CN104388580A - Method for identifying homozygotic type or heterozygous type NK603 on basis of digital PCR (polymerase chain reaction) - Google Patents
Method for identifying homozygotic type or heterozygous type NK603 on basis of digital PCR (polymerase chain reaction) Download PDFInfo
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Abstract
The invention discloses a method for identifying homozygotic type or heterozygous type NK603 on the basis of digital PCR (polymerase chain reaction), and relates to the technical field of molecular biology and transgenic test. The method comprises steps as follows: 1), collection: NK603 specificity transformation events verified by European Union and a corn internal standard gene adh1 quantitative determination method are adopted; 2), digital PCR amplification: a DNA (deoxyribonucleic acid) of NK603 is taken as a research template; 3), data analysis: if 95% of confidence intervals of target molecular number calculated values of NK603 specificity transformation events and corn internal standard genes adh1are overlapped, NK603 is judged as homozygotic type NK603; and if two times of 95% of confidence intervals of target molecular number calculated values of NK603 specificity transformation events and 95% of confidence intervals corn internal standard genes adh1are overlapped, NK603 is judged as heterozygous type NK603. The method can rapidly, accurately and reliably identify absolute quantification of homozygotic type and heterozygous type herbicide-resistant genetically-modified corn strain NK603.
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 NK603 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, supervise the increasing challenge brought to 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 and a kind ofly identify method that is homozygous or heterozygous transgenic herbicide-resistant corn strain NK603, 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 NK603 based on digital pcr, described NK603 is transgenosis herbicide-resistant corn variety.
The object of the present invention is achieved like this:
One, method that is homozygous or heterozygous NK603 is identified based on digital pcr
1. collect
European Union is adopted to pass through NK603 specificity transformation event and corn internal standard gene adh 1 quantitative detecting method of checking,
NK603 primer sequence:
NK603F：5’-ATGAATGACCTCGAGTAAGCTTGTTAA-3’，
NK603R：5’-AAGAGATAACAGGATCCACTCAAACACT-3’；
NK603 probe sequence:
5’-FAM-TGGTACCACGCGACACACTTCCACTC-TAMRA-3’；
Internal standard gene adh 1 primer sequence:
adh1F：5’-CCAGCCTCATGGCCAAAG-3’，
adh1R：5’-CCTTCTTGGCGGCTTATCTG-3’；
Internal standard gene adh 1 probe sequence:
adh1P：5’-FAM-CTTAGGGGCAGACTCCCGTGTTCCCT-TAMRA-3’；
2. digital pcr amplification
With the DNA of NK603 for research template, above-mentioned primed probe is utilized to be combined into line number word pcr amplification respectively, NK603 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 NK603 specificity transformation event and corn internal standard gene adh 1 target molecule number calculated value respectively; If 95% fiducial interval of specificity transformation event and corn internal standard gene adh 1 target molecule number calculated value has overlap in NK603 strain, be judged to be homozygous NK603; 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 adh 1 target molecule number calculated value in NK603 strain, be then judged to be heterozygous NK603.
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 homozygous or heterozygous transgenic herbicide-resistant corn strain NK603 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 transgenosis herbicide-resistant corn strain NK603 homozygosity;
2. avoid and utilize traditional breeding way to screen problem that is homozygous or heterozygous transgenic herbicide-resistant corn strain NK603 length consuming time;
3. avoid utilize real time fluorescence quantifying PCR method to identify homozygous or heterozygous transgenic herbicide-resistant corn strain NK603 error is large, the problem of result difficult judgment.
In a word, the present invention be a kind of fast, accurately, the method for the homozygous and heterozygous transgenic herbicide-resistant corn strain NK603 absolute quantitation of qualification reliably.
Accompanying drawing explanation
The homozygous NK603 of Fig. 1 to be template amount be 6ng, the amplification curve of corn internal standard gene adh 1 on microfluid panel, repeats 1;
The homozygous NK603 of Fig. 2 to be template amount be 6ng, the amplification curve of corn internal standard gene adh 1 on microfluid panel, repeats 2;
The homozygous NK603 of Fig. 3 to be template amount be 6ng, the amplification curve of corn internal standard gene adh 1 on microfluid panel, repeats 3;
The homozygous NK603 of Fig. 4 to be template amount be 6ng, the amplification curve of corn internal standard gene adh 1 on microfluid panel, repeats 4;
The homozygous NK603 of Fig. 5 to be template amount be 6ng, the amplification curve of corn internal standard gene adh 1 on microfluid panel, repeats 5;
The homozygous NK603 of Fig. 6 to be template amount be 6ng, the amplification curve of corn internal standard gene adh 1 on microfluid panel, repeats 6;
The amplification curve of homozygous NK603, NK603 specificity transformation event on microfluid panel of Fig. 7 to be template amount be 6ng, repeats 1;
The amplification curve of homozygous NK603, NK603 specificity transformation event on microfluid panel of Fig. 8 to be template amount be 6ng, repeats 2;
The amplification curve of homozygous NK603, NK603 specificity transformation event on microfluid panel of Fig. 9 to be template amount be 6ng, repeats 3;
The amplification curve of homozygous NK603, NK603 specificity transformation event on microfluid panel of Figure 10 to be template amount be 6ng, repeats 4;
The amplification curve of homozygous NK603, NK603 specificity transformation event on microfluid panel of Figure 11 to be template amount be 6ng, repeats 5;
The amplification curve of homozygous NK603, NK603 specificity transformation event on microfluid panel of Figure 12 to be template amount be 6ng, repeats 6;
The homozygous NK603 of Figure 13 to be template amount be 9.6ng, the amplification curve of corn internal standard gene adh 1 on microfluid panel, repeats 1;
The homozygous NK603 of Figure 14 to be template amount be 9.6ng, the amplification curve of corn internal standard gene adh 1 on microfluid panel, repeats 2;
The NK603 that isozygotys of Figure 15 to be template amount be 9.6ng, the amplification curve of corn internal standard gene adh 1 on microfluid panel, repeats 3;
The homozygous NK603 of Figure 16 to be template amount be 9.6ng, the amplification curve of corn internal standard gene adh 1 on microfluid panel, repeats 4;
The homozygous NK603 of Figure 17 to be template amount be 9.6ng, the amplification curve of corn internal standard gene adh 1 on microfluid panel, repeats 5;
The homozygous NK603 of Figure 18 to be template amount be 9.6ng, the amplification curve of corn internal standard gene adh 1 on microfluid panel, repeats 6;
The amplification curve of homozygous NK603, NK603 specificity transformation event on microfluid panel of Figure 19 to be template amount be 9.6ng, repeats 1;
The amplification curve of homozygous NK603, NK603 specificity transformation event on microfluid panel of Figure 20 to be template amount be 9.6ng, repeats 2;
The amplification curve of homozygous NK603, NK603 specificity transformation event on microfluid panel of Figure 21 to be template amount be 9.6ng, repeats 3;
The amplification curve of homozygous NK603, NK603 specificity transformation event on microfluid panel of Figure 22 to be template amount be 9.6ng, repeats 4;
The amplification curve of homozygous NK603, NK603 specificity transformation event on microfluid panel of Figure 23 to be template amount be 9.6ng, repeats 5;
The amplification curve of homozygous NK603, NK603 specificity transformation event on microfluid panel of Figure 24 to be template amount be 9.6ng, repeats 6;
The heterozygous NK603 of Figure 25 to be template amount be 6ng, the amplification curve of corn internal standard gene adh 1 on microfluid panel, repeats 1;
The heterozygous NK603 of Figure 26 to be template amount be 6ng, the amplification curve of corn internal standard gene adh 1 on microfluid panel, repeats 2;
The heterozygosis NK603 of Figure 27 to be template amount be 6ng, the amplification curve of corn internal standard gene adh 1 on microfluid panel, repeats 3;
The heterozygous NK603 of Figure 28 to be template amount be 6ng, the amplification curve of corn internal standard gene adh 1 on microfluid panel, repeats 4;
The heterozygous NK603 of Figure 29 to be template amount be 6ng, the amplification curve of corn internal standard gene adh 1 on microfluid panel, repeats 5;
The heterozygous NK603 of Figure 30 to be template amount be 6ng, the amplification curve of corn internal standard gene adh 1 on microfluid panel, repeats 6;
The heterozygous NK603 of Figure 31 to be template amount be 6ng, the amplification curve of NK603 specificity transformation event on microfluid panel, repeats 1;
The heterozygous NK603 of Figure 32 to be template amount be 6ng, the amplification curve of NK603 specificity transformation event on microfluid panel, repeats 2;
The heterozygous NK603 of Figure 33 to be template amount be 6ng, the amplification curve of NK603 specificity transformation event on microfluid panel, repeats 3;
The heterozygous NK603 of Figure 34 to be template amount be 6ng, the amplification curve of NK603 specificity transformation event on microfluid panel, repeats 4;
The heterozygous NK603 of Figure 35 to be template amount be 6ng, the amplification curve of NK603 specificity transformation event on microfluid panel, repeats 5;
The heterozygous NK603 of Figure 36 to be template amount be 6ng, the amplification curve of NK603 specificity transformation event on microfluid panel, repeats 6;
The heterozygous NK603 of Figure 37 to be template amount be 9.6ng, the amplification curve of corn internal standard gene adh 1 on microfluid panel, repeats 1;
The heterozygous NK603 of Figure 38 to be template amount be 9.6ng, the amplification curve of corn internal standard gene adh 1 on microfluid panel, repeats 2;
The heterozygous NK603 of Figure 39 to be template amount be 9.6ng, the amplification curve of corn internal standard gene adh 1 on microfluid panel, repeats 3;
The heterozygous NK603 of Figure 40 to be template amount be 9.6ng, the amplification curve of corn internal standard gene adh 1 on microfluid panel, repeats 4;
The heterozygous NK603 of Figure 41 to be template amount be 9.6ng, the amplification curve of corn internal standard gene adh 1 on microfluid panel, repeats 5;
The heterozygous NK603 of Figure 42 to be template amount be 9.6ng, the amplification curve of corn internal standard gene adh 1 on microfluid panel, repeats 6;
The heterozygous NK603 of Figure 43 to be template amount be 9.6ng, the amplification curve of NK603 specificity transformation event on microfluid panel, repeats 1;
The heterozygous NK603 of Figure 44 to be template amount be 9.6ng, the amplification curve of NK603 specificity transformation event on microfluid panel, repeats 2;
The heterozygous NK603 of Figure 45 to be template amount be 9.6ng, the amplification curve of NK603 specificity transformation event on microfluid panel, repeats 3;
The heterozygous NK603 of Figure 46 to be template amount be 9.6ng, the amplification curve of NK603 specificity transformation event on microfluid panel, repeats 4;
The heterozygous NK603 of Figure 47 to be template amount be 9.6ng, the amplification curve of NK603 specificity transformation event on microfluid panel, repeats 5;
The heterozygous NK603 of Figure 48 to be template amount be 9.6ng, the amplification curve of NK603 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 NK603
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 NK603 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 NK603 to 100ng/ μ l, the light absorption value measured value of 260/280 is respectively 1.82 and 1.84, for subsequent use.
2, digital pcr detects
1) have chosen the specificity through the NK603 transformant of European Union's checking and internal standard gene adh 1 quantitative detecting method, wherein NK603 specificity transformation event amplified fragments size is 108bp, the amplified fragments size of internal standard gene adh 1 is 70bp, NK603 primer sequence:
NK603F：5’-ATGAATGACCTCGAGTAAGCTTGTTAA-3’，
NK603R：5’-AAGAGATAACAGGATCCACTCAAACACT-3’；
NK603 probe sequence:
NK603P：5’-FAM-TGGTACCACGCGACACACTTCCACTC-TAMRA-3’；
Internal standard gene adh 1 primer sequence:
adh1F：5’-CCAGCCTCATGGCCAAAG-3’，
adh1R：5’-CCTTCTTGGCGGCTTATCTG-3’；
Internal standard gene adh 1 probe sequence:
adh1P：5’-FAM-CTTAGGGGCAGACTCCCGTGTTCCCT-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 NK603 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 NK603.
The homozygous NK603 being 6ng with DNA amount is for template, the primer of corn internal standard gene adh 1 and NK603 specificity of transformant event and probe is adopted to carry out digital pcr amplified reaction respectively, internal standard gene adh 1 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 321-342 each panel having the quantity of the reaction chamber of signal concentrate on, mean value is 332, the target molecule number mean value detected is 435.2, as table 1, NK603 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 297-340 each panel having the quantity of the reaction chamber of signal concentrate on, mean value is 322.3, the target molecule number mean value detected is 418.5, as table 1.95% fiducial interval of NK603 specificity transformation event and corn internal standard gene adh 1 target molecule number calculated value has overlap, and result meets genetic development, can be judged to be homozygous transgenic herbicide-resistant corn NK603.
The homozygous NK603 being 9.6ng with DNA amount is for template, the primer of corn internal standard gene adh 1 and NK603 specificity of transformant event and probe is adopted to carry out digital pcr amplified reaction respectively, internal standard gene adh 1 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 432-474 each panel having the quantity of the reaction chamber of signal concentrate on, mean value is 456.8, the target molecule number mean value detected is 694.3, as table 1, NK603 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 427-473 each panel having the quantity of the reaction chamber of signal concentrate on, mean value is 446.5, the target molecule number mean value detected is 669.2, as table 1.95% fiducial interval of NK603 specificity transformation event and corn internal standard gene adh 1 target molecule number calculated value has overlap, and result meets genetic development, can be judged to be homozygous transgenic herbicide-resistant corn NK603.
The heterozygous NK603 being 6ng with DNA amount is template, the primer of corn internal standard gene adh 1 and NK603 specificity of transformant event and probe is adopted to carry out digital pcr amplified reaction respectively, internal standard gene adh 1 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 310-356 each panel having the quantity of the reaction chamber of signal concentrate on, mean value is 335.5, the target molecule number mean value detected is 441.5, as table 1, NK603 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 161-212 each panel having the quantity of the reaction chamber of signal concentrate on, mean value is 178.3, the target molecule number mean value detected is 203.5, as table 1.Two times of 95% fiducial interval of NK603 specificity transformation event target molecule number calculated value have overlap with 95% fiducial interval of corn internal standard gene target molecule number calculated value, result meets genetic development, can be judged to be heterozygous transgenic herbicide-resistant corn NK603.
The heterozygous NK603 being 9.6ng with DNA amount is template, the primer of corn internal standard gene adh 1 and NK603 specificity of transformant event and probe is adopted to carry out digital pcr amplified reaction respectively, internal standard gene adh 1 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 448-467 each panel having the quantity of the reaction chamber of signal concentrate on, mean value is 456.2, the target molecule number mean value detected is 692.0, as table 1, NK603 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 242-291 each panel having the quantity of the reaction chamber of signal concentrate on, mean value is 274.0, the target molecule number mean value detected is 339.7, as table 1.Two times of 95% fiducial interval of NK603 specificity transformation event target molecule number calculated value have overlap with 95% fiducial interval of corn internal standard gene target molecule number calculated value, result meets genetic development, can be judged to be heterozygous transgenic herbicide-resistant corn NK603.
Above result can be found out, the present invention is that the qualification of transgenosis herbicide-resistant corn strain NK603 homozygosity provides a kind of accurate, reliable absolute quantitation measuring method detected based on digital pcr, can identify transgenosis herbicide-resistant corn strain NK603 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 adh 1 and NK603 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 NK603 based on digital pcr, it is characterized in that:
1. collect
European Union is adopted to pass through NK603 specificity transformation event and corn internal standard gene adh 1 quantitative detecting method of checking,
NK603 primer sequence:
NK603F：5’-ATGAATGACCTCGAGTAAGCTTGTTAA-3’，
NK603R：5’-AAGAGATAACAGGATCCACTCAAACACT-3’；
NK603 probe sequence:
5’-FAM-TGGTACCACGCGACACACTTCCACTC-TAMRA-3’；
Internal standard gene adh 1 primer sequence:
adh1F：5’-CCAGCCTCATGGCCAAAG-3’，
adh1R：5’-CCTTCTTGGCGGCTTATCTG-3’；
Internal standard gene adh 1 probe sequence:
adh1P：5’-FAM-CTTAGGGGCAGACTCCCGTGTTCCCT-TAMRA-3’；
2. digital pcr amplification
With the DNA of NK603 for research template, above-mentioned primed probe is utilized to be combined into line number word pcr amplification respectively, NK603 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 NK603 specificity transformation event and corn internal standard gene adh 1 target molecule number calculated value respectively; If 95% fiducial interval of specificity transformation event and corn internal standard gene adh 1 target molecule number calculated value has overlap in NK603 strain, be judged to be homozygous NK603; 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 adh 1 target molecule number calculated value in NK603 strain, be then judged to be heterozygous NK603;
Described NK603 is transgenosis herbicide-resistant corn variety.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105112536A (en) * | 2015-09-15 | 2015-12-02 | 中国检验检疫科学研究院 | Double digital PCR fluorescent quantitative detection method for transgenic maize NK603 |
| CN109182472A (en) * | 2018-10-12 | 2019-01-11 | 上海市农业科学院 | A kind of ddPCR detection method for identifying transgene abrotanum genotype |
| WO2023081614A3 (en) * | 2021-11-02 | 2023-06-15 | Monsanto Technology Llc | Transgenic corn event zm_bcs216090 and methods for detection and uses thereof |
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| CN101445826A (en) * | 2008-06-19 | 2009-06-03 | 新疆农业科学院核技术生物技术研究所 | Simple and new method for identifying copy numbers of transgenic plants |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105112536A (en) * | 2015-09-15 | 2015-12-02 | 中国检验检疫科学研究院 | Double digital PCR fluorescent quantitative detection method for transgenic maize NK603 |
| CN109182472A (en) * | 2018-10-12 | 2019-01-11 | 上海市农业科学院 | A kind of ddPCR detection method for identifying transgene abrotanum genotype |
| WO2023081614A3 (en) * | 2021-11-02 | 2023-06-15 | Monsanto Technology Llc | Transgenic corn event zm_bcs216090 and methods for detection and uses thereof |
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