CN105255874B - A kind of kit and detection method of accurate quantification detection Transgenic corn lines T25 - Google Patents
A kind of kit and detection method of accurate quantification detection Transgenic corn lines T25 Download PDFInfo
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Abstract
The invention discloses the kits and detection method of a kind of accurate quantification detection Transgenic corn lines T25.In particular it relates to which one group for detecting the primer and probe of Transgenic corn lines T25, with nucleotide sequence shown in sequence table SEQ ID No.1 to SEQ ID No.6, and the kit containing these primer and probes.The present invention also provides a kind of detection methods using dual droplet type digital pcr technology accurate quantification detection Transgenic corn lines T25, this method is without relying on certified reference material or other standards product, with higher accuracy, sensitivity and repeatability, it is easy to standardize.
Description
Technical field
The present invention relates to the kits and detection method of a kind of quantitative detection Transgenic corn lines T25, belong to molecular biosciences
Field.
Background technique
With extensive plantation of the genetically modified crops in worldwide, edible safety and environmental security always by
To the majority of consumers, the great attention of national governments and related international organization.Transgenosis has been worked out one after another in many countries and regions
Biosafety management regulation implements mark system.The Ministry of Agriculture, China has issued " agriculture genetically modified organism on January 5th, 2002
Mark management method ", it is specified that sale is included in the agriculture genetically modified organism of catalogue within Chinese territory, there should be apparent mark.It is modern
New " the law of food safety " that October in year implements further has made the clearly rule that " must significantly indicate " to GM food
Fixed, this will necessarily propose higher demand to the detection of transgene component.However it is big different from European Union (identification thresholds 0.9), Australia
The countries such as Leah (identification thresholds 1.0), South Korea's (identification thresholds 3.0), Japanese (identification thresholds 5.0), China, which is not yet put into effect, turns base
Because of identification thresholds regulation, qualitative i.e. zero threshold value of mark is taken, this is with the existing detection GMOs in China mainly with qualitative method
Based on, the standard method that lacking can quantify has some relations.Therefore, the accurate quantitative detecting method of transgenosis is established in research, can be
China's transgenic product quantitative identifying and threshold management lay the foundation, and promotion is in line with international standards, and in maintenance international trade
National interests provide technical guarantee.
The content of transgene component in food is usually to be indicated with the ratio of number of transgene and endogenous gene, real
When fluorescent PCR (qPCR) be general quantitative detecting method in the world now.This method is examined in real time using the variation of fluorescence signal
The variation for surveying amplified production amount in pcr amplification reaction, passes through the certified reference material (Certified to gradient dilution
Reference Materials, CRM) detection, obtain endogenous and foreign gene standard curve respectively, and then pass through circulation threshold
Endogenous in sample to be tested and foreign gene content is calculated in value (Ct).As it can be seen that the inhibition ingredient of nucleic acids in samples amplification, reality
The quality of amplification efficiency and standard curve can be directly affected by testing environment and the technical ability of operator etc. all, thus interfere it is quantitative accurate
Property.
The droplet type digital pcr technology (droplet digital PCR, ddPCR) newly risen in recent years, by dilution
It is distributed in 10000~20000 droplets to certain density DNA molecular, makes the quantity 1 of DNA molecular in most of droplet
Or 0, positive reaction unit number is then read by the accumulation of PCR amplification and positive signal, is calculated further according to Poisson distribution
DNA molecular copy number in sample.Currently, transgenosis quantitative detection is carried out still in its infancy using ddPCR, research report
Quantity is few, primarily directed to Mon810 transgenic corns, to the validity and practicability of remaining transgenic strain quantitative detection
It is on the knees of the gods, it development space and has a extensive future.
Transgenic corns are most wide one of the genetically modified crops of grown worldwide area, choose wherein representative strain
T25 is research object, analyzes its gene composition, selects the external source assortment of genes in suitable, design high special primer and
Probe is established and is suitable for the quantitative dual ddPCR detection method of T25 transgenosis.Through specificity, detection dynamic range, LOD,
LOQ, accuracy and repeatability verifying, it was demonstrated that this method is met in the world to the technical requirements of transgenosis quantitative detection, thus
It lays the foundation for the accurate quantitative study of other genetically modified crops.
Summary of the invention
The technical problem to be solved in the present invention is to provide one group for detecting primer and the spy of Transgenic corn lines T25
Needle.
The invention solves another technical problem be to provide a kind of accurate quantification detection Transgenic corn lines T25's
Kit.
The invention solves third technical problem be to provide a kind of accurate quantification detection Transgenic corn lines T25's
Detection method.
To achieve the above object, the invention adopts the following technical scheme:
It is by outer for expanding the present invention provides one group for detecting the primer and probe of Transgenic corn lines T25
The primer and probe of source gene pat-T35S, and the primer and probe composition for expanding endogenous gene zein, sequence are shown in Table
1.Wherein, the end of probe 5 ' the label HEX of foreign gene pat-T35S is expanded, 3 ' end label BHQ expand the spy of endogenous gene zein
Needle 5 ' holds flag F AM, 3 ' end label BHQ.
1 ddPCR primer of table/probe sequence
The present invention also provides the kits of accurate quantification detection Transgenic corn lines T25 a kind of, which includes upper
State the primer and probe for detecting Transgenic corn lines T25.Further, which further includes completing dual number
PCR reacts material requested and reagent, such as positive control, ddPCR master mix, magnesium acetate, droplet generate oil, droplet life
Cheng Ka, 96 orifice plates and aluminium foil heat-sealing film etc., above-mentioned material and reagent are preferably individually packed.
It is especially a kind of the present invention also provides the detection method of accurate quantification detection Transgenic corn lines T25 a kind of
Using the method for dual digital pcr technology accurate quantification detection Transgenic corn lines T25, this method comprises:
(1) sample to be tested DNA is extracted, extracting method or commercial kit well known in the prior art can be used and carry out
It extracts；
(2) dual ddPCR reaction system is prepared, wherein the reaction system includes that SEQ ID NO:1 is extremely in sequence table
SEQ ID NO:4 in primer and probe and sequence table shown in SEQ ID NO:3 for expanding foreign gene pat-T35S
To the primer and probe for being used to expand endogenous gene zein shown in SEQ ID NO:6；Preferably, specific at of the invention one
In embodiment, dual ddPCR is shown in reaction system such as table 2；
The 2 dual ddPCR reaction system of corn gene strain T25 of table
| Component | Working solution concentration | Sample-adding amount μ L | Reaction system final concentration |
| ddPCR master mix | 2× | 10 | 1× |
| Magnesium acetate | 25mM | 0.8 | 1mM |
| T25-F | 10μM | 1.5 | 750nM |
| T25-R | 10μM | 1.5 | 750nM |
| T25-P | 10μM | 1.5 | 750nM |
| Zein-F | 10μM | 1.5 | 750nM |
| Zein-R | 10μM | 0.7 | 350nM |
| Zein-P | 10μM | 0.5 | 250nM |
| DNA profiling | 100~60ng/ μ L | 1 | / |
| Deionized water | / | 1 | / |
(3) the dual ddPCR reaction system and droplet prepared step (2) generate oil and are added in droplet generation card, are placed in
Droplet generates in instrument and generates droplet；
(4) droplet of the Water-In-Oil of generation is transferred in 96 orifice plates, is carried out amplification reaction, reaction condition is shown in Table 3；
3 corn gene strain T25ddPCR reaction condition of table
Note: warming and cooling rate answers≤2.5 DEG C/s
(5) result judgement.
96 orifice plates after amplification are placed in droplet to read in instrument (QX100, Bio-Rad, Pleasanton, CA), are utilized
QuantaSoft software carries out result reading and analysis.Droplet reads two signal paths in instrument and reads endogenous gene zein respectively
FAM and foreign gene pat-T35S HEX fluorescence signal, the positive droplet containing amplified production be free of amplified production yin
Property droplet can show the difference of fluorescence signal intensity, can the highest point of negative droplet cluster fluorescence amplitude be that boundary carrys out given threshold
Line.The copy number for obtaining the inside and outside source gene of corn is calculated according to Poisson distribution principle, and then is turned by the calculating of following formula
The percentage composition of gene corn T25.
The invention has the advantages that 1) caused by avoiding the absolute quantitation of nucleic acid molecules by PCR amplification efficiency variance
Deviation；2) without relying on certified reference material or other standards product；3) there is higher accuracy, sensitivity and repeatability, easily
In standardization；4) it is more suitable for the detection of nucleic acids of low copy number, suitable for the accurate quantitative of low abundance transgene component；5) due to
DdPCR is end point determination, higher to the tolerance level of inhibitor, therefore the detection error caused by can reducing by matrix type.6)
It is marked by different fluorescence signals, is easier to realize the dual amplified reaction of the interior foreign gene in same reaction system, thus
Reduce testing cost.
The invention will be further described with specific embodiment with reference to the accompanying drawings of the specification, all to disclose according to the present invention
The equivalent replacement of any this field that content is done, all belongs to the scope of protection of the present invention.
Detailed description of the invention
The ddPCR testing result of zein (A) and pat-T35S (B) under the conditions of Fig. 1 different annealing temperature.
Detection dynamic range of the dual ddPCR of Fig. 2 to endogenous gene zein (A) and foreign gene pat-T35S (B).
The ddPCR measurement of %T25 content in Fig. 3 various concentration AOCS 0306-H6.
Specific embodiment
1 ddPCR primer of embodiment, probe design
To realize specific detection and absolute quantification analysis to Transgenic corn lines T25 and Maize genome, we
Respectively choose insertion pat gene 3 ' end with CaMV35S terminator join domain (Genebank no.DQ156557.1) and
3 ' the ends (GenBank no.JQ083080.1) that corn list copies endogenous gene zein are used as target sequence, pass through the online work of NCBI
Tool carries out sequence analysis and comparison, is designed using Prime Express software V3.0 (ABI, Foster City, CA, USA)
More than 50 combine primer and probe, draw by the final acquisition high specificity of screening, suitable for the interior external source of ddPCR amplification condition
Each 1 set of object/probe combinations, sequence is shown in Table 1.The end of foreign gene pat-T35S probe 5 ' label HEX, endogenous gene zein probe 5 '
Flag F AM is held, 3 ' ends of two probes mark BHQ, so as to complete internal foreign gene in same ddPCR reaction system
Synchronous amplification detection, lay the foundation for the relative quantitative assay of Maize genome transgenic corn strain T25 content.By giving birth to
The synthesis of work bioengineering (Shanghai) limited liability company.
2 kit forms of embodiment
The kit includes the primer and probe for being used to detect Transgenic corn lines T25 that embodiment 1 designs, positive right
According to (T25 transgenic corns leaves genomic DNA is certified reference material, is purchased from American Oil Chemists association), ddPCR
Master mix (being purchased from U.S. BioRad company), droplet generate oil (being purchased from U.S. BioRad company), 25mM magnesium acetate (is purchased from
Sigma Co., USA), droplet generate card (be purchased from U.S. BioRad company), (purchase of 96 orifice plate of Twin Tec Semi-Skirted
From German Eppendorf company), aluminium foil heat-sealing film (be purchased from U.S. BioRad company).
The foundation of 3 detection method of embodiment
1.DNA is extracted
The DNA in sample is extracted using the CTAB method of improvement.Specific step is as follows: taking 100mg sample, 300 μ L are added and go out
Bacterium deionized water, abundant homogeneous mix；The CTAB buffer that 700 μ L are preheating to 65 DEG C is added, 10 μ L RNase are added after mixing
Solution, oscillation；65 DEG C of heating 30min；Heat enters 10 μ L Proteinase K Solutions, mixes gently, 65 DEG C of heating 30min；12000g centrifugation
Supernatant is transferred in the 1.5mL centrifuge tube containing 500 μ L chloroforms by 10min, vibrates 30s；12000g is centrifuged 15min, directly
To the apparent layering of appearance；Upper phase is transferred in the 1.5mL centrifuge tube containing 500 μ L chloroforms, is vibrated；12000g
It is centrifuged 5min, upper phase is transferred in a new 1.5mL centrifuge tube；The CTAB precipitation buffering liquid of 2 times of volumes is added, blows
It beats and mixes；60min is placed at room temperature；12000g is centrifuged 5min, discards supernatant；350 μ L sodium chloride (1.2M) dissolution precipitating is added；
30s is vibrated after 350 μ L chloroforms are added；12000g is centrifuged 10min, and upper phase is transferred in a new centrifuge tube；It is added
The isopropanol of 0.6 times of volume after being mixed by inversion, is placed at room temperature for 20min；12000g is centrifuged 10min, discards supernatant；It is added 70%
500 μ L of ethyl alcohol is mixed；12000g is centrifuged 10min, discards supernatant；After drying precipitated, with the molten DNA of 100 μ L sterilizing TE weight.To mentioning
The nucleic acid taken carries out the measurement of concentration and purity with nucleic acid-protein analyzer (Beckman Coulter, DU800), it is ensured that extracts
For the A260/A280 of nucleic acid between 1.8-2.0, concentration answers > 100ng/ μ L, saves backup in -20 DEG C.
2. preparing the ddPCR reaction system of 20 μ L according to table 2
3. droplet generates
The ddPCR reaction system of 20 μ L and 70 μ L droplets are generated oil to be added separately in 8 hole droplets generation card, are placed in micro-
Drop, which generates, generates droplet in instrument (QX100, Bio-Rad, Pleasanton, CA).
4. amplified reaction
The droplet (40 μ L) of the Water-In-Oil of generation is slowly transferred to Eppendorf Twin Tec Semi-Skirted
In 96 orifice plates, it is enterprising that sealer is placed on PCR instrument (GeneAmp 9700, Applied BioSystems, Foster City, CA)
Row amplified reaction, amplification condition are as shown in table 3.
5. result judgement
96 orifice plates after amplification are placed in droplet to read in instrument (QX100, Bio-Rad, Pleasanton, CA), are utilized
QuantaSoft software carries out result reading and analysis.Droplet reads two signal paths in instrument and reads endogenous gene zein respectively
FAM and foreign gene pat-T35S HEX fluorescence signal, the positive droplet containing amplified production be free of amplified production yin
Property droplet can show the difference of fluorescence signal intensity, can the highest point of negative droplet cluster fluorescence amplitude be that boundary carrys out given threshold
Line.The copy number for obtaining the inside and outside source gene of corn is calculated according to Poisson distribution principle, and then is turned by the calculating of following formula
The percentage composition of gene corn T25.
The present invention is also optimized ddPCR testing conditions, such as annealing temperature.It takes sample S2, S3 as template, divides
It is not carried out amplification reaction under 50 DEG C, 53 DEG C and 55 DEG C of annealing temperature, compares the testing result of ddPCR under different condition.By
For Fig. 1 as it can be seen that the amplification efficiency of endogenous gene zein is relatively low under the conditions of 50 DEG C, positive droplet and negative droplet boundary are unobvious；53
DEG C and 55 DEG C under the conditions of zein and pat-T35S amplification efficiency it is higher, may occur in which obvious positive droplet cluster, and 53 DEG C
Under the conditions of the fluorescence signal that expands be slightly above 55 DEG C, therefore, by 53 DEG C as best most fiery temperature.
The present invention also compares mono-/bis-weight reaction effect.Zein, pat-T35S, zein/pat- are prepared respectively
Tri- kinds of reaction systems of T35S, every kind of system set 8 detection multiple holes, wherein a hole is used as negative control, remaining 7 multiple holes then contains
There is the DNA profiling 75ng of sample U6 (it is expected that the copy of zein gene 27523, the copy of pat-T35S gene 430, T25 content
1.56%) ddPCR detection, is carried out.As a result, it has been found that substance and dual ddPCR detect zein copy number (deviation=1.1%),
Pat-T35S copy number (deviation=4.3%) and T25 percentage composition (deviation=2.8%) are not significantly different (table 4), and double
The measured value coefficient of variation reacted again reacts (table 4) less than normal compared with substance, illustrates there is preferably repeatability.
The definite value effect of 4 substance of table and dual ddPCR compare
4 detection method Evaluation on specificity of embodiment
Standard items/reference sample
Certified reference materials (the certified such as T25 maize leaf genomic DNA (AOCS 0306-H6,120ng/ μ L)
Reference materials, CRM) it is purchased from American Oil Chemists association (AOCS) or the joint study of executive committee, European Union
Heart standard and measuring study institute (IRMM), other transgenic positive samples such as Bioscein tomato is by Guangdong inspection and quarantining for import/export
Office (GDCIQ) provides, what transgenic paddy rice BT63, section rich No. 6 and Kemingdao were organized from China Inst. of Quarantine Inspection Sciences
Co-verification tests (CAIQ-CT), and in addition non-transgenic cotton seed and wheat flour are then Beijing Administration for Entry-Exit Inspection and Quarantine
(BJCIQ) sample is saved, details are shown in Table 5.
Sample list is used in the experiment of table 5
Include the transgenic corns of other strains using listed sample in table 5, non-transgenic corn, genetically engineered soybean, turn
Trans-genetic hybrid rice, transgene tomato, transgenic potato, non-transgenic cotton and wheat flour etc., take its DNA, adjust concentration to
70ng/ μ L is respectively used to ddPCR detection, and each sample sets 2 parallel repetitions, while replacing template as feminine gender using sterile water
Control, using AOCS 0306-H6 (60ng/ μ L) as positive control, determines the specificity of this method.As a result, it has been found that AOCS
There are positive amplification signal, but these in the zein gene of 0306-H6 and the transgenic corns of other strains, non-transgenic corn
Only have the pat-T35S genetic test of AOCS 0306-H6 to be positive in corn sample；In addition, the zein of other non-corn samples
It is negative with pat-T35S genetic test, no positive droplet (result is not shown).Prove that this method has good specificity.
Embodiment 5 detects dynamic range, minimum detection limit, minimum quantitative limit
Maize leaf genomic DNA (AOCS 0306-H6) is 120ng/ μ L, and 1 copy Maize genome is 2.725pg
(Arumuganathan&Earle, 1991), therefore corn endogenous gene zein and foreign gene pat- in AOCS 0306-H6
The content of T35S is 44037 copies/μ L.AOCS 0306-H6 is diluted by a certain percentage with TE buffer, obtains series
The standard items S1-S6 (table 6) of various concentration, each dilute sample carry out dual ddPCR detection respectively, and each sample sets 5 repetitions,
1 blank control of setting simultaneously (replaces template DNA with deionized water).
The dilution and preparation list of 6 standard sample of table
As a result, it has been found that in the dual ddPCR reaction system of 20 μ L, zein and pat-T35S gene content respectively 8.8~
Extraordinary linear relationship, coefficient R are showed within the scope of 29904 and 8.6~26376 copy numbers2It reaches respectively
0.9999 and 1 (Fig. 2).It is more than 4 orders of magnitude that it, which detects dynamic range, and covering transgenosis Routine Test Lab detects (usual external source
Gene is with endogenous gene copy number ratio within the scope of 0.1%-100%) required for target gene concentration range.
Minimum detection limit (LOQ) refers to that in acceptable precision and levels of accuracy, template to be measured can be by reliable definite value
Minimum copy number it is horizontal.It specifically refers to measure the minimum of coefficient of variation CV≤25% of copy number in detection dynamic range
Detection level.According to this standard, the dual ddPCR LOQ of corn endogenous gene zein and foreign gene pat-T35S are respectively
23 and 25 copies (table 7 and table 8).
Minimum detection limit (LOD), which refers in a sample, reliably to be detected, and the not necessarily minimum copy number of definite value.
When being usually all larger than by 5 duplicate positive droplet numbers or be equal to 2, corresponding minimum concentration is set to LOD.Therefore, it is originally grinding
The LOD for studying carefully middle zein and pat-T35S is respectively 8.8 and 8.6 copies (table 7 and table 8).
7 ddPCR of table detects dynamic range, LOD and LOQ and analyzes result I.
Note: adding No. * for LOD value, adds No. # for LOQ value, 5 retests (including NTC) are carried out to each dilution.
8 ddPCR of table detects dynamic range, LOD and LOQ and analyzes result II.
Note: adding No. * for LOD value, adds No. # for LOQ value, 5 retests (including NTC) are carried out to each dilution.
6 accuracy of embodiment
Accuracy (truness) refers to the consistency journey between the average value and acceptable reference value of one group of test result
Degree.According to the standard of European Union and Codex, the criterion of accuracy is the measured value Ying Can in entirely detection dynamic range
It examines in ± 25% range of value.
Utilize AOCS 0306-H6 (GMO content > 99.99%) and non-transgenic corn leaf DNA standard items AOCS
0306-C2 (GMO content < 0.01%) is mixed in a certain ratio, and preparing a series of nucleic acid concentrations is 75ng/ μ L, different T25
The transgenic sample U1-U8 (table 6) of percentage composition, each sample carry out dual ddPCR detection respectively, and each sample sets 7 repetitions,
1 blank control of setting simultaneously (replaces template DNA with deionized water).
As a result, it has been found that in the T25 content range of 0.11%-50%, corn endogenous gene zein copy number, foreign gene
The ddPCR measured value of pat-T35S copy number and T25 percentage composition is very close with predicted value, standard deviation substantially all <
10% (table 9 and table 10), the criterion much smaller than 25% illustrate that this method has extraordinary accuracy, and to the corn
The minimum quantitative concentrations of strain meet up to 0.11% in the world to the quantitative detection demand of GMO.
In addition, equally being shown in the result that the AOCS 0306-H6 to gradient dilution carries out ddPCR detection, entirely detecting
In dynamic range, the percentage composition of T25 measured by various concentration template all standard reference point (being similar to 100%) ±
In 25% range (Fig. 3), meet the accuracy criterion in the world to quantitative detecting method.
The ddPCR testing result I of 9 difference %T25 content transgenic corns of table
The ddPCR testing result II of 10 difference %T25 content transgenic corns of table
7 repeatability of embodiment
The testing result of above-mentioned analysis dynamic range, the experimental data of LOQ and LOD and difference %T25 content sample U1-U8
It all can be used to assess the repeatability of whole detection.By table 7 to table 10 as it can be seen that in entire quantitative detection dynamic range, for jade
The measurement coefficient of variation CV of rice endogenous gene zein copy number, foreign gene pat-T35S copy number and T25 percentage composition is small
In 25%, meet the verifying requirement in the world to transgenosis measuring method, it was demonstrated that this method is quantitatively examined for T25 transgenic corns
There is good repeatability when survey.
Obviously, the above embodiment of the present invention be only to clearly illustrate example of the present invention, and not be pair
The restriction of embodiments of the present invention.For those of ordinary skill in the art, may be used also on the basis of the above description
To make other variations or changes in different ways.Here all embodiments can not be exhaustive.It is all to belong to this hair
The obvious changes or variations that bright technical solution is extended out are still in the scope of protection of the present invention.
Claims (1)
1. a kind of detection method of accurate quantification detection Transgenic corn lines T25, which is characterized in that this method comprises:
(1) sample to be tested DNA is extracted；
(2) dual ddPCR reaction system is prepared, wherein the reaction system includes SEQ ID NO:1 to SEQ ID in sequence table
SEQ ID NO:4 to SEQ in primer and probe and sequence table shown in NO:3 for expanding foreign gene pat-T35S
For expanding the primer and probe of endogenous gene zein shown in ID NO:6；For expanding shown in sequence table SEQ ID No.3
The end of probe 5 ' the label HEX of foreign gene pat-T35S, 3 ' end label BHQ；For expanding shown in sequence table SEQ ID No.6
The probe 5 ' of endogenous gene zein holds flag F AM, 3 ' end label BHQ；
(3) the dual ddPCR reaction system and droplet prepared step (2) generate oil and are added in droplet generation card, are placed in droplet
It generates in instrument and generates droplet；
(4) droplet that step (3) generate is transferred in 96 orifice plates, is carried out amplification reaction；
Reaction condition is
(5) result judgement: the copy number for obtaining the inside and outside source gene of corn is calculated according to Poisson distribution principle, and then by as follows
Formula calculates the percentage composition for obtaining transgenic corns T25:
。
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| CN107312817A (en) * | 2016-04-25 | 2017-11-03 | 中国检验检疫科学研究院 | Primed probe, kit and the method precisely quantitatively detected for genetically modified rice Kemingdao strain-specific gene composition |
| CN107312820A (en) * | 2016-04-25 | 2017-11-03 | 中国检验检疫科学研究院 | Primed probe, kit and the method precisely quantitatively detected for genetically modified rice TT51-1 strain-specific gene compositions |
| CN106222300B (en) * | 2016-08-04 | 2019-11-26 | 北京出入境检验检疫局检验检疫技术中心 | A kind of kit and detection method of accurate quantification detection A rotavirus |
| CN106596489B (en) * | 2016-12-19 | 2019-06-28 | 中国科学院苏州生物医学工程技术研究所 | Processing method for fluorescence intensity data in fluorescence drop detection |
| CN108830046B (en) * | 2018-05-08 | 2021-09-17 | 中国农业科学院生物技术研究所 | Method for estimating DNA content of biotechnological product |
| CN109913571A (en) * | 2019-04-03 | 2019-06-21 | 深圳出入境检验检疫局食品检验检疫技术中心 | Transgenic corn lines MON87427 detection method and reagent |
| CN109825632A (en) * | 2019-04-03 | 2019-05-31 | 深圳出入境检验检疫局食品检验检疫技术中心 | Transgenic corn lines DAS40278-9 detection method and reagent |
| CN109825635A (en) * | 2019-04-03 | 2019-05-31 | 深圳出入境检验检疫局食品检验检疫技术中心 | Method, reagent and the kit that Transgenic corn lines 98140 detect |
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| Title |
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| 利用微滴数字PCR分析转基因生物外源基因拷贝数;姜羽等;《农业生物技术学报》;20140914;第22卷(第10期);1.3.5；2.2.1文字部分 |
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