CN104480218A - Method for authenticating homozygotic type or heterozygous type MON810 on basis of digital PCR - Google Patents
Method for authenticating homozygotic type or heterozygous type MON810 on basis of digital PCR Download PDFInfo
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Abstract
The invention discloses a method for authenticating the homozygotic type or heterozygous type MON810 on the basis of a digital PCR, and relates to the technical field of molecular biology and transgenic tests. The method includes the steps of firstly, conducting collection, wherein the MON810 specificity conversion event verified through the European Union and the corn endogenous reference gene hmgA quantitative detection method are adopted; secondly, conducting digital PCR amplification, wherein the DNA of the MON810 serves as the research template; thirdly, conducting data analysis, wherein if the 95% confidence interval of the MON810 specificity conversion event and the 95% confidence interval of the corn endogenous reference gene hmgA target molecular number value overlap, the MON810 is judged to be of the homozygotic type, and if the area two times as large as the 95% confidence interval of the MON810 specificity conversion event target molecular number value and the 95% confidence interval of the corn endogenous reference gene hmgA target molecular number value overlap, the MON810 is judged to be of the heterozygous type. The method can rapidly, accurately and reliably authenticate the absolute quantification of the homozygotic type insect-resistant transgenic maize strain MON810 and the heterozygous type insect-resistant transgenic maize strain MON810.
Description
Technical field
The present invention relates to molecular biology and detection GMOs technical field, particularly relate to a kind of method based on the homozygous or heterozygous MON810 of digital pcr qualification.
Background technology
In recent years, the genetically modified crops such as corn, soybean, cotton, rape and tomato have been got permission plantation in a lot of country and have produced, by the end of 2013, there are 27 kinds of crops to relate to 336 transformant in world wide and got permission plantation 36 different countries and regions or as processing raw material, cultivated area has reached 1.75 hundred million hectares, wherein the area of 27% plants the gene stacking kind (James, 2013, International Agriculture biotechnology applications Servers Organization) comprising multiple transformation event.Along with the continuous increase of transgenic event, bring increasing challenge to the supervision of various countries' GMO bio-safety.
GMO detection reference material is the important substance basis of genetically modified organism and products thereof composition detection, in the program and link process of preparation transgenosis reference material, the qualification of starting material homozygosity is the key link of preparation standard material, current American Oil chemist association (AOCS) and EU criteria material and tolerance institute (IRMM) are identified and the method for homozygosity are mainly adopted the method for traditional breeding mode or real-time fluorescence quantitative PCR (Li Yun waits quietly, 2013, Scientia Agricultura Sinica).Meanwhile, research finds to adopt traditional breeding method mode, and need by inbreeding of more generation for many years, could obtain homozygous transgenic material, length consuming time, wastes time and energy; And the error of real time fluorescence quantifying PCR method own is large, when carrying out homozygosity qualification to genetically modified crops, application condition is large, judges to bring very large difficulty to result.
Digital pcr (digital polymerase chain reaction, dPCR), as the quantitative new technology of DNA, achieves unique DNA absolute quantitation.Research at present in clinical diagnosis, quantitative, the unicellular genetic expression of transgene component, environmental microorganism detection and order-checking of future generation etc. has played vital role (Li Liang etc., 2012, Progress in Biochemistry and Biophysics).The external source of digital pcr technical Analysis transgenic insect-resistant corn MON810 seed powder that adopts Corbisier etc. detect gene and
hmgthe copy number of A gene, finds the copy number ratio absolute quantitation result adopting digital pcr to detect, with adopt the method for real-time fluorescence quantitative PCR and utilize plasmid DNA to do result ratio that reference material carries out relative quantification to seed powder is consistent.Therefore proposing digital pcr and there is meter characteristic, can be used for measuring the DNA copy number ratio of transgenosis relevant criterion material. unit molecule amplification efficiency is for the estimated bias significance of the copy number and short-movie section global DNA that improve STb gene fragment.Target dna molecule is the key of digital pcr accurate quantitative analysis in the Stochastic sum independent distribution of reaction chamber, Bhat etc. think, the capacity of reaction chamber is the main source of uncertainty, and the relative uncertainty degree of evaluation is below 6%, this discovery may be used for the confidence level research that other digital pcrs are measured.
We wish by utilizing digital pcr technology, set up a kind of method identifying the pest-resistant corn strain MON810 of homozygous or heterozygous transgenic, and then lay the foundation for preparation standard material or raw-material qualification breeding work, ensure the true and reliable of material Background.
Summary of the invention
Object of the present invention is just the difficulty overcoming existing employing traditional breeding method and the existence of application real time fluorescence quantifying PCR method qualification transgenic line homozygosity, there is provided a kind of and identify method that is homozygous or heterozygous MON810 based on digital pcr, described MON810 is transgenic insect-resistant corn kind.
The object of the present invention is achieved like this:
One, method that is homozygous or heterozygous MON810 is identified based on digital pcr
1. collect
European Union is adopted to pass through MON810 specificity transformation event and the corn internal standard gene hmgA quantitative detecting method of checking,
MON810 primer sequence:
MON810F：5’-TCGAAGGACGAAGGACTCTAACGT-3’，
MON810R：5’-GCCACCTTCCTTTTCCACTATCTT-3’；
MON810 probe sequence:
MON810P：5’-FAM-AACATCCTTTGCCATTGCCCAGC-TAMRA-3’；
Internal standard gene hmgA primer sequence:
hmgAF：5’-TTGGACTAGAAATCTCGTGCTGA-3’，
hmgAR：5’-GCTACATAGGGAGCCTTGTCCT-3’；
Internal standard gene hmgA probe sequence:
hmgAP：5’-FAM-CAATCCACACAAACGCACGCGTA-TAMRA-3’；
2. digital pcr amplification
With the DNA of MON810 for research template, above-mentioned primed probe is utilized to be combined into line number word pcr amplification respectively, MON810 is diluted with water to the concentration of 5ng/ μ l or 8ng/ μ l, the digit chip of 48.770 carries out digital pcr amplification to two parameters respectively, and collects target molecule fluorescent signal;
3. data analysis
Utilize digital pcr instrument data analysis software to add up detection data, calculate 95% fiducial interval of MON810 specificity transformation event and corn internal standard gene hmgA target molecule number calculated value respectively; If 95% fiducial interval of specificity transformation event and corn internal standard gene hmgA target molecule number calculated value has overlap in MON810 strain, be judged to be homozygous MON810; If two times of 95% fiducial interval of specificity transformation event target molecule number calculated value have overlap with 95% fiducial interval of corn internal standard gene hmgA target molecule number calculated value in MON810 strain, be then judged to be heterozygous MON810.
Working mechanism:
Present method utilizes digital pcr technology to carry out the basic functional principle of absolute quantitation to unique DNA, adopt the microfluidic methods of the popular research field of present analysis chemistry, be dispersed in the microreactor of chip by the nucleic acid solution after Macrodilution, the nucleic acid-templated number of each reactor is less than or equals 1.Like this after PCR circulation, there is the reactor of a nucleic acid templates to provide fluorescent signal, do not have the reactor of template just there is no fluorescent signal.According to the volume of relative proportion and reactor, the nucleic acid concentration of original solution just can be extrapolated.The foreign gene of genetically modified crops and internal standard gene are independently increased respectively, according to the target fluorescent molecule number collected, counting statistics foreign gene and internal standard gene target molecule number respectively, and whether 95% fiducial interval of specificity transformation event and internal standard gene target molecule number calculated value has overlap, identifies the homozygosity of genetically modified crops.
Two, apply
When template amount is 6ng and 9.6ng, present method can go out the homozygous or pest-resistant corn strain MON810 of heterozygous transgenic by successful identification.
Compared with art methods, the present invention has following advantages and positively effect:
1. provide first and utilize digital pcr technology to identify the method for transgenic insect-resistant corn strain MON810 homozygosity;
2. avoid and utilize traditional breeding way to screen problem that is homozygous or heterozygous transgenic pest-resistant corn strain MON810 length consuming time;
3. avoid utilize real time fluorescence quantifying PCR method to identify homozygous or heterozygous transgenic pest-resistant corn strain MON810 error is large, the problem of result difficult judgment.
In a word, the present invention is a kind of method fast, accurately and reliably identifying homozygous or heterozygous transgenic pest-resistant corn strain MON810 absolute quantitation.
Accompanying drawing explanation
The homozygous MON810 of Fig. 1 to be template amount be 6ng, the amplification curve of corn internal standard gene hmgA on microfluid panel, repeats 1;
The homozygous MON810 of Fig. 2 to be template amount be 6ng, the amplification curve of corn internal standard gene hmgA on microfluid panel, repeats 2;
The homozygous MON810 of Fig. 3 to be template amount be 6ng, the amplification curve of corn internal standard gene hmgA on microfluid panel, repeats 3;
The homozygous MON810 of Fig. 4 to be template amount be 6ng, the amplification curve of corn internal standard gene hmgA on microfluid panel, repeats 4;
The homozygous MON810 of Fig. 5 to be template amount be 6ng, the amplification curve of corn internal standard gene hmgA on microfluid panel, repeats 5;
The homozygous MON810 of Fig. 6 to be template amount be 6ng, the amplification curve of corn internal standard gene hmgA on microfluid panel, repeats 6;
The amplification curve of homozygous MON810, MON810 specificity transformation event on microfluid panel of Fig. 7 to be template amount be 6ng, repeats 1;
The amplification curve of homozygous MON810, MON810 specificity transformation event on microfluid panel of Fig. 8 to be template amount be 6ng, repeats 2;
The amplification curve of homozygous MON810, MON810 specificity transformation event on microfluid panel of Fig. 9 to be template amount be 6ng, repeats 3;
The amplification curve of homozygous MON810, MON810 specificity transformation event on microfluid panel of Figure 10 to be template amount be 6ng, repeats 4;
The amplification curve of homozygous MON810, MON810 specificity transformation event on microfluid panel of Figure 11 to be template amount be 6ng, repeats 5;
The amplification curve of homozygous MON810, MON810 specificity transformation event on microfluid panel of Figure 12 to be template amount be 6ng, repeats 6;
The homozygous MON810 of Figure 13 to be template amount be 9.6ng, the amplification curve of corn internal standard gene hmgA on microfluid panel, repeats 1;
The homozygous MON810 of Figure 14 to be template amount be 9.6ng, the amplification curve of corn internal standard gene hmgA on microfluid panel, repeats 2;
The MON810 that isozygotys of Figure 15 to be template amount be 9.6ng, the amplification curve of corn internal standard gene hmgA on microfluid panel, repeats 3;
The homozygous MON810 of Figure 16 to be template amount be 9.6ng, the amplification curve of corn internal standard gene hmgA on microfluid panel, repeats 4;
The homozygous MON810 of Figure 17 to be template amount be 9.6ng, the amplification curve of corn internal standard gene hmgA on microfluid panel, repeats 5;
The homozygous MON810 of Figure 18 to be template amount be 9.6ng, the amplification curve of corn internal standard gene hmgA on microfluid panel, repeats 6;
The amplification curve of homozygous MON810, MON810 specificity transformation event on microfluid panel of Figure 19 to be template amount be 9.6ng, repeats 1;
The amplification curve of homozygous MON810, MON810 specificity transformation event on microfluid panel of Figure 20 to be template amount be 9.6ng, repeats 2;
The amplification curve of homozygous MON810, MON810 specificity transformation event on microfluid panel of Figure 21 to be template amount be 9.6ng, repeats 3;
The amplification curve of homozygous MON810, MON810 specificity transformation event on microfluid panel of Figure 22 to be template amount be 9.6ng, repeats 4;
The amplification curve of homozygous MON810, MON810 specificity transformation event on microfluid panel of Figure 23 to be template amount be 9.6ng, repeats 5;
The amplification curve of homozygous MON810, MON810 specificity transformation event on microfluid panel of Figure 24 to be template amount be 9.6ng, repeats 6;
The heterozygous MON810 of Figure 25 to be template amount be 6ng, the amplification curve of corn internal standard gene hmgA on microfluid panel, repeats 1;
The heterozygous MON810 of Figure 26 to be template amount be 6ng, the amplification curve of corn internal standard gene hmgA on microfluid panel, repeats 2;
The heterozygosis MON810 of Figure 27 to be template amount be 6ng, the amplification curve of corn internal standard gene hmgA on microfluid panel, repeats 3;
The heterozygous MON810 of Figure 28 to be template amount be 6ng, the amplification curve of corn internal standard gene hmgA on microfluid panel, repeats 4;
The heterozygous MON810 of Figure 29 to be template amount be 6ng, the amplification curve of corn internal standard gene hmgA on microfluid panel, repeats 5;
The heterozygous MON810 of Figure 30 to be template amount be 6ng, the amplification curve of corn internal standard gene hmgA on microfluid panel, repeats 6;
The heterozygous MON810 of Figure 31 to be template amount be 6ng, the amplification curve of MON810 specificity transformation event on microfluid panel, repeats 1;
The heterozygous MON810 of Figure 32 to be template amount be 6ng, the amplification curve of MON810 specificity transformation event on microfluid panel, repeats 2;
The heterozygous MON810 of Figure 33 to be template amount be 6ng, the amplification curve of MON810 specificity transformation event on microfluid panel, repeats 3;
The heterozygous MON810 of Figure 34 to be template amount be 6ng, the amplification curve of MON810 specificity transformation event on microfluid panel, repeats 4;
The heterozygous MON810 of Figure 35 to be template amount be 6ng, the amplification curve of MON810 specificity transformation event on microfluid panel, repeats 5;
The heterozygous MON810 of Figure 36 to be template amount be 6ng, the amplification curve of MON810 specificity transformation event on microfluid panel, repeats 6;
The heterozygous MON810 of Figure 37 to be template amount be 9.6ng, the amplification curve of corn internal standard gene hmgA on microfluid panel, repeats 1;
The heterozygous MON810 of Figure 38 to be template amount be 9.6ng, the amplification curve of corn internal standard gene hmgA on microfluid panel, repeats 2;
The heterozygous MON810 of Figure 39 to be template amount be 9.6ng, the amplification curve of corn internal standard gene hmgA on microfluid panel, repeats 3;
The heterozygous MON810 of Figure 40 to be template amount be 9.6ng, the amplification curve of corn internal standard gene hmgA on microfluid panel, repeats 4;
The heterozygous MON810 of Figure 41 to be template amount be 9.6ng, the amplification curve of corn internal standard gene hmgA on microfluid panel, repeats 5;
The heterozygous MON810 of Figure 42 to be template amount be 9.6ng, the amplification curve of corn internal standard gene hmgA on microfluid panel, repeats 6;
The heterozygous MON810 of Figure 43 to be template amount be 9.6ng, the amplification curve of MON810 specificity transformation event on microfluid panel, repeats 1;
The heterozygous MON810 of Figure 44 to be template amount be 9.6ng, the amplification curve of MON810 specificity transformation event on microfluid panel, repeats 2;
The heterozygous MON810 of Figure 45 to be template amount be 9.6ng, the amplification curve of MON810 specificity transformation event on microfluid panel, repeats 3;
The heterozygous MON810 of Figure 46 to be template amount be 9.6ng, the amplification curve of MON810 specificity transformation event on microfluid panel, repeats 4;
The heterozygous MON810 of Figure 47 to be template amount be 9.6ng, the amplification curve of MON810 specificity transformation event on microfluid panel, repeats 5;
The heterozygous MON810 of Figure 48 to be template amount be 9.6ng, the amplification curve of MON810 specificity transformation event on microfluid panel, repeats 6;
Figure 49 is digital pcr reaction thermal map.
Embodiment
Describe in detail below in conjunction with drawings and Examples:
1, CTAB method is adopted to extract the genomic dna of transgenic corns MON810
1) first freezing more than the 10h at-20 DEG C by mortar and pestle, heats 100 DEG C by 1.5 × CTAB extracting solution simultaneously, 10%CTAB is preheating to 56 DEG C; The fresh blade of transgenic corns MON810 of liquid nitrogen grinding 8.0 about g, by powder transfer in the centrifuge tube of 50ml; In centrifuge tube, add 1.5 × CTAB extracting solution of 20 ml preheatings, after stirring rapidly, be put into 56 DEG C of water-bath 40 min with glass stick, period puts upside down evenly frequently gently; After water-bath, be cooled to room temperature; Adding 20 ml chloroform/primary isoamyl alcohol (24:1), add a cover, putting upside down evenly to forming milkiness shape, centrifugal 20 min of 4000 r/min under room temperature; Transfer supernatant, in 50 new ml centrifuge tubes, adds the 10%CTAB solution of 1/10 volume, 56 DEG C of preheatings, then adds equal-volume chloroform/primary isoamyl alcohol (24:1), puts upside down centrifugal 20 min of mixing 30min, 4000 r/min; Transfer supernatant, in new 50ml centrifuge tube, adds equal-volume 1%CTAB lye, and be mixed to gently and form DNA floss, 3000 r/min make DNA be sunken at the bottom of pipe in centrifugal 10 minutes; Abandoning supernatant, tips upside down on centrifuge tube on thieving paper that several minutes is with air-dry precipitation, and add 5 ml 1 M NaCl solution and 5 μ l RNase, 56 DEG C of water-bath dissolving DNAs, spend the night; Add 95% ethanol of 10 ml precoolings, precipitated dna, DNA is ticked to be put in 76% ethanol and soaks 30min; 5 min in transfer DNA to 95% ethanol, centrifugal remove 95% ethanol, vacuum-drying; After drying, use 1mlTE dissolving DNA.
2) Nanodrop 2000 ultramicrospectrophotometer is adopted to carry out concentration determination to the sample extracted, with the λ DNA of 100ng/ μ l as reference, by the concentration of the concentration quantitative of homozygous and heterozygous transgenic corn MON810 to 100ng/ μ l, the light absorption value measured value of 260/280 is respectively 1.84 and 1.83, for subsequent use.
2, digital pcr detects
1) have chosen the specificity through the MON810 transformant of European Union's checking and internal standard gene hmgA quantitative detecting method, wherein MON810 specificity transformation event amplified fragments size is 92bp, the amplified fragments size of internal standard gene hmgA is 79bp, MON810 primer sequence:
MON810F：5’-TCGAAGGACGAAGGACTCTAACGT-3’，
MON810R：5’-GCCACCTTCCTTTTCCACTATCTT-3’；
MON810 probe sequence:
MON810P：5’-FAM-AACATCCTTTGCCATTGCCCAGC-TAMRA-3’；
Internal standard gene hmgA primer sequence:
hmgAF：5’-TTGGACTAGAAATCTCGTGCTGA-3’，
hmgAR：5’-GCTACATAGGGAGCCTTGTCCT-3’；
Internal standard gene hmgA probe sequence:
hmgAP：5’-FAM-CAATCCACACAAACGCACGCGTA-TAMRA-3’；
2) digital pcr analysis is a BioMark HD System (Fluidigm, BioMark HD, USA)
On carry out, adopt 48.770 digit chips, detect and signal collect software be BioMark Data Collection, analysis software is Fluidigm Digital PCR analysis.
Digital pcr reaction volume is 4 μ l, wherein DNA profiling amount is 6ng or 9.6ng, other component concentrations are: TaqMan Gene Expression Master Mix (Applied Biosystem, PN 4369016) 2 μ l, 20 × GE Sample Loading Reagent (Fluidigm, PN 85000746) 0.4 μ l, 20 × gene-specific assays 0.2 μ l, DNA-free water 0.2 μ l, DNA solution amount is 1.2 μ l.
Digital pcr response procedures is: 50 ° of C UNG process 120 seconds, 95 ° of C denaturations 600 seconds, carries out 40 PCR circulations: 95 ° of C sex change 15 seconds, 60 ° of C annealing in 1 minute and extending, and collects fluorescent signal.
With water by transgenic corns MON810 genomic dna gradient dilution to 5ng/ μ l and 8ng/ μ l two concentration, with 1.2 μ L genomic dnas for template, carry out digital pcr reaction, each concentration each parameter reaction repetition 6 times.
3, experimental result
The method provided in the present invention is utilized to carry out Analysis and Identification to homozygous or heterozygous MON810.
The homozygous MON810 being 6ng with DNA amount is for template, the primer of corn internal standard gene hmgA and MON810 specificity of transformant event and probe is adopted to carry out digital pcr amplified reaction respectively, internal standard gene hmgA amplification curve diagram is as Fig. 1 ~ 6, thermal map reaction is as the Panel01-S01 in Figure 49, Panel02-S02, Panel03-S03, Panel07-S07, Panel08-S08, Panel09-S09, all can observe amplification curve on each microfluid panel intact, Ct value concentrates between 20-35, and between the 363-391 each panel having the quantity of the reaction chamber of signal concentrate on, mean value is 381.5, the target molecule number mean value detected is 527.2, as table 1, MON810 specificity of transformant event amplification curve is as Fig. 7 ~ 12, thermal map reaction is as Panel04-S04, Panel05-S05, Panel06-S06, Panel10-S10, Panel11-S11, the Panel12-S12 in Figure 49, all can observe amplification curve on each microfluid panel intact, Ct value concentrates between 20-35, and between the 340-368 each panel having the quantity of the reaction chamber of signal concentrate on, mean value is 353.8, the target molecule number mean value detected is 474.3, as table 1.95% fiducial interval of MON810 specificity transformation event and corn internal standard gene hmgA target molecule number calculated value has overlap, and result meets genetic development, can be judged to be the pest-resistant corn MON810 of homozygous transgenic.
The homozygous MON810 being 9.6ng with DNA amount is for template, the primer of corn internal standard gene hmgA and MON810 specificity of transformant event and probe is adopted to carry out digital pcr amplified reaction respectively, internal standard gene hmgA amplification curve diagram is as Figure 13 ~ 18, thermal map reaction is as the Panel13-S13 in Figure 49, Panel14-S14, Panel15-S15, Panel19-S19, Panel20-S20, Panel21-S21, all can observe amplification curve on each microfluid panel intact, Ct value concentrates between 20-35, and between the 476-495 each panel having the quantity of the reaction chamber of signal concentrate on, mean value is 484.2, the target molecule number mean value detected is 764.0, as table 1, MON810 specificity of transformant event amplification curve is as Figure 19 ~ 24, thermal map reaction is as Panel16-S16, Panel17-S17, Panel18-S18, Panel22-S22, Panel23-S23, the Panel24-S24 in Figure 49, all can observe amplification curve on each microfluid panel intact, Ct value concentrates between 20-35, and between the 456-483 each panel having the quantity of the reaction chamber of signal concentrate on, mean value is 468.7, the target molecule number mean value detected is 723.5, as table 1.95% fiducial interval of MON810 specificity transformation event and corn internal standard gene hmgA target molecule number calculated value has overlap, and result meets genetic development, can be judged to be the pest-resistant corn MON810 of homozygous transgenic.
The heterozygous MON810 being 6ng with DNA amount is template, the primer of corn internal standard gene hmgA and MON810 specificity of transformant event and probe is adopted to carry out digital pcr amplified reaction respectively, internal standard gene hmgA amplification curve diagram is as Figure 25 ~ 30, thermal map reaction is as the Panel25-S25 in Figure 49, Panel26-S26, Panel27-S27, Panel31-S31, Panel32-S32, Panel33-S33, all can observe amplification curve on each microfluid panel intact, Ct value concentrates between 20-35, and between the 338-372 each panel having the quantity of the reaction chamber of signal concentrate on, mean value is 347.7, the target molecule number mean value detected is 463.2, as table 1, MON810 specificity of transformant event amplification curve is as Fig. 7 ~ 12, thermal map reaction is as Panel28-S28, Panel29-S29, Panel30-S30, Panel34-S34, Panel35-S35, the Panel36-S36 in Figure 49, all can observe amplification curve on each microfluid panel intact, Ct value concentrates between 20-35, and between the 194-214 each panel having the quantity of the reaction chamber of signal concentrate on, mean value is 200.3, the target molecule number mean value detected is 232.3, as table 1.Two times of 95% fiducial interval of MON810 specificity transformation event target molecule number calculated value have overlap with 95% fiducial interval of corn internal standard gene hmgA target molecule number calculated value, result meets genetic development, can be judged to be the pest-resistant corn MON810 of heterozygous transgenic.
The heterozygous MON810 being 9.6ng with DNA amount is template, the primer of corn internal standard gene hmgA and MON810 specificity of transformant event and probe is adopted to carry out digital pcr amplified reaction respectively, internal standard gene hmgA amplification curve diagram is as Figure 37 ~ 42, thermal map reaction is as the Panel37-S37 in Figure 49, Panel38-S38, Panel39-S39, Panel43-S43, Panel44-S44, Panel45-S45, all can observe amplification curve on each microfluid panel intact, Ct value concentrates between 20-35, and between the 475-501 each panel having the quantity of the reaction chamber of signal concentrate on, mean value is 489.3, the target molecule number mean value detected is 778.7, as table 1, MON810 specificity of transformant event amplification curve is as Figure 43 ~ 48, thermal map reaction is as Panel40-S40, Panel41-S41, Panel42-S42, Panel46-S46, Panel47-S47, the Panel48-S48 in Figure 49, all can observe amplification curve on each microfluid panel intact, Ct value concentrates between 20-35, and between the 285-358 each panel having the quantity of the reaction chamber of signal concentrate on, mean value is 318.3, the target molecule number mean value detected is 412.3, as table 1.Two times of 95% fiducial interval of MON810 specificity transformation event target molecule number calculated value have overlap with 95% fiducial interval of corn internal standard gene hmgA target molecule number calculated value, result meets genetic development, can be judged to be the pest-resistant corn MON810 of heterozygous transgenic.
Above result can be found out, the present invention is that the qualification of transgenic insect-resistant corn strain MON810 homozygosity provides a kind of accurate, reliable absolute quantitation measuring method detected based on digital pcr, can identify transgenic insect-resistant corn strain MON810 that is homozygous or heterozygous.The present invention is that the qualification of transformed variety and control provide necessary means.
The statistical analysis table of table 1 corn internal standard gene hmgA and MON810 specificity transformation event target molecule number
Note: each data are 6 mean values repeated.
Claims (1)
1. identify a method that is homozygous or heterozygous MON810 based on digital pcr, it is characterized in that:
1. collect
European Union is adopted to pass through MON810 specificity transformation event and the corn internal standard gene hmgA quantitative detecting method of checking,
MON810 primer sequence:
MON810F：5’-TCGAAGGACGAAGGACTCTAACGT-3’，
MON810R：5’-GCCACCTTCCTTTTCCACTATCTT-3’；
MON810 probe sequence:
MON810P：5’-FAM-AACATCCTTTGCCATTGCCCAGC-TAMRA-3’；
Internal standard gene hmgA primer sequence:
hmgAF：5’-TTGGACTAGAAATCTCGTGCTGA-3’，
hmgAR：5’-GCTACATAGGGAGCCTTGTCCT-3’；
Internal standard gene hmgA probe sequence:
hmgAP：5’-FAM-CAATCCACACAAACGCACGCGTA-TAMRA-3’；
2. digital pcr amplification
With the DNA of MON810 for research template, above-mentioned primed probe is utilized to be combined into line number word pcr amplification respectively, MON810 is diluted with water to the concentration of 5ng/ μ l or 8ng/ μ l, the digit chip of 48.770 carries out digital pcr amplification to two parameters respectively, and collects target molecule fluorescent signal;
3. data analysis
Utilize digital pcr instrument data analysis software to add up detection data, calculate 95% fiducial interval of MON810 specificity transformation event and corn internal standard gene hmgA target molecule number calculated value respectively; If 95% fiducial interval of specificity transformation event and corn internal standard gene hmgA target molecule number calculated value has overlap in MON810 strain, be judged to be homozygous MON810; If two times of 95% fiducial interval of specificity transformation event target molecule number calculated value have overlap with 95% fiducial interval of corn internal standard gene hmgA target molecule number calculated value in MON810 strain, be then judged to be heterozygous MON810;
Described MON810 is transgenic insect-resistant corn kind.
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| CN109825635A (en) * | 2019-04-03 | 2019-05-31 | 深圳出入境检验检疫局食品检验检疫技术中心 | Method, reagent and the kit that Transgenic corn lines 98140 detect |
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| CN109825635A (en) * | 2019-04-03 | 2019-05-31 | 深圳出入境检验检疫局食品检验检疫技术中心 | Method, reagent and the kit that Transgenic corn lines 98140 detect |
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