulating hybridization 16h (spending the night), speed 500 μ l/min, make the fully combination (hybridization) of probe on CY3 marked product and chip; After hybridization finishes, with 1ml elution buffer (500 μ l hybridization buffers, 500 μ lH
2o, 20 μ L10%SDS) 32 DEG C of wash cycles 20min (100 μ l/min), then use 40 DEG C of wash cycles 20min of 1ml elution buffer (100 μ l/min); Take out chip, with Microarry Scanner Genepix4000B scanning, scanning resolution is 10 μ m, and wavelength is 635nm, reads scanning result, preserves picture post analysis chip data.
Remarks explanation:
The preparation method of described hybridization buffer is: in 6 × SSPE of every L, add the methane amide of 250ml, and utilize NaOH that concentration is 0.4mol/L and the HCl solution of 0.2mol/L to be adjusted to pH6.6-6.8.
The preparation method of described elution buffer is: in 500 μ l hybridization buffers, add 500 μ lH
2o, then add the SDS solution that the mass concentration of 20 μ L is 10%.
6), judge whether sample is transgenic Fructus Lycopersici esculenti kind:
Obtain fluorescence signal intensity and the standard error of each probe with chip analysis software, signal value, after background noise subduction, is normalized data to same level by the average signal value of adjusting under this scan channel; When chip Quality Control probe hybridization result is correct, we think that crossover process operation is errorless, chip results is credible.For each detection gene, in the time having more than 3 probe signals intensity to exceed 500 and 4 signal errors P<0.01 between repetition, we think that this gene is detected.When one or more tomato kind internal reference genes are detected, promotor, terminator, reporter gene are all undetected, think that this sample is the negative tomato sample of transgenosis.When one or more tomato kind internal reference genes are detected, one or more promotors, terminator, reporter gene are detected simultaneously, think that this sample is transgenic positive tomato sample.
Net result is as shown in Figure 1:
Method A--shows that in regular-PCR marking method, (Fig. 1 a), foreign gene 35s, 35s-EFE, no and nptII are detected, detect probe number and account for respectively 87.1% (54), 53.3% (8), 73.3% (33) and 66.5% (133) for the designed probe number of these genes, front 10 probe average signal strength that each gene detects are respectively 16219,15267,8591 and 13881; Reference gene fru, apx, mcpi and lat52 are detected, detect probe number and account for respectively 20.8% (16), 19.6% (20), 54.2% (13) and 51.9% (28) for the designed probe number of these genes, front 10 probe average signal strength that each gene detects are respectively 10735,10298,4077 and 5313; None probe signals of foreign gene acc and cry1AC detects, and confirms that " Bioscein " do not contain this 2 foreign genes, and chip false drop rate is 0%.
Method B--is multiple-and (Fig. 1 is b) in the method for PCR mark, foreign gene 35s, 35s-EFE, no and nptII are detected, detect probe number and account for respectively 50% (31), 40% (6), 31.1% (14) and 10.5% (21) for the designed probe number of these genes, front 10 probe average signal strength that each gene detects are respectively 3325,2831,1854 and 3573; Reference gene fru, apx, mcpi and lat52 are detected, detect probe number and account for respectively 10.4% (8), 7.8% (8), 37.5% (9) and 42.6% (23) for the designed probe number of these genes, front 10 probe average signal strength that each gene detects are respectively 7189,12846,7699 and 3093; None probe signals of foreign gene acc and cry1AC detects, and confirms that " Bioscein " do not contain this 2 foreign genes, and chip false drop rate is 0%.
In the method for method C--random primer labelling, (Fig. 1 c), foreign gene 35s, 35s-EFE and nptII are detected, detect probe number and account for respectively 12.9% (8), 20% (3) and 6.5% (13) for the designed probe number of these genes, front 10 probe average signal strength that each gene detects are respectively 946,1198 and 1240; Reference gene fru and apx are detected, detect probe number and account for respectively 5.2% (4) and 13.7% (14) for the designed probe number of these genes, front 10 probe average signal strength that each gene detects are respectively 3487 and 5346; None probe signals of foreign gene acc and cry1AC detects, and confirms that " Bioscein " do not contain this 2 foreign genes, and chip false drop rate is 0%.
The above results explanation:
(1) regular-PCR method mark sample hybridization signal is more, and strength of signal is good, is secondly the method for multiple-PCR mark, and method signal value and the number of signals of random primer labelling are far smaller than the above two.
(2) 3 kinds of methods " Bioscein " tomato sample reference gene, foreign gene all detect, and illustrate that it is transgenic positive tomato sample.
Confirmatory experiment 1:
By " Bioscein " tomato sample genomic dna described in embodiment 1, adopt regular-PCR amplification, agarose gel electrophoresis detects amplified band;
Acquired results is: result is identical with embodiment 1, and " Bioscein " is transgenic positive sample.
Contrast experiment 1:
By step 5 in embodiment 1) hybridization time 16h make 8h into, other conditions are constant.The conclusion of final gained is: hybridization signal intensity obviously reduces, using method A) recall rate of foreign gene is 8-22.5%, the recall rate of reference gene is 5-37.5%.
Embodiment 2 is made into the sample to be tested in embodiment 1 " beautiful spring ZN " by " Bioscein ", all the other are all with embodiment 1.
Remarks explanation: step 4) in only adopted method A (regular-PCR) and method B (multiplex PCR).
Net result is as shown in Figure 2:
Method A--shows that in regular-PCR marking method, (Fig. 2 a), foreign gene 35s, acc, no and nptII are detected, detect probe number and account for respectively 79% (49), 32% (16), 66.7% (30) and 43% (86) for the designed probe number of these genes, front 10 probe average signal strength that each gene detects are respectively 20081,19509,7382 and 13305; Reference gene fru, apx, mcpi and lat52 are detected, detect probe number and account for respectively 16.9% (13), 19.6% (20), 54.2% (13) and 51.9% (28) for the designed probe number of these genes, front 10 probe average signal strength that each gene detects are respectively 6186,11339,4754 and 3727; Foreign gene 35s-EFE and cry1AC without detecting, confirm " beautiful spring ZN " do not contain this 2 foreign genes, chip false drop rate is 0%.
Method B--is multiple-and (Fig. 2 is b) in the method for PCR mark, foreign gene 35s, acc, no and nptII are detected, detect probe number and account for respectively 77.4% (48), 46% (23), 51.1% (23) and 29.5% (59) for the designed probe number of these genes, front 10 probe average signal strength that each gene detects are respectively 11301,10888,8280 and 15696; Reference gene fru, apx, mcpi and lat52 are detected, detect probe number and account for respectively 16.9% (13), 16.7% (17), 54.2% (13) and 51.9% (28) for the designed probe number of these genes, front 10 probe average signal strength that each gene detects are respectively 9762,11002,3260 and 16458; Foreign gene 35s-EFE and cry1AC without detecting, confirm " beautiful spring ZN " do not contain this 2 foreign genes, chip false drop rate is 0%.
The above results explanation:
(1) regular-PCR, multiplex PCR marking method affect not quite " beautiful spring ZN " tomato sample chip detected result, the two hybridization signal band, and strength of signal is suitable.
(2) 2 kinds of methods " beautiful spring ZN " tomato sample reference gene, foreign gene all detect, and illustrate that it is transgenic positive sample.
Confirmatory experiment 2:
By " beautiful spring ZN " the tomato sample genomic dna described in embodiment 2, adopt regular-PCR amplification, agarose gel electrophoresis detects amplified band;
Acquired results is: result is identical with embodiment 2, and " beautiful spring ZN " is transgenic positive sample.
Embodiment 3 is made into the sample to be tested in embodiment 1 " cooperation 903 " by " Bioscein ", all the other are all with embodiment 1.
Remarks explanation: step 4) in only adopted method A (regular-PCR).
Net result is as shown in Figure 3: reference gene fru, apx, mcpi and lat52 are all detected, detect probe number and account for respectively 16.9% (13), 15.6% (16), 50% (12) and 42.6% (23) for the designed probe number of these genes, front 10 probe average signal strength that each gene detects are respectively 5372,8490,4517 and 3102; Foreign gene, without detecting, illustrates that " cooperation 903 " tomato sample is transgenosis negative sample.
Confirmatory experiment 3:
By " cooperation 903 " the tomato sample genomic dna described in embodiment 3, adopt regular-PCR amplification, agarose gel electrophoresis detects amplified band;
Acquired results is: result is identical with embodiment 3, and tomato reference gene has amplified band, but foreign gene is without amplified band, illustrates that " cooperation 903 " is for transgenosis negative sample.
Finally, it is also to be noted that, what more than enumerate is only several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, can also have many distortion.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention, all should think protection scope of the present invention.
Claims (6)
1. utilize the synthetic micro-fluid chip of original position to detect the method for transgenic Fructus Lycopersici esculenti, it is characterized in that comprising the following steps:
1) design of the chip probe that, transgenic Fructus Lycopersici esculenti foreign gene detects; 895 oligonucleotide probes as described in Table 1 have been synthesized in design;
2), prepare oligonucleotide microarray;
3), the extraction of genomic dna in tomato sample to be measured;
4), the specific fragment of genome or whole genome fluorescent mark;
5), chip hybridization;
6), judge whether sample is transgenic Fructus Lycopersici esculenti kind.
2. the method for utilizing the synthetic micro-fluid chip of original position to detect transgenic Fructus Lycopersici esculenti according to claim 1, is characterized in that described step 2) be:
Prepare oligonucleotide microarray: first synthesize in position on micro-fluid chip 895 of synthetic chemistry modified sequence oligonucleotide probes successively; Then synthetic chip is carried out on chip scanner fluorometric analysis, with the fluorescence background of detection chip itself; Carry out, after fluorescent mark, hybridizing with chip again analysis chip specificity, sensitivity and repeatability, assessment chip quality with Quality Control cDNA or the DNA fragmentation that comes from plasmid.
3. the method for utilizing the synthetic micro-fluid chip of original position to detect transgenic Fructus Lycopersici esculenti according to claim 2, is characterized in that described step 3) be the extraction of genomic dna in tomato sample to be measured:
Get 0.1-0.2g tomato sample, liquid nitrogen grinding, packs in the aseptic eppendorf pipe of 1.5ml; Add 1ml CTAB extracting solution, in 65 DEG C of water-bath 60-90min; Centrifuging and taking supernatant, adds phenol/chloroform/primary isoamyl alcohol, and volume ratio is 25:24:1, and amounting to consumption is 2/3 of supernatant volume, fully mixes centrifuging and taking supernatant; Add chloroform/primary isoamyl alcohol, centrifuging and taking supernatant; Add NaAC solution and dehydrated alcohol precipitation; The centrifugal supernatant of abandoning, the alcohol washing precipitation that volumetric concentration is 70%; The centrifugal supernatant of abandoning, vacuum-drying; Add 50-100 μ 1TE solution dissolving DNA.
4. the method for utilizing the synthetic micro-fluid chip of original position to detect transgenic Fructus Lycopersici esculenti according to claim 3, is characterized in that described step 4) be the specific fragment of genome or whole genome fluorescent mark, any one in the following 3 kinds of methods of optional use:
Method A, taking tomato sample DNA to be checked as template, design and filter out the primer of promotor conventional in transgenic Fructus Lycopersici esculenti, terminator, reporter gene and tomato kind internal reference gene, carry out PCR reaction, and it carried out to CY3 fluorescent mark; In PCR reaction system, replace common dCTP with the dCTP of CY3 mark; Concrete primer information: 35s-F/R:5 '-GCTCCTACAAATGCCATCA-3 '/5 '-GATAGTGGGATTGTGCGTCA-3 '; Nos-F/R:5 '-GAATCCTGTTGCCGGTCTTG-3 '/5 '-TTATCCTAGTTTGCGCGCTA-3 '; 35s-EFE-F/R:5 '-TGGGATCTAAGCACTTGCAATTGGA-3 '/5 '-GCACAAACAGACGGGACACGAAT-3 '; NptII-F/R:5 '-GAAAAATACCGCTGCGTAAAAG-3 '/5 '-TCAGGGCTTTGTTCATCTTCAT-3 '; Acc-F/R:5 '-TTTTGCCTCGCTGATCCTGGC-3 '/5 '-GGTTTCGCCTTTCGGCTTCTGT-3 '; Cry1AC-F/R:5 '-TCATCCCTTACGTCAGTGGAG-3 '/5 '-CCATCATTGCGATAAAGGAAA-3 '; Lat52-F/R:5 '-ATGCAGGAACATCAGCACAGAGGC-3 '/5 '-TCTTTGCAGTCCTCCCTTGGGCTT-3 '; Mcpi-F/R:5 '-TGGCACAAGATGCAACTCTGACGA-3 '/5 '-ACAGGCCTGACAGAACGTACCA-3 '; Apx-F/R:5 '-CTCTTGGAGCCCATTAGGGAGCA-3 '/5 '-TTGGAAGTAAGCACTACTTAGTAGTCATCAC-3 '; Fru-F/R:5 '-GGTAGTTCAGTACCTGTGTTGGACG-3 '/5 '-TGACATCTAATCCTAAGGTAGGGCG-3 ';
Method B, insert joint Head-F at 5 ' end of method A forward primer used, sequence is GCCTTGTTCTACCATTACGC, and reverse primer 5 ' end inserts joint Head-R, and sequence is TCAGCCACCTCTTTGTCCTT; Taking tomato sample DNA to be checked as template, carry out first round multiplex PCR amplification by the combination of primers of belt lacing, amplification system is after totally 11 circulations; Then taking the PCR product of the first round as template, taking the general header sequence Head-F/Head-R that adds as primer, carry out second and take turns multi-PRC reaction; In PCR reaction system, replace common dCTP with the dCTP of CY3 mark;
Method C, by tomato sample DNA to be checked first with restriction enzyme A ccII digestion, enzyme tangent condition is 37 DEG C, 60min; Enzyme cuts into and carries out random primer labelling reaction with Random Primer DNA Labeling Kit Ver.2.0 afterwards; In reaction system, replace common dCTP with the dCTP of CY3 mark.
5. a kind of method of utilizing the synthetic micro-fluid chip of original position to detect transgenic Fructus Lycopersici esculenti according to claim 4, is characterized in that described step 5) chip hybridization is:
CY3 marked product after purifying is added to isopyknic hybridization buffer, and described hybridization buffer is 6 × SSPE, 25% methane amide, pH6.6-6.8; 95 DEG C of sex change 5min are placed on precooling 3min on ice; Join again in sample feeding pipe, 30 DEG C, bidirectional circulating hybridization 16h, speed 500 μ l/min; After hybridization finishes, with 32 DEG C of wash cycles 20min of 1ml elution buffer, elution buffer is by 500 μ l hybridization buffers, 500 μ lH
2o, 20 μ L10%SDS compositions; Use again 40 DEG C of wash cycles 20min of 1ml elution buffer; Take out chip, with Microarry Scanner Genepix4000B scanning, scanning resolution is 10 μ m, and wavelength is 635nm, reads scanning result, preserves picture post analysis chip data.
6. the method for utilizing the synthetic micro-fluid chip of original position to detect transgenic Fructus Lycopersici esculenti according to claim 5, is characterized in that described step 6) for to judge whether sample is transgenic Fructus Lycopersici esculenti kind:
Obtain fluorescence signal intensity and the standard error of each probe with chip analysis software, signal value, after background noise subduction, is normalized data to same level by the average signal value of adjusting under this scan channel; Last according to the signal value of promotor, terminator, reporter gene, tomato kind internal reference gene and chip Quality Control probe, the confidence level of analysis chip results of hybridization, judges whether this sample belongs to transgenic positive tomato sample; Judging criterion is: if reference gene is detected, foreign gene is detected, and explanation is tomato transgenic positive sample; If reference gene is detected, foreign gene does not detect, and explanation is non-transgenic sample.
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