CN107937595A - A kind of method, primer and probe and application that feed transgenic corn IE034 is quantitatively detected based on ddPCR - Google Patents
A kind of method, primer and probe and application that feed transgenic corn IE034 is quantitatively detected based on ddPCR Download PDFInfo
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Abstract
The invention discloses a kind of method, primer and probe and application that feed transgenic corn IE034 is quantitatively detected based on ddPCR, it is related to molecular biology and detection GMOs technical field.The primer and probe includes the primer and probe of foreign gene Cry1Ie and reference gene rubisco respectively, and the quantitative analysis method includes：1) Feed Sample genomic DNA is extracted；2) specific primer and probe of foreign gene Cry1Ie and reference gene rubisco is utilized, ddPCR amplifications are carried out to the genomic DNA after dilution；3) detection and analysis of PCR product.This method independent of standard curve, from the influence of PCR mortifiers and PCR amplification efficiency, the sensitivity with higher and stability compared with Standard PCR, the copy Particle density of feed transgenic precisely can be quantitatively detected, can be to feed transgenic corn IE034 quantitative analyses.
Description
Technical field
The invention belongs to molecular biology and detection GMOs detection technique field, and in particular to feed transgenic corn
The droplet type digital pcr quantitative detecting method of IE034.
Background technology
2016, the cultivated areas of global genetically modified crops increased to 1.851 hundred million hectares from 1,700,000 hectares of 1996,
Wherein the cultivated area of transgenic corns is 60,600,000 hectares, accounts for the 64% of the total cultivated area of global corn, utility ratio 26%.
By the end of 2016, the regulator approval genetically modified crops for sharing 40 country /regions were used as grain and/or feed and release
It is put into environment, there are 1238 to be related to feed applications in 3768 regulatory approvals being approved, including direct purposes or adds
Work purposes, the genetically modified crops accounting for feed is about 33%.Corn is important feedstuff, with feed processing and work
Growth of the industry alcohol to corn demand, transgenic corns proportion in genetically modified crops are planted can also increase year by year.China
It is transgenic corns import big country, import volume in 2015 is 3,300,000 tons of corns, currently accelerates examining for genetically modified crops plantation
Batch, to improve maize production rate and reduce the dependence to the ever-increasing import transgenic corns of quantity.
Genetically modified crops commercial growth brings huge social and economic benefit, while its potential safety to the whole world
Problem also result in the very big concern of people.At present, existing more than 60 a countries have been formulated to containing transgene component or by turning base
Because of the system that the product that crop is process is identified, and increasingly the carrying to transgenic product attention rate with consumer
Height, more highlights the importance that quantitative detection is carried out to transgene component, how accurately, be accurately determined genetically modified crops and
Foreign gene in its product, has become a key point of GMO detection technical research now, and transgenosis
The important parameter of bio-safety evaluation.
At present, generally use real-time fluorescence quantitative PCR (qPCR) carries out the transgene component in transgenic corns IE034
Quantitative detection.Through retrieval, the prior art has issued for relevant technical solution, and China Patent No. 201410457457.5 is public
Open day be 2014.12.10 application case disclose transgenic corns IE034 specificity of transformant quantitative PCR detection primer and
Probe and method, it devises upstream primer sequence, downstream primer sequence and fluorescence probe sequence and carries out PCR amplification, although should
The method of real-time fluorescence quantitative PCR can carry out specific quantification detection to transgenic corns IE034 and its derived varieties, but
Still remain the defects of certain：When analyzing copy number of foreign gene, real-time fluorescence quantitative PCR be necessarily dependent upon standard curve and
The gene of known copy number, simply a kind of relative quantitation method, and the quality of standard curve is vulnerable to DNA purity, primer and spy
The factors such as the concentration of pin, the response inhabitation factor influence so that detection sensitivity and accuracy all do not reach accurate quantitative analysis
It is required that the content of too low or excessive estimation transgenosis may be caused.Be additionally, since foreign gene be typically with low copy number (1~
2) it is incorporated into acceptor gene group, during transgene component in detection of complex material, qPCR is easily done be subject to background dna
Disturb.And the droplet type digital pcr (ddPCR) of rising in recent years is a kind of new absolute quantitation technology, realized by extreme dilution
Theoretic unimolecule amplification so that rare target nucleic acid sequence is separated with substantial amounts of background dna, improves its relative amount,
And then sensitivity and the repeatability of detection are improved, the technology is not answered in the detection field of transgenic corns IE034 at present
With.
Therefore, based on prior art the defects of, precisely quantitatively examine feed transgenic corn IE034 it is urgent to provide a kind of
The method of survey.
The content of the invention
1. the technical problem solved：
The existing round pcr of detection sensitivity and accuracy based on to(for) feed transgenic corn IE034 all do not reach
The requirement of accurate quantitative analysis, the present invention provide a kind of method precisely quantitatively detected to feed transgenic corn IE034.
2. technical solution：
The purpose of the present invention is what is be achieved through the following technical solutions：
The present invention provides a kind of primer and probe that feed transgenic corn IE034 is quantitatively detected based on ddPCR, bag
Include：
Foreign gene Cry1Ie forward primers：5′-AGAGCTGGGTGCGCTACAAC-3′
Foreign gene Cry1Ie reverse primers：5′-ACACCAGGGTGTCGTAGCTG-3′
Foreign gene Cry1Ie probes：5′-FAM-TTCCGCAAGGACATGACCCTGATGGT-TAMAR-3′
Reference gene rubisco forward primers：5′-ATGTAGCTTATCCATTAGAC-3′
Reference gene rubisco reverse primers：5′-TTGAAACCAAATACGTTACC-3′
Reference gene rubisco probes：5′-FAM-ATTTGAAGAGGGTTCTGTTACTAACAT-TAMAR-3′
As further improvement of the present invention, PCR amplification is carried out using primer and probe.
As further improvement of the present invention, feed transfer is quantitatively detected based on ddPCR present invention also offers one kind
The method of gene corn IE034, comprises the following steps：
1) genomic DNA of Feed Sample, is extracted；
2), ddPCR is expanded：Using the genomic DNA described in step 1) as template, the primer and probe, difference are utilized
Corresponding digital pcr amplification reaction system and response procedures are set according to foreign gene Cry1Ie and reference gene rubisco, into
Row amplified reaction, obtains pcr amplification product；
3), the pcr amplification product that step 2) obtains is detected and analyzed.
It is when amplification gene is foreign gene Cry1Ie, genomic DNA concentration is dilute as further improvement of the present invention
Release to 10~100ng/ μ L；When amplification gene is corn reference gene rubisco, by genomic DNA concentration dilution to 1~
0.1ng/μL。
As further improvement of the present invention, primer concentration is 10 μ in primer and probe combination in the step 2)
Mol/L, concentration and probe concentration are 5 μm of ol/L.
As further improvement of the present invention, the digital pcr amplification reaction system bag of the foreign gene Cry1Ie
Include：10 μ L of 2X ddPCRSupermix for Probes, each 0.4 μ L of upstream and downstream primer, concentration is 10 μm of ol/L, 0.6 μ of probe
L, concentration are 5 μm of ol/L, 2.5 μ L of DNA profiling, and aseptic deionized water complements to 20 μ L；The number of the reference gene rubisco
Word pcr amplification reaction system includes：13.5 μ L of 2X ddPCRSupermix for Probes, upstream and downstream primer each 0.3 μ L are dense
Spend for 10 μm of ol/L, 0.2 μ L of probe, concentration is 5 μm of ol/L, 3 μ L of DNA profiling, and aseptic deionized water complements to 20 μ L.
As further improvement of the present invention, the digital pcr amplified reaction program of foreign gene Cry1Ie is：95 DEG C pre-
120s is denatured, 95 DEG C of denaturation 10s, 59 DEG C of annealing 30s, 72 DEG C of extension 30s, 47 circulate altogether；Expand reference gene rubisco
Digital pcr amplified reaction program be：95 DEG C of pre-degenerations 600s, 95 DEG C of denaturation 15s, 54 DEG C of annealing 30s, 72 DEG C of extension 30s, altogether
45 circulations.
As further improvement of the present invention, the step 3) comprises the following steps：
A) PCR product of gained is placed in droplet analyzer, and set on QuantSoft softwares detection pattern as
ABS, detects the fluorescence signal of FAM；
B) droplet analyzer carries out fluoroscopic examination and analysis one by one to each droplet；
C) the copy Particle density of foreign gene and reference gene in each Feed Sample is calculated by QuantSoft.
As further improvement of the present invention, the method is applied to the quantitative inspection of feed transgenic corn IE034
Survey.
3. beneficial effect：
Compared with prior art, the beneficial effects of the present invention are：
(1) primer and probe that feed transgenic corn IE034 is quantitatively detected based on ddPCR of the invention, Neng Goushi
Now to the specific amplification of foreign gene in feed and corn reference gene, with transgenic corns IE034's of the prior art
PCR amplification is compared, and the accuracy and sensitivity of detection method is greatly improved.
(2) method that feed transgenic corn IE034 is quantitatively detected based on ddPCR of the invention, is directed to existing skill
When fluorescence quantitative PCR detection feed transgenic corn IE034 is used in art, easily inhibitor is disturbed in by background dna, with
And different samples and the influence of PCR amplification efficiency change during experiment, cause the defects of sensitivity is low, accuracy is poor, it is real
The ddPCR amplifications of feed transgenic corn IE034 genomic DNAs are showed, pcr amplification product meets under the conditions of this method
The testing conditions of ddPCR, finally realize the absolute quantitation detection of feed transgenic corn IE034 foreign genes, detected
PCR amplification efficiency during standard curve, the interference from inhibitor in background dna and different samples are not depended in journey and is tested
The influence of change, has the sensitivity and accuracy of higher.
(3) method that feed transgenic corn IE034 is quantitatively detected based on ddPCR of the invention, according to foreign gene
The characteristics of with reference gene rubisco, genomic DNA is diluted, when amplification gene is foreign gene Cry1Ie, gene
Group DNA concentration scope is 10~100ng/ μ L；When amplification gene is corn reference gene rubisco, genomic DNA concentration range
1~0.1ng/ μ L, the amplification times of ddPCR are can reach in the range of said gene group DNA concentration, meet testing conditions, the party
Method is simple to genomic DNA processing method, beneficial to popularization.
(4) method that feed transgenic corn IE034 is quantitatively detected based on ddPCR of the invention, respectively according to external source
The characteristics of primer and probe of gene C ry1Ie and reference gene rubisco, sets ddPCR amplification reaction systems and response procedures,
Ensure that the ddPCR amplification reaction systems of use and response procedures are suitable for the primer and probe that the present invention designs, in suitable base
Because realizing the amplified reaction of ddPCR under the conditions of group DNA profiling, have the characteristics that with strong points, further improve what is finally detected
Accuracy and sensitivity.
(5) method that feed transgenic corn IE034 is quantitatively detected based on ddPCR of the invention, this method can be real
The quantitative detection of foreign gene in existing transgenic corns IE034 and products thereof, one is provided for the analysis and detection of transgenic product
Fixed guiding value.
Brief description of the drawings
Fig. 1 is the ddPCR amplifications of foreign gene when experimental group Feed Sample genomic DNA concentration is 100ng/ μ L；
Fig. 2 is the ddPCR amplifications of foreign gene when experimental group Feed Sample genomic DNA concentration is 10ng/ μ L；
Fig. 3 is the ddPCR amplifications of foreign gene when experimental group Feed Sample genomic DNA concentration is 1ng/ μ L；
Fig. 4 is the ddPCR amplifications of reference gene when experimental group Feed Sample genomic DNA concentration is 10ng/ μ L；
Fig. 5 is the ddPCR amplifications of reference gene when experimental group Feed Sample genomic DNA concentration is 1ng/ μ L；
Fig. 6 is the ddPCR amplifications of reference gene when experimental group Feed Sample genomic DNA concentration is 0.1ng/ μ L；
Fig. 7 is the ddPCR amplifications of foreign gene when control group Feed Sample genomic DNA concentration is 100ng/ μ L；
Fig. 8 is the ddPCR amplifications of foreign gene when control group Feed Sample genomic DNA concentration is 10ng/ μ L；
Fig. 9 is the ddPCR amplifications of foreign gene when control group Feed Sample genomic DNA concentration is 1ng/ μ L；
Figure 10 is the ddPCR amplifications of reference gene when control group Feed Sample genomic DNA concentration is 10ng/ μ L；
Figure 11 is the ddPCR amplifications of reference gene when control group Feed Sample genomic DNA concentration is 1ng/ μ L；
Figure 12 is the ddPCR amplifications of reference gene when control group Feed Sample genomic DNA concentration is 0.1ng/ μ L.
Embodiment
The embodiment of the present invention will be described in further detail below.
Embodiment 1
1st, experiment forage compounding：
Experiment is divided into two kinds of experimental group and control group with feed, and experimental group field corn component is transgenic corns IE034,
Control group field corn component is the non-transgenic corn comprehensive 31 of near isogenic lines, and experimental group and control group forage compounding scheme are such as
Shown in table 1.
1 experimental group of table and control group forage compounding scheme
Wherein, fish meal producer is the peru fish meal of Shandong Tiancheng Biological Technology Co., Ltd.'s production；Casein producer is
sigma；The producer of gelatin is sigma；The producer of cod-liver oil is Norway AxellusAS, Orkla；Multi-vitamins mineral matter
Science and Technology Ltd. is herded for Xinxiang eight by producer；The producer of Carophyll red 10% is Royal DSM nutrition product method Co., Ltd.
The used transgenic corns IE034 of this experiment and its parent's non-transgenic corn comprehensive 31 are by Scientia Agricultura Sinica
Institute's crop research institute provides.
2nd, feed extracting genome DNA
Using animal derived plant feed genome DNA extracting reagent kit DP323, which buys from Tiangeng biochemistry section
Skill Co., Ltd, experimental group and control group feed genomic DNA are extracted using DNA extraction kit DP323 respectively：
1) prepared feed is ground, takes the Feed Sample after 50mg crushing to add 320 μ L buffer solution GA, vibration
Suspend to thorough；
2) 20 μ L Proteinase K Solutions are added, are mixed, 20~30min is placed at 65 DEG C, mix sample therebetween 2~3 times；
3) 340 μ L buffer solution GB are added, fully reverse to mix, 65 DEG C of placement 10min, mix sample 2~3 times therebetween；
4) 2min is centrifuged with the rotating speed of 12000rpm, takes 440 μ L of supernatant liquid into new pipe；
5) 220 μ L absolute ethyl alcohols are added into supernatant, it is fully reverse to mix 6~8 times；
6) by (adsorption column is put into collecting pipe in previous step resulting solution and flocculent deposit all one adsorption column CB3 of addition
In), 12000rpm centrifugation 2min, outwell waste liquid, adsorption column CB3 are put back in collecting pipe；
7) 500 μ L buffer solutions GD, 12000rpm centrifugation 30s are added into adsorption column CB3, waste liquid are outwelled, by adsorption column CB3
Put back in collecting pipe；
8) 600 μ L rinsing liquids PW, 12000rpm centrifugation 30s are added into adsorption column CB3, outwell waste liquid.By adsorption column CB3
It is put into collecting pipe；
9) 500 μ L buffer solutions GD, 12000rpm centrifugation 30s are added into adsorption column CB3, waste liquid are outwelled, by adsorption column CB3
Put back in collecting pipe；
10) adsorption column CB3 is put back in collecting pipe, 12000rpm centrifugation 2min, outwell waste liquid, adsorption column CB3 is placed in
Room temperature is placed several minutes, thoroughly to dry rinsing liquid remaining in sorbing material；
11) adsorption column CB3 is transferred in a clean centrifuge tube, 50 μ L is vacantly added dropwise to the middle part of adsorbed film and wash
De- buffer solution TE, room temperature place 2~5min, 12000rpm centrifugations 2min.
3rd, primer and probe
Digital pcr amplification is carried out with the combination of following primer and probe, primer and probe has by precious bioengineering (Dalian)
Limit company synthesizes, and the probe 5 ' of wherein foreign gene Cry1Ie marks luminophore FAM, and 3 ' mark quenching group TAMAR, internal reference
Gene rubisco probes 5 ' mark luminophore FAM, 3 ' mark quenching group TAMAR：
Foreign gene Cry1Ie forward primers：5′-AGAGCTGGGTGCGCTACAAC-3′
Foreign gene Cry1Ie reverse primers：5′-ACACCAGGGTGTCGTAGCTG-3′
Foreign gene Cry1Ie probes：5′-FAM-TTCCGCAAGGACATGACCCTGATGGT-TAMAR-3′
Reference gene rubisco forward primers：5′-ATGTAGCTTATCCATTAGAC-3′
Reference gene rubisco reverse primers：5′-TTGAAACCAAATACGTTACC-3′
Reference gene rubisco probes：5′-FAM-ATTTGAAGAGGGTTCTGTTACTAACAT-TAMAR-3′
4th, ddPCR is expanded
Using the experimental group of ultraviolet specrophotometer quantitative analysis extraction and the concentration of control group Feed Sample genomic DNA
And purity, the results showed that, the OD260nm/OD280nm ratios of experimental group and control group Feed Sample genomic DNA 1.7 to
Between 1.9, the concentration of experimental group and control group Feed Sample genomic DNA is each about 100ng/ μ L.
Experimental group and control group Feed Sample genomic DNA are diluted before ddPCR amplifications, expand foreign gene
Sample gene group DNA is diluted to 100ng/ μ L, 10ng/ μ L and 1ng/ μ tri- concentration gradients of L respectively during Cry1Ie；Amplification is beautiful
Sample gene group DNA is diluted to 10ng/ μ L, 1ng/ μ L and 0.1ng/ μ tri- concentration gradients of L during rice reference gene rubisco.
Above-mentioned primer and probe is subjected to dissolved dilution with TE buffer solutions, the concentration after primer dilution is 10 μm of ol/
L, the concentration after probe dilution is 5 μm of ol/L.
Using the genomic DNA after dilution as template, combined using the primer and probe after dilution, prepare corresponding ddPCR
Amplification reaction system, sets response procedures, and every group of sample sets 3 parallel progress ddPCR amplified reactions respectively, obtains PCR expansions
Increase production thing.The correspondence of genomic DNA template concentration and amplification gene after dilution is as shown in table 2.
2 genomic DNA template concentration of table and amplification gene correspondence
The digital pcr amplification reaction system of foreign gene Cry1Ie is as shown in table 3.
The digital pcr amplification reaction system of 3 foreign gene Cry1Ie of table
The digital pcr amplification reaction system of reference gene rubisco is as shown in table 4.
The digital pcr amplification reaction system of 4 reference gene rubisco of table
Prepared example reaction system is added in the sample well of droplet card, pay attention to avoiding producing during sample-adding
Bubble, then oil is occurred into for the droplet of 70 μ L and is added in reaction oilhole, by sealing gasket cover on card base occurs, it is placed in droplet hair
The droplet of Water-In-Oil is generated in raw device, occurs to draw 40 μ L droplets in the reacting hole of card upper strata from droplet, is transferred to 96 hole PCR reactions
In one reacting hole of plate, pay attention to avoiding the destruction and aggregation of droplet, 180 DEG C of heat-sealings of sealer instrument, after heat-sealing aluminium film sealing
It is placed in PCR instrument and sets response procedures progress PCR amplification.
The pcr amplification reaction program of foreign gene is：95 DEG C of pre-degenerations 120s, 95 DEG C of denaturation 10s, 59 DEG C of 30s that anneal, 72
DEG C extension 30s, altogether 47 circulation；The pcr amplification reaction program of reference gene is：95 DEG C of pre-degeneration 600s, 95 DEG C of denaturation
15s, 54 DEG C of annealing 30s, 72 DEG C of extension 30s, totally 45 circulate.
5th, ddPCR results read and analyze
DdPCR is placed in droplet analyzer (QX200, BIO-RAD, USA) after reaction, by the PCR reaction plates after amplification
It is interior, QuantSoft softwares are opened, click on Flush System key rinse-systems, then click on Setup key input sample messages,
Detection pattern is set as ABS, detects the fluorescence signal of FAM, and Run is clicked on after the completion of input, and droplet analyzer can be automatically to each
Droplet carries out fluoroscopic examination and analysis, QuantSoft softwares according to the positive droplet detected and the ratio of feminine gender droplet, according to
Poisson distribution principle, which calculates, obtains the copy Particle density of foreign gene and reference gene in sample.
Droplet generation is the committed step of droplet type digital pcr, when only droplet number is more than 11000, the distribution of DNA molecular
The Principle of Statistics of Poisson distribution can just be met, system can just count positive droplet and negative droplet.
Fig. 1 is experimental group Feed Sample genomic DNA concentration when being 100ng/ μ L, the amplification figure of foreign gene Cry1Ie,
The black signal point of top half represents the droplet that PCR amplification occurs in figure, and system interpretation is positive signal, and positive droplet number is
2883；The black signal point of the latter half represents the droplet that PCR amplification does not occur, and system interpretation is negative signal, negative droplet
Number is 13171.
Fig. 2 is experimental group Feed Sample genomic DNA concentration when being 10ng/ μ L, the amplification figure of foreign gene Cry1Ie, figure
The black signal point of middle top half represents the droplet that PCR amplification occurs, and system interpretation is positive signal, and positive droplet number is
306；The black signal point of the latter half represents the droplet that PCR amplification does not occur, and system interpretation is negative signal, negative droplet number
For 15281.
Fig. 3 is experimental group Feed Sample genomic DNA concentration when being 1ng/ μ L, and the amplification figure of foreign gene Cry1Ie, is
System is not detected by positive signal, shows that positive droplet number is 0 in figure；Black signal point represents the droplet that PCR amplification does not occur, is
System interpretation is negative signal, and negative droplet number is 15938.
Fig. 4 is experimental group Feed Sample genomic DNA concentration when being 10ng/ μ L, the amplification figure of reference gene rubisco,
System is not detected by positive signal, shows that positive droplet number is 0 in figure；Black signal point represents the droplet that PCR amplification does not occur,
System interpretation is negative signal, and negative droplet number is 15656.
Fig. 5 is experimental group Feed Sample genomic DNA concentration when being 1ng/ μ L, the amplification figure of reference gene rubisco, figure
The black signal point of middle top half represents the droplet that PCR amplification occurs, and system interpretation is positive signal, and positive droplet number is
15454；The black signal point of the latter half represents the droplet that PCR amplification does not occur, and system interpretation is negative signal, negative droplet
Number is 3344.
Fig. 6 is experimental group Feed Sample genomic DNA concentration when being 0.1ng/ μ L, the amplification figure of reference gene rubisco,
The black signal point of top half represents the droplet that PCR amplification occurs in figure, and system interpretation is positive signal, and positive droplet number is
2158；The black signal point of the latter half represents the droplet that PCR amplification does not occur, and system interpretation is negative signal, negative droplet
Number is 12620.
Fig. 7 is control group Feed Sample genomic DNA concentration when being 100ng/ μ L, the amplification figure of foreign gene Cry1Ie,
System is not detected by positive signal, shows that positive droplet number is 0 in figure；Black signal point represents the droplet that PCR amplification does not occur,
System interpretation is negative signal, and negative droplet number is 15110.
Fig. 8 is control group Feed Sample genomic DNA concentration when being 10ng/ μ L, and the amplification figure of foreign gene Cry1Ie, is
System is not detected by positive signal, shows that positive droplet number is 0 in figure；Black signal point represents the droplet that PCR amplification does not occur, is
System interpretation is negative signal, and negative droplet number is 15034.
Fig. 9 is control group Feed Sample genomic DNA concentration when being 1ng/ μ L, and the amplification figure of foreign gene Cry1Ie, is
System is not detected by positive signal, shows that positive droplet number is 0 in figure；Black signal point represents the droplet that PCR amplification does not occur, is
System interpretation is negative signal, and negative droplet number is 14442.
Figure 10 is control group Feed Sample genomic DNA concentration when being 10ng/ μ L, the amplification figure of reference gene rubisco,
System is not detected by positive signal, shows that positive droplet number is 0 in figure；Black signal point represents the droplet that PCR amplification does not occur,
System interpretation is negative signal, and negative droplet number is 13600.
Figure 11 is control group Feed Sample genomic DNA concentration when being 1ng/ μ L, the amplification figure of reference gene rubisco,
The black signal point of top half represents the droplet that PCR amplification occurs in figure, and system interpretation is positive signal, and positive droplet number is
14016；The black signal point of the latter half represents the droplet that PCR amplification does not occur, and system interpretation is negative signal, negative droplet
Number is 2087.
Figure 12 is control group Feed Sample genomic DNA concentration when being 0.1ng/ μ L, the amplification of reference gene rubisco
Figure, the black signal point of top half represents the droplet that PCR amplification occurs in figure, and system interpretation is positive signal, positive droplet
Number is 3439；The black signal point of the latter half represents the droplet that PCR amplification does not occur, and system interpretation is negative signal, negative
Droplet number is 14499.
The total droplet number formed during the present embodiment ddPCR is reacted counts, and statistical result is as shown in table 5.
5 experimental group of table and control group Feed Sample ddPCR expand the total droplet number to be formed
As shown in Table 5, the droplet number of generation is between 12856~18930, is all higher than 11000, meets droplet type numeral
The analysis requirement of PCR droplets.
The copy of foreign gene and reference gene in experimental group and control group Feed Sample is read according to QuantSoft softwares
Particle density is as shown in table 6, wherein, No call represent not detect.
The copy number of foreign gene and reference gene in 6 experimental group of table and control group Feed Sample
As shown in Table 6, when genomic DNA concentration is 100ng/ μ L, external source gene C ry1Ie in experimental group Feed Sample
Copy number mean concentration is 240.33copies/ μ L；When genomic DNA concentration is 10ng/ μ L, foreign gene Cry1Ie's copies
Shellfish Particle density average value is 23.63copies/ μ L；When genomic DNA concentration is 1ng/ μ L, do not detected in experimental group Feed Sample
To foreign gene；The result shows that when quantitatively being detected to the foreign gene Cry1Ie progress ddPCR in Feed Sample, template DNA is dense
Degree should be 10~100ng/ μ L.Foreign gene is not detected by control group Feed Sample, illustrates result and the expection of digital pcr
It is consistent.
When genomic DNA concentration is 10ng/ μ L, corn internal reference base is not detected by experimental group and control group Feed Sample
Because of rubisco；When genomic DNA concentration is 1ng/ μ L, the copy number of reference gene is dense in experimental group and control group Feed Sample
It is respectively 2099.67copies/ μ L and 2221.67copies/ μ L to spend average value；It is interior when genomic DNA concentration is 0.1ng/ μ L
The copy number mean concentration for joining gene is respectively 216.67copies/ μ L and 219.33copies/ μ L；As a result illustrate to feed
When corn reference gene rubisco progress ddPCR in sample is quantitatively detected, the concentration of template DNA should be 0.1~1ng/ μ L.
The present invention the result shows that, ddPCR precisely can quantitatively detect foreign gene and reference gene in Feed Sample,
With good repeatability and stability.
Schematically the present invention and embodiments thereof are described above, this describes no restricted, institute in attached drawing
What is shown is also one of embodiments of the present invention, and actual flow is not limited thereto.So if common skill of this area
Art personnel are enlightened by it, without departing from the spirit of the invention, are not inventively designed and the technical solution
Similar frame mode and embodiment, are within the scope of protection of the invention.
Claims (9)
- A kind of 1. primer and probe that feed transgenic corn IE034 is quantitatively detected based on ddPCR, it is characterised in that：Including：Foreign gene Cry1Ie forward primers：5 '-AGAGCTGGGTGCGCTACAAC-3 ',Foreign gene Cry1Ie reverse primers：5 '-ACACCAGGGTGTCGTAGCTG-3 ',Foreign gene Cry1Ie probes：5′-FAM-TTCCGCAAGGACATGACCCTGATGGT-TAMAR-3′；Reference gene rubisco forward primers：5 '-ATGTAGCTTATCCATTAGAC-3 ',Reference gene rubisco reverse primers：5 '-TTGAAACCAAATACGTTACC-3 ',Reference gene rubisco probes：5′-FAM-ATTTGAAGAGGGTTCTGTTACTAACAT-TAMAR-3′.
- A kind of 2. method that feed transgenic corn IE034 is quantitatively detected based on ddPCR, it is characterised in that：Usage right will The primer and probe described in 1 is asked to carry out PCR amplification.
- 3. the method according to claim 2 that feed transgenic corn IE034 is quantitatively detected based on ddPCR, its feature It is：Comprise the following steps：1) genomic DNA of Feed Sample, is extracted；2), ddPCR is expanded：Using the genomic DNA described in step 1) as template, using the primer and probe, basis respectively Foreign gene Cry1Ie and reference gene rubisco sets corresponding digital pcr amplification reaction system and response procedures, carries out DdPCR amplified reactions, obtain pcr amplification product；3), the pcr amplification product that step 2) obtains is detected and analyzed.
- 4. the method according to claim 3 that feed transgenic corn IE034 is quantitatively detected based on ddPCR, its feature It is：When amplification gene is foreign gene Cry1Ie, by genomic DNA concentration dilution to 10~100ng/ μ L；Amplification gene is During corn reference gene rubisco, by genomic DNA concentration dilution to 1~0.1ng/ μ L.
- 5. the method according to claim 3 that feed transgenic corn IE034 is quantitatively detected based on ddPCR, its feature It is：Primer concentration is 10 μm of ol/L in primer and probe combination in the step 2), and concentration and probe concentration is 5 μm of ol/L.
- 6. the method according to claim 3 that feed transgenic corn IE034 is quantitatively detected based on ddPCR, its feature It is：The digital pcr amplification reaction system of the foreign gene Cry1Ie includes：2X ddPCRSupermix for Probes10 μ L, each 0.4 μ L of upstream and downstream primer, concentration are 10 μm of ol/L, 0.6 μ L of probe, and concentration is 5 μm of ol/L, DNA profiling 2.5 μ L, aseptic deionized water complement to 20 μ L；The digital pcr amplification reaction system of the reference gene rubisco includes： 13.5 μ L of 2XddPCRSupermix for Probes, each 0.3 μ L of upstream and downstream primer, concentration are 10 μm of ol/L, 0.2 μ L of probe, Concentration is 5 μm of ol/L, 3 μ L of DNA profiling, and aseptic deionized water complements to 20 μ L.
- 7. the method that feed transgenic corn IE034 is quantitatively detected based on ddPCR according to claim 3 or 6, it is special Sign is：The digital pcr amplified reaction program of the foreign gene Cry1Ie is：95 DEG C of pre-degeneration 120s, 95 DEG C of denaturation 10s, 59 DEG C of annealing 30s, 72 DEG C of extension 30s, 47 circulate altogether；The digital pcr amplification of the reference gene rubisco is anti- The program is answered to be：95 DEG C of pre-degeneration 600s, 95 DEG C of denaturation 15s, 54 DEG C of annealing 30s, 72 DEG C of extension 30s, totally 45 circulate.
- 8. the method according to claim 3 that feed transgenic corn IE034 is quantitatively detected based on ddPCR, its feature It is：The step 3) comprises the following steps：A) PCR product of gained is placed in droplet analyzer, and detection pattern is set as ABS on QuantSoft softwares, examined Survey the fluorescence signal of FAM；B) droplet analyzer carries out fluoroscopic examination and analysis one by one to each droplet；C) the copy Particle density of foreign gene and reference gene in Feed Sample is calculated by QuantSoft.
- 9. the application of the method according to claim 2 that feed transgenic corn IE034 is quantitatively detected based on ddPCR, It is characterized in that：The method is applied to the quantitative detection of feed transgenic corn IE034.
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| CN109971879A (en) * | 2019-03-18 | 2019-07-05 | 中国检验检疫科学研究院 | Detect the PCR primer combination and application of transgenic soybean lines MON87705 |
| CN110616271A (en) * | 2019-10-12 | 2019-12-27 | 生态环境部南京环境科学研究所 | Specific primer for quantitatively detecting biomass of red-eared turtle in environment and detection method and application thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109971879A (en) * | 2019-03-18 | 2019-07-05 | 中国检验检疫科学研究院 | Detect the PCR primer combination and application of transgenic soybean lines MON87705 |
| CN110616271A (en) * | 2019-10-12 | 2019-12-27 | 生态环境部南京环境科学研究所 | Specific primer for quantitatively detecting biomass of red-eared turtle in environment and detection method and application thereof |
| CN110616271B (en) * | 2019-10-12 | 2022-05-03 | 生态环境部南京环境科学研究所 | Specific primer for quantitatively detecting biomass of red-eared turtle in environment and detection method and application thereof |
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