CN106868137A - Transgenic rice multiple digital pcr quantitative detecting method - Google Patents
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Abstract
The invention discloses a kind of transgenic rice multiple digital pcr quantitative detecting method, belong to the quantitative detection field of transgenic paddy rice.The present invention discloses the multiple digital pcr quantitative determination primer pair and probe for detecting transgenic paddy rice KF2, KF6, KF8, KMD1, M12, TT51 1 and G6H1 first.On this basis, the present invention further established a kind of multiple digital pcr quantitative detecting method of transgenic paddy rice KF2, KF6, KF8, KMD1, M12, TT51 1 and G6H1.The inventive method can realize that the sample pair simultaneously containing KF2, KF6, KF8, KMD1, M12, TT51 1 and 7 kinds of G6H1 or wherein several transgenic paddy rices carries out quantitative determination, and it is high to the stability of transgene component quantitative determination, it is a kind of effective ways of Transgenic Rice component quantifying detection.
Description
Technical field
The present invention relates to transgenic rice multiple digital pcr quantitative detecting method, belong to the quantitative determination of transgenic paddy rice
Field.
Background technology
Latest data shows that global genetically modified crops cultivated area reached 1.81 hundred million hectares in 2015, than 1996 170
Ten thousand hectares increase more than 100 times (James, Clive. " 20th Anniversary (1996 to 2015) of the
global commercialization of biotech crops and biotech crop highlights in
2015."ISAAA Brief 51(2015).).Modern biotechnology with genetic engineering as core has penetrated into agricultural extensively
Every field, has been altered in steps research and the production model of traditional agriculture.Genetically modified crops by it is quick, continuously for business
Metaplasia is produced.No matter in developed country or developing country, genetically modified crops all produce to environment, economy, the energy, health and society
Tremendous influence is given birth to.Because transgenic technology is related to the importances such as social public security, safety management and social forest, because
This genetically modified crops is not only national governments, scientific research personnel and plantation peasant's focus of attention, also result in each row in the whole world each
The extensive concern of industry.Therefore, the numerous and confused mark system for setting up transgene component of various countries and area, on world community man and ground
In the transgene component mark system that area formulates, quantitative identifying system occupies larger specific gravity, wherein with European Union and Japan and Korea S most generation
The quantitative threshold value of table, such as European Union is 0.9% (EURL-GMFF.Definition of Minimum Performance
Requirements for Analytical Methods of GMO Testing European Network of GMO
Laboratories(ENGL),European Commission,(2003a)Commission Regulation(EC)
No.1829/2003 of September 22,2003,Concerning on genetically modified food and
feed.Off.J.Eur.Commun L268:1–23；European Commission,(2003b)Commission
Regulation(EC)No.1830/2003 of September 22,2003,Concerning the traceability
and labelling of genetically modified organisms and the traceability of food
and feed products produced from genetically modified organisms and amending
Directive 2001/18/EC.Off.J.Eur.Commun L268:24–28.)；Japan and South Korea are respectively：5% and 3%
(Matsuoka T(2001)GMO labeling and detection methods in Japan.APEC-JIRCAS
Joint Symposium and Workshop on Agricultural Biotechnology；Ministry of
Agriculture and Forestry(2000)Guidelines for Labeling of Genetically Modified
Agricultural Products.MAF Notification No.31.).China mainly adopts in terms of transgene component mark
Qualitative mark system is taken, China is made in the presence of different at aspects such as detection techniques due to qualitative mark system and quantitative identifying system
Easily restricted by international standard in terms of crop import and export, it is therefore desirable to greatly develop transgene component quantitative measurement technology
And set up the quantitative identifying system of correlation.
The concept of digital pcr (Digital PCR) proposed (Sykes, P.J., et al. " Quantitation in 1992
of targets for PCR by use of limiting dilution."BioTechniques13.3(1992):444-
9.), its general principle is by the way that micro-example to be carried out big times of dilution and point liquid, until each amplification region contains target expansion
It is more than 0,1 or 1 to increase molecule, then enters performing PCR amplification, according to the presence or absence of amplified signal, using Poisson distribution (Poisson
distribution)(Pinheiro,Leonardo B.,et al."Evaluation of a droplet digital
polymerase chain reaction format for DNA copy number quantification."
Analytical chemistry 84.2(2011):1003-1011.) calculate the original copy number or concentration of sample.Numeral
PCR is independent of amplification efficiency and quantifying based on standard curve, and higher to the tolerance of some PCR inhibiting factors.Should
Technology all achieves breakthrough development at aspects such as gene copy number analysis, gene expression analysis, Genotypings.In transgenosis
Context of detection, also there is corresponding research report (Demeke, Tigst, et al. " Assessment of droplet digital
PCR for absolute quantification of genetically engineered OXY235 canola and
DP305423 soybean samples."Food Control 46(2014):470-474；Morisset,Dany,et al."
Quantitative analysis of food and feed samples with droplet digital PCR."PLoS
One 8.5(2013):E62583.), feasibility and reliability of the digital pcr in terms of detection GMOs are shown.But at present this
A little researchs are more based on 2 weight digital pcr detections, and once genetically modified crops transformation event quantitatively can only be divided
Analysis, and the requirement of quantitative identifying at present is directed to single component quantifying rather than single transformation event, such as European Union is to quantitative mark
The definition of knowledge is：The material produced containing genetically modified organism in food, its ratio must not be higher than the food composition of single component
0.9%.Therefore, current digital pcr detection method is not suitable for determining while various transformation events of single component in sample
Amount detection.At present, the transgenic paddy rice of China's research and development mainly has KF2, KF6, KF8, KMD1, M12, TT51-1, G6H1 this 7 product
System, therefore exploitation one kind detects this multiple digital pcr quantitative detecting method of 7 kinds of transgenic paddy rice contents simultaneously, turns for paddy rice
The quantitative determination of gene element will have great importance.
The content of the invention
First technical problem to be solved by this invention be to provide for detect 7 kinds of transgenic paddy rice KF2, KF6, KF8,
The multiple digital pcr quantitative determination primer pair and probe of KMD1, M12, TT51-1 and G6H1；
Second technical problem to be solved by this invention be set up a kind of transgenic paddy rice KF2, KF6, KF8, KMD1,
The multiple digital pcr quantitative detecting method of M12, TT51-1 and G6H1.
In order to solve the above technical problems, the technical solution used in the present invention is：
The present invention is disclosed for detecting transgenic paddy rice KF2, KF6, KF8, KMD1, M12, TT51-1 and G6H1 first
Multiple digital pcr quantitative determination primer pair and probe, including：
Sense primer shown in SEQ ID No.1 is classified as by nucleotides sequence and anti-sense primer shown in SEQ ID No.2 is constituted
Transgenic paddy rice KF2 specificity of transformant primer pairs；The nucleotides sequence of transgenic paddy rice KF2 specificity of transformant probes is classified as
Shown in SEQ ID No.3；
Sense primer shown in SEQ ID No.4 is classified as by nucleotides sequence and anti-sense primer shown in SEQ ID No.5 is constituted
Transgenic paddy rice KF6 specificity of transformant primer pairs；The nucleotides sequence of transgenic paddy rice KF6 specificity of transformant probes is classified as
Shown in SEQ ID No.6；
Sense primer shown in SEQ ID No.7 is classified as by nucleotides sequence and anti-sense primer shown in SEQ ID No.8 is constituted
Transgenic paddy rice KF8 specificity of transformant primer pairs；The nucleotides sequence of transgenic paddy rice KF8 specificity of transformant probes is classified as
Shown in SEQ ID No.9；
Sense primer shown in SEQ ID No.10 is classified as by nucleotides sequence and anti-sense primer shown in SEQ ID No.11 is constituted
Transgenic paddy rice KMD1 specificity of transformant primer pairs；The nucleotide sequence of transgenic paddy rice KMD1 specificity of transformant probes
Shown in SEQ ID No.12；
Sense primer shown in SEQ ID No.13 is classified as by nucleotides sequence and anti-sense primer shown in SEQ ID No.14 is constituted
Transgenic paddy rice M12 specificity of transformant primer pairs；The nucleotides sequence of transgenic paddy rice M12 specificity of transformant probes is classified as
Shown in SEQ ID No.15；
Sense primer shown in SEQ ID No.16 is classified as by nucleotides sequence and anti-sense primer shown in SEQ ID No.17 is constituted
Transgenic paddy rice TT51-1 specificity of transformant primer pairs；The nucleotides of transgenic paddy rice TT51-1 specificity of transformant probes
Sequence is shown in SEQ ID No.18；And,
Sense primer shown in SEQ ID No.19 is classified as by nucleotides sequence and anti-sense primer shown in SEQ ID No.20 is constituted
Transgenic paddy rice G6H1 specificity of transformant primer pairs；The nucleotide sequence of transgenic paddy rice G6H1 specificity of transformant probes
Shown in SEQ ID No.21.
Multiple digital pcr quantitative determination primer pair of the present invention and probe can be applied to detect transgenic paddy rice
Any one or more in KF2, KF6, KF8, KMD1, M12, TT51-1 and G6H1.
The present invention further discloses many of a kind of transgenic paddy rice KF2, KF6, KF8, KMD1, M12, TT51-1 and G6H1
Weight digital pcr quantitative detecting method, comprises the following steps：(1) DNA of paddy rice sample to be detected is extracted；(2) DNA to extract
It is template, with multiple digital pcr quantitative determination primer pair of the present invention and probe, and paddy rice reference gene detection primer
Pair and probe set up digital pcr system go forward side by side performing PCR amplification；(3) data analysis is carried out to amplification, foreign gene is calculated and is copied
The ratio between shellfish number and reference gene copy number, obtain the quantitative values of transgene component；That is, each reacting hole is analyzed using QuantaSoft
The fluorescence signal value of middle foreign gene and reference gene, foreign gene and internal reference in each reaction are calculated by Poisson distribution formula
The copy number of gene, copy number of foreign gene is designated as：Ne, reference gene copy number is designated as：Ni, by the ratio between both copy numbers
The quantitative values of transgene component are obtained, i.e.,：Gm content=Ne/Ni × 100%.
In the multiple digital pcr quantitative detecting method of the present invention, the digital pcr system is：The cumulative volume of reaction system is
20 μ L, wherein, 2xddPCR Supermix for probe 10 μ L and are visited the specific detection primer pair of 7 kinds of transgenic paddy rices
The primer of pin and paddy rice reference gene detection primer pair and probe composition and the μ L of mixture 6, the μ L of DNA profiling 4 of probe.Its
In, in the mixture of the primer and probe, the upstream and downstream primer concentration of paddy rice reference gene detection primer pair is respectively
3000nmol/L, the concentration of paddy rice reference gene probe is 600nmol/L；7 kinds of specific detection primers pair of transgenic paddy rice
Each upstream and downstream primer concentration be respectively 1500nmol/L；The concentration of 7 kinds of specific probes of transgenic paddy rice is respectively
300nmol/L.The program of the PCR amplifications includes：95 DEG C of predegeneration 5min；95 DEG C of denaturation 30sec, 60 DEG C of annealing and extension
60sec, totally 40 circulations；98 DEG C of 10min, 4 DEG C of preservations.
The multiple digital pcr quantitative detecting method of the present invention, from rice sucrose phosphatase (Sucrose phosphate
Synthase, SPS), used as paddy rice reference gene, the paddy rice reference gene detection primer by nucleotides sequence to being classified as SEQ for gene
Sense primer shown in ID No.22 and anti-sense primer shown in SEQ ID No.23 are constituted；The nucleosides of the paddy rice reference gene probe
Acid sequence is 5 ' end mark fluorescent group HEX of probe shown in SEQ ID No.24, and 3 ' hold mark fluorescent quenching group BHQ1.
5 ' the end difference mark fluorescent group FAM of 7 kinds of specific probes of transgenic paddy rice of the present invention, mark fluorescent is quenched respectively at 3 ' ends
Group BHQ1.
The inventive method can according to actual needs increase and decrease corresponding transgenic paddy rice event, and flexibility is strong, while operating letter
Just, it is a kind of effective ways of transgene component quantitative determination.
The invention also discloses a kind of multiplicity of transgenic paddy rice KF2, KF6, KF8, KMD1, M12, TT51-1 and G6H1
Word PCR immue quantitative detection reagent boxes, including：2xddPCR Supermix for probe, paddy rice reference gene detection primer pair and
Probe, the multiple digital pcr quantitative determination primer pair of transgenic paddy rice and probe；Wherein, the multiplicity of the transgenic paddy rice
Word PCR quantitative determinations primer pair and probe are multiple digital pcr quantitative determination primer pair of the present invention and probe, the spy
5 ' end difference mark fluorescent group FAM of pin, 3 ' hold mark fluorescent quenching group BHQ1 respectively；The paddy rice reference gene detection
Primer pair is classified as sense primer shown in SEQ ID No.22 by nucleotides sequence and anti-sense primer shown in SEQ ID No.23 is constituted；Institute
The nucleotides sequence for stating paddy rice reference gene probe is classified as shown in SEQ ID No.24,5 ' end mark fluorescent group HEX of probe, 3 '
Hold mark fluorescent quenching group BHQ1.
The present invention is utilized respectively the multiple digital PCR method that other genetically modified crops and the checking of non-transgenic crop are set up
Specificity, as a result show that all other genetically modified crops and the false positive rate of non-transgenic crop are below 5%, show this hair
The specificity of the multiple digital PCR method of bright foundation preferably, meets the quantitative determination needs to transgenic paddy rice.
The determination result of quantitative limit and test limit shows, when transgenic paddy rice content is between 5%-0.05%, multiplicity
RSD between word PCR quantitative value and theoretical value is both less than 25%, meets the requirement (EURL-GMFF) of quantitative determination, while
Show that the repeatability and stability of the multiple digital pcr of present invention foundation are preferable.When transgenosis content is 0.01%, multiplicity
RSD between word PCR quantitative value and theoretical value is 39.25%.Therefore, quantifying for the inventive method is determined according to data above
Be limited to 0.05%, detection is limited to 0.01%, meet transgene component it is actually detected the need for.
Technical solution of the present invention compared with prior art, has the advantages that：
The present invention establishes a kind of for 7 kinds of transgenic paddy rices (KF2, KF6, KF8, KMD1, M12, TT51-1, G6H1)
Multiple digital pcr quantitative detecting method, sample of the realization pair simultaneously containing 7 kinds above or wherein several transgenic paddy rices is determined
Amount detection, eliminates the difference quantitative determination to single transgenic paddy rice composition, while also eliminate conventional PCR method quantitatively examining
Survey the steps such as standard curve drafting.In addition, transgene component is by copy number of foreign gene and reference gene copy number
Obtained from ratio is calculated, 7 foreign genes and 1 reference gene amplification are in same reaction system in the methods of the invention
In, so as to improve the stability to transgene component quantitative determination.
Brief description of the drawings
Fig. 1 is multiple digital pcr amplification.
Specific embodiment
Further describe the present invention with reference to specific embodiment, advantages of the present invention and feature will be with description and
It is apparent.It should be understood that the embodiment is only exemplary, any limitation is not constituted to the scope of the present invention.This area
Technical staff should be understood that without departing from the spirit and scope of the invention can to the details of technical solution of the present invention and
Form is modified or is replaced, but these modifications or replacement each fall within protection scope of the present invention.
The foundation of the transgenic rice multiple digital pcr quantitative detecting method of embodiment 1
1st, materials and methods
1.1 experiment materials
Experiment transgenic line used is respectively：KF2, KF6, KF8, KMD1, M12, TT51-1, G6H1 (transgenosis water
Rice)；GTS40-3-2 (genetically engineered soybean)；MON15985 (transgene cotton)；Oxy235 (transgene rape)；MON863、
MON89034 (transgenic corns).Non-transgenic paddy rice, soybean, corn, cotton, rape DNA are used as negative Quality Control.
1.2 DNA are extracted
Weigh 200mg pulverized specimens according to kit (DNeasy Plant Mini Kit, Qiagen, Hilden,
Germany) illustrate to extract DNA.Determined at spectrophotometer 260-280nm and obtain DNA mass and purity, and it is solidifying with agarose
Gel electrophoresis determine its integrality.The 7 kinds of transgenic paddy rice DNA for extracting are mixed in equal volume, then uses non-transgenic rice genome
DNA is diluted to following corresponding concentration：5%th, 1%, 0.5%, 0.1%, 0.05%, 0.01%.
1.3 primers and probe and its preparation
From rice sucrose phosphatase (Sucrose phosphate synthase, SPS) gene as paddy rice internal reference base
Cause, its probe 5 ' end mark fluorescent group HEX, 3 ' ends mark BHQ1,7 kinds of specific sequences of the transformation event of transgenic paddy rice
The end of probe 5 ' all mark fluorescent group FAM, 3 ' end mark BHQ1,8 groups of primed probes are mixed, corresponding primer name
Sequence and each primed probe concentration in the mixture is claimed to be shown in Table 1.
The multiple digital PCR primer of table 1 and detecting probe information
1.4 digital pcrs are expanded and data analysis
Digital pcr system：The mixture of 10 μ L 2xddPCR Supermix for probe, 6 μ L primers and probe, 4 μ L
DNA profiling or ddH2O, cumulative volume is 20 μ L.
Sample (20 μ L) general pipettor in reaction system laboratory is added to 8 of a row in the middle of DG8 cartridge
Individual hole, each in DG8 cartridge most one row of bottom, 8 holes to add 70 μ L droplets that oil occurs, instrument is automatically performed droplet generation.
The droplet of generation is transferred in 96 orifice plates, heat-sealing film is sealed up, performing PCR is entered in placement Standard PCR instrument.PCR amplification programs are：95
DEG C predegeneration 5min；95 DEG C of denaturation 30sec, 60 DEG C of annealing and extension 60sec, totally 40 circulations；98 DEG C of 10min, 4 DEG C of preservations.
96 orifice plates for completing PCR are put into droplet reading instrument, the droplet that instrument is automatically performed each sample reads, it is desirable to per Kong Wei
Drop generation number just includes statistical analysis more than 8000, and the positive droplet containing amplified signal and the negative droplet not expanded are with glimmering
Photo threshold is distinguished, and the fluorescence signal value of foreign gene and reference gene in each reacting hole is analyzed using QuantaSoft, is passed through
Poisson distribution formula calculates the copy number (see Fig. 1) of foreign gene and reference gene in each reaction, copy number of foreign gene note
For：Ne, reference gene copy number is designated as：Ni, the quantitative values of transgene component is obtained by the ratio between both copy numbers, i.e.,：Turn
Gene element content=Ne/Ni × 100%.
2nd, result and analysis
2.1 specificity verifications
The specificity of digital pcr is typically evaluated with false positive rate (false positive rate), it is now recognized that false sun
Property rate less than 5% show set up digital pcr specificity preferably, meet detection the need for (Holst-Jensen, A.；
Crespo,T.；Simplicio,A.；Richl,P.；Welsche,M.；Dobnik,D.；Dreo,T.Deliverable 6.1
MPP Decathlon,http://www.decathlon-project.eu/sites/default/files/
Deliverable6.1_MPP_Decathlon.pdf.).In the methods of the invention, other genetically modified crops and non-are utilized respectively
The specificity of the multiple digital pcr that genetically modified crops checking is set up, the result is shown in Table 2.Result shows, all other transgenosis
The false positive rate of crop and non-transgenic crop is below 5%, shows the specificity of the multiple digital PCR method of present invention foundation
Preferably, the quantitative determination needs to transgenic paddy rice are met.
The multiple digital pcr false positive rate detection of table 2
The determination of the quantitative limit and test limit of 2.2 multiple digital pcrs
In order to determine the quantitative limit and test limit of the multiple digital pcr set up, determine respectively transgenosis content be 5%,
1%th, the content of 0.5%, 0.1%, 0.05%, 0.01% sample, the difference between analysis quantitative result and theoretical value, each sample
Product are repeated 4 times, and 4 parallel, totally 16 reactions are set every time.
The results are shown in Table 3, when transgenic paddy rice content is between 5%-0.05%, the quantitative value of multiple digital pcr with it is theoretical
RSD between value is both less than 25%, meets the requirement (EURL-GMFF) of quantitative determination, while also indicating that the multiple numeral of foundation
The repeatability and stability of PCR are preferable.When transgenosis content is 0.01%, the quantitative value of multiple digital pcr and theoretical value it
Between RSD be 39.25%.Therefore according to data above, quantifying for the inventive method is defined to 0.05%, detection is limited to
0.01%.
The multiple digital pcr test limit of table 3 determines with quantitative limit
SEQUENCE LISTING
<110>Zhejiang Academy of Agricultural Science
<120>Transgenic rice multiple digital pcr quantitative detecting method
<130> ZJ-2001-170102A
<160> 24
<170> PatentIn version 3.5
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gctcgtctgg ctaagatcg 19
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gcacggacta tacaagttgt gat 23
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gtcagataaa ctgattggtc tgat 24
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cgaccatgat gctgttctgc 20
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cgttatttat gagatgggtg atctcaccca tgcttg 36
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tggtgagcgt tttgcagtct 20
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ctgatccact agcaggaggt cc 22
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tgttgtgctg ccaatgtggc ctg 23
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agagactggt gatttcagcg gg 22
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aagcgtcaat ttgtttacac c 21
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atggatgtat cgccaccagc acc 23
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Claims (10)
1. it is used to detect that the multiple digital pcr of transgenic paddy rice KF2, KF6, KF8, KMD1, M12, TT51-1 and G6H1 is quantitatively examined
Survey primer pair and probe, it is characterised in that including：
Sense primer shown in SEQ ID No.1 is classified as by nucleotides sequence and anti-sense primer shown in SEQ ID No.2 is constituted turns base
Because of paddy rice KF2 specificity of transformant primer pairs；The nucleotides sequence of transgenic paddy rice KF2 specificity of transformant probes is classified as SEQ ID
Shown in No.3；
Sense primer shown in SEQ ID No.4 is classified as by nucleotides sequence and anti-sense primer shown in SEQ ID No.5 is constituted turns base
Because of paddy rice KF6 specificity of transformant primer pairs；The nucleotides sequence of transgenic paddy rice KF6 specificity of transformant probes is classified as SEQ ID
Shown in No.6；
Sense primer shown in SEQ ID No.7 is classified as by nucleotides sequence and anti-sense primer shown in SEQ ID No.8 is constituted turns base
Because of paddy rice KF8 specificity of transformant primer pairs；The nucleotides sequence of transgenic paddy rice KF8 specificity of transformant probes is classified as SEQ ID
Shown in No.9；
Sense primer shown in SEQ ID No.10 is classified as by nucleotides sequence and anti-sense primer shown in SEQ ID No.11 is constituted turns
Trans-genetic hybrid rice KMD1 specificity of transformant primer pairs；The nucleotides sequence of transgenic paddy rice KMD1 specificity of transformant probes is classified as
Shown in SEQ ID No.12；
Sense primer shown in SEQ ID No.13 is classified as by nucleotides sequence and anti-sense primer shown in SEQ ID No.14 is constituted turns
Trans-genetic hybrid rice M12 specificity of transformant primer pairs；The nucleotides sequence of transgenic paddy rice M12 specificity of transformant probes is classified as SEQ
Shown in ID No.15；
Sense primer shown in SEQ ID No.16 is classified as by nucleotides sequence and anti-sense primer shown in SEQ ID No.17 is constituted turns
Trans-genetic hybrid rice TT51-1 specificity of transformant primer pairs；The nucleotide sequence of transgenic paddy rice TT51-1 specificity of transformant probes
Shown in SEQ ID No.18；And,
Sense primer shown in SEQ ID No.19 is classified as by nucleotides sequence and anti-sense primer shown in SEQ ID No.20 is constituted turns
Trans-genetic hybrid rice G6H1 specificity of transformant primer pairs；The nucleotides sequence of transgenic paddy rice G6H1 specificity of transformant probes is classified as
Shown in SEQ ID No.21.
2. the multiple digital pcr quantitative determination primer pair and probe described in claim 1 detection transgenic paddy rice KF2, KF6,
Application in any one or more in KF8, KMD1, M12, TT51-1 or G6H1.
3. the multiple digital pcr quantitative determination side of a kind of transgenic paddy rice KF2, KF6, KF8, KMD1, M12, TT51-1 and G6H1
Method, it is characterised in that comprise the following steps：
(1) DNA of paddy rice sample to be detected is extracted；(2) it is template with the DNA for extracting, with the multiple numeral described in claim 1
PCR quantitative determinations primer pair and probe, and paddy rice reference gene detection primer pair and probe are set up digital pcr system and are carried out
PCR is expanded；(3) data analysis is carried out to amplification, the ratio between copy number of foreign gene and reference gene copy number is calculated, is obtained
The quantitative values of transgene component.
4. according to the multiple digital pcr quantitative detecting method described in claim 3, it is characterised in that the digital pcr system
For：The cumulative volume of reaction system is 20 μ L, wherein, the μ L of 2xddPCR Supermix for probe 10, described in claim 1
The primer of multiple digital pcr quantitative determination primer pair and probe and paddy rice reference gene detection primer pair and probe composition and spy
The μ L of mixture 6 of pin, the μ L of DNA profiling 4.
5. according to the multiple digital pcr quantitative detecting method described in claim 3, it is characterised in that the paddy rice reference gene
It is rice sucrose phosphatase gene；The paddy rice reference gene detection primer as nucleotides sequence to being classified as shown in SEQ ID No.22
Sense primer and anti-sense primer shown in SEQ ID No.23 are constituted；
The nucleotides sequence of the paddy rice reference gene probe is classified as shown in SEQ ID No.24.
6. according to the multiple digital pcr quantitative detecting method described in claim 4, it is characterised in that：The primer and probe
In mixture, the upstream and downstream primer concentration of paddy rice reference gene detection primer pair is respectively 3000nmol/L, paddy rice reference gene
The concentration of probe is 600nmol/L；
The upstream and downstream primer concentration of multiple digital pcr quantitative determination primer pair is respectively 1500nmol/L described in claim 1；
The concentration of probe described in claim 1 is respectively 300nmol/L.
7. according to the multiple digital pcr quantitative detecting method described in claim 3, it is characterised in that the program of the PCR amplifications
Including：95 DEG C of predegeneration 5min；95 DEG C of denaturation 30sec, 60 DEG C of annealing and extension 60sec, totally 40 circulations；98℃ 10min.
8. according to the multiple digital pcr quantitative detecting method described in claim 3, it is characterised in that the paddy rice reference gene
5 ' end mark fluorescent group HEX of probe, 3 ' hold mark fluorescent quenching group BHQ1；
5 ' end difference mark fluorescent group FAM of probe described in claim 1,3 ' hold mark fluorescent quenching group BHQ1 respectively.
9. the multiple digital pcr quantitative determination examination of a kind of transgenic paddy rice KF2, KF6, KF8, KMD1, M12, TT51-1 and G6H1
Agent box, including：2xddPCR Supermix for probe, paddy rice reference gene detection primer pair and probe, transgenic paddy rice
Multiple digital pcr quantitative determination primer pair and probe；Characterized in that, the multiple digital pcr of the transgenic paddy rice is quantified
Detection primer pair and probe are the multiple digital pcr quantitative determination primer pair and probe described in claim 1；At 5 ' ends of probe
Difference mark fluorescent group FAM, 3 ' hold mark fluorescent quenching group BHQ1 respectively.
10. according to the multiple digital pcr immue quantitative detection reagent box described in claim 9, it is characterised in that the paddy rice internal reference base
Because detection primer is to being classified as sense primer shown in SEQ ID No.22 and anti-sense primer shown in SEQ ID No.23 as nucleotides sequence
Composition；The nucleotides sequence of the paddy rice reference gene probe is classified as shown in SEQ ID No.24,5 ' end mark fluorescent bases of probe
Group HEX, 3 ' hold mark fluorescent quenching group BHQ1.
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