CN105255874A - Kit for accurately and quantitatively detecting transgenic maize line T25 and detection method thereof - Google Patents
Kit for accurately and quantitatively detecting transgenic maize line T25 and detection method thereof Download PDFInfo
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Abstract
The invention discloses a kit for accurately and quantitatively detecting a transgenic maize line T25 and a detection method thereof. The invention in particular relates to a group of primers and probes which are used for detecting the transgenic maize line T25 as well as a kit containing the primers and probes, wherein the primers and probes have nucleotide sequences shown in SEQ ID No.1 to SEQ ID No.6 in a sequence table. The invention also provides a detection method for accurately and quantitatively detecting the transgenic maize line T25 by utilizing a dual microdroplet type digital PCR technology. The detection method provided by the invention is independent of a certified reference material or other standard substances, has higher accuracy, sensitivity and repeatability and is easy to be standardized.
Description
Technical field
The present invention relates to test kit and the detection method of a kind of detection by quantitative Transgenic corn lines T25, belong to biology field.
Background technology
Along with the extensive plantation of genetically modified crops in worldwide, its edible safety and environmental security are subject to the great attention of consumers in general, national governments and relevant international organization always.A lot of countries and regions have worked out GMO bio-safety laws and regulations on the management one after another, implement mark system.The Ministry of Agriculture of China, on January 5th, 2002, has issued " agriculture genetically modified organism identity management way ", is defined in the agriculture genetically modified organism sold and list catalogue within Chinese territory, should has obvious mark.New " the law of food safety " implemented October in this year has made the clear stipulaties that must " significantly indicate " to genetically modified food again further, and this will inevitably propose higher demand to the detection of transgene component.But be different from the countries such as European Union's (identification thresholds 0.9), Australia (identification thresholds 1.0), Korea S's (identification thresholds 3.0), Japan's (identification thresholds 5.0), China not yet puts into effect transgenic labeling threshold value regulation, take qualitative mark i.e. zero threshold value, the existing detection GMOs of this and China, mainly based on qualitative method, lacks and can quantitative standard method have some relations.Therefore, the accurate quantitative detecting method of transgenosis is set up in research, can be China's transgenic product quantitative identifying and threshold management lays the foundation, and promotes in line with international standards, and for safeguarding that the national interests in international trade provide technical guarantee.
The content of transgene component in food normally represents with transgene and the ratio of the quantity of native gene, and real-time fluorescence PCR (qPCR) is quantitative detecting method general in the world now.The method utilizes the change of fluorescent signal to detect the change of amplified production amount in pcr amplification reaction in real time, by the certified reference material (CertifiedReferenceMaterials to gradient dilution, CRM) detection, obtain endogenous and foreign gene typical curve respectively, and then calculate content that is endogenous in testing sample and foreign gene by cycle threshold (Ct).Visible, the suppression composition of nucleic acids in samples amplification, the technical ability etc. of experimental situation and operator all directly can affect the quality of amplification efficiency and typical curve, thus disturb quantitative accuracy.
The new droplet type digital pcr technology (dropletdigitalPCR risen in recent years, ddPCR), by being distributed to be diluted to certain density DNA molecular in 10000 ~ 20000 droplets, the quantity of DNA molecular in most of droplet is made to be 1 or 0, then read positive reaction unit number by the accumulation of pcr amplification and positive signal, then calculate the DNA molecular copy number in sample according to Poisson's distribution.Current, utilize ddPCR to carry out transgenosis detection by quantitative and be still in the starting stage, research report quantity is few, mainly for Mon810 transgenic corns, to the validity of all the other transgenic strain detection by quantitative and practicality on the knees of the gods, development space and having a extensive future.
Transgenic corns is one of the widest genetically modified crops of grown worldwide area, choosing wherein representative strain T25 is research object, analyze its gene to form, select the suitable interior external source assortment of genes, design primer and the probe of high special, set up and be applicable to the quantitative dual ddPCR detection method of T25 transgenosis.Through specificity, detect dynamicrange, LOD, LOQ, accuracy and repeatability checking, proves that the method meets in the world to the technical requirements of transgenosis detection by quantitative, thus be that the accurate quantitative examination of other genetically modified crops lays the foundation.
Summary of the invention
The technical problem to be solved in the present invention is to provide one group for detecting primer and the probe of Transgenic corn lines T25.
Another technical problem that the present invention will solve is to provide the test kit that a kind of accurate quantification detects Transgenic corn lines T25.
The 3rd technical problem that the present invention will solve is to provide the detection method that a kind of accurate quantification detects Transgenic corn lines T25.
For achieving the above object, the present invention is by the following technical solutions:
The invention provides one group for detecting primer and the probe of Transgenic corn lines T25, is by for the primer of the foreign gene pat-T35S that increases and probe, and for the primer of the native gene zein that increases and probe composition, sequence is in table 1.Wherein, mark HEX held by the probe 5 ' of amplification foreign gene pat-T35S, 3 ' end mark BHQ, and flag F AM held by the probe 5 ' of amplification native gene zein, 3 ' end mark BHQ.
Table 1ddPCR primer/probe sequence
The present invention also provides a kind of accurate quantification to detect the test kit of Transgenic corn lines T25, and this test kit comprises above-mentioned primer for detecting Transgenic corn lines T25 and probe.Further, this test kit has also comprised dual digital pcr reaction material requested and reagent, such as positive control, ddPCRmastermix, magnesium acetate, droplet generate oil, droplet generation card, 96 orifice plates and aluminium foil thermal sealer etc., and above-mentioned materials and reagent are preferably packaging separately.
Present invention also offers the detection method that a kind of accurate quantification detects Transgenic corn lines T25, particularly a kind of method utilizing dual digital pcr technology accurate quantification to detect Transgenic corn lines T25, the method comprises:
(1) extract testing sample DNA, extracting method well known in the prior art or commercial kit can be used to extract;
(2) dual ddPCR reaction system is prepared, wherein, described reaction system comprises the primer for the foreign gene pat-T35S that increases shown in SEQ ID NO:1 to SEQIDNO:3 and probe, and the primer for the native gene zein that increases shown in SEQ ID NO:4 to SEQIDNO:6 and probe; Preferably, in a specific embodiments of the present invention, dual ddPCR reaction system is as shown in table 2;
The dual ddPCR reaction system of table 2 corn gene strain T25
| Component | Working fluid concentration | Application of sample 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/μL | 1 | / |
| Deionized water | / | 1 | / |
(3) dual ddPCR reaction system step (2) prepared and droplet generate oil and add in droplet generation card, are placed in droplet instrument for generating and generate droplet;
(4) be transferred in 96 orifice plates by the water in oil droplet generated, carry out amplified reaction, reaction conditions is in table 3;
Table 3 corn gene strain T25ddPCR reaction conditions
Note: warming and cooling rate should≤2.5 DEG C/s
(5) result judges.
96 orifice plates after amplification are placed in droplet reader (QX100, Bio-Rad, Pleasanton, CA), utilize QuantaSoft software to carry out result reading and analysis.In droplet reader, two signalling channels read the FAM of native gene zein and the HEX fluorescent signal of foreign gene pat-T35S respectively, positive droplet containing amplified production and the difference that can not present fluorescence signal intensity containing the negative droplet of amplified production can the vertex of negative droplet bunch fluorescence amplitude be that boundary is to set threshold line.Calculate the copy number obtaining corn inside and outside source gene according to Poisson's distribution principle, and then obtain the percentage composition of transgenic corns T25 by following formulae discovery.
Advantage of the present invention is: 1) absolute quantitation of nucleic acid molecule is avoided to the deviation caused by pcr amplification efficiency variance; 2) without the need to relying on certified reference material or other standards product; 3) there is higher tolerance range, susceptibility and repeatability, be easy to stdn; 4) be more suitable for the detection of nucleic acids of low copy number, be applicable to the precisely quantitative of low abundance transgene component; 5) because ddPCR is end point determination, higher to the tolerance level of inhibitor, therefore can reduce the detection error caused by matrix type.6) by different fluorescent signals mark, more easily realize the dual amplified reaction of foreign gene in same reaction system, thus reduce testing cost.
Below in conjunction with specification drawings and specific embodiments, the invention will be further described, and the equivalent replacement of all any this areas of doing according to the disclosure of invention, all belongs to protection scope of the present invention.
Accompanying drawing explanation
The ddPCR detected result of zein (A) and pat-T35S (B) under Fig. 1 different annealing temperature condition.
The dual ddPCR of Fig. 2 is to the detection dynamicrange of native gene zein (A) and foreign gene pat-T35S (B).
In Fig. 3 different concns AOCS0306-H6, the ddPCR of %T25 content measures.
Embodiment
Embodiment 1ddPCR primer, probe design
For realizing specific detection to Transgenic corn lines T25 and Maize genome and absolute quantification analysis, we choose insertion pat gene 3 ' end respectively and hold (GenBankno.JQ083080.1) as target sequence with the connecting zone (Genebankno.DQ156557.1) of CaMV35S terminator and 3 ' of corn list copy native gene zein, sequential analysis and comparison is carried out by NCBI online tool, utilize PrimeExpress software V3.0 (ABI, FosterCity, CA, USA) more than 50 are designed to primer and probe combinations, finally high specificity is obtained through screening, be applicable to each 1 cover of interior exogeneous primer/probe combinations of ddPCR amplification condition, sequence is in table 1.Foreign gene pat-T35S probe 5 ' end mark HEX, native gene zein probe 5 ' end flag F AM, 3 ' end of two probes all marks BHQ, thus the synchronous amplification that can complete internal foreign gene in same ddPCR reaction system detects, in Maize genome, the relative quantitative assay of Transgenic corn lines T25 content lays the foundation.Synthesized by Sangon Biotech (Shanghai) Co., Ltd..
Embodiment 2 test kit forms
This test kit comprises primer and the probe for detecting Transgenic corn lines T25 that embodiment 1 designs, positive control (T25 transgenic corns leaves genomic DNA, for certified reference material, purchased from American Oil Chemists association), ddPCRmastermix (purchased from American BioRad company), droplet generates oil (purchased from American BioRad company), 25mM magnesium acetate (purchased from American Sigma company), droplet generates card (purchased from American BioRad company), TwinTecSemi-Skirted96 orifice plate (purchased from German Eppendorf company), aluminium foil thermal sealer (purchased from American BioRad company).
The foundation of embodiment 3 detection method
1.DNA extracts
Adopt the DNA in improved method of CTAB extraction sample.Concrete steps are as follows: get 100mg sample, add 300 μ L sterilizing deionized waters, abundant homogeneous mixing; Add the CTAB damping fluid that 700 μ L are preheating to 65 DEG C, after mixing, add 10 μ LRNase solution, vibration; 65 DEG C of heating 30min; Heat enters 10 μ L Proteinase K Solution, mixes gently, 65 DEG C of heating 30min; The centrifugal 10min of 12000g, transfers to one containing in the 1.5mL centrifuge tube of 500 μ L chloroforms, vibration 30s by supernatant; , until there is obvious layering in the centrifugal 15min of 12000g; Upper phase is transferred to one containing in the 1.5mL centrifuge tube of 500 μ L chloroforms, vibration; The centrifugal 5min of 12000g, transfers in a new 1.5mL centrifuge tube by upper phase; Add the CTAB precipitation buffering liquid of 2 times of volumes, piping and druming mixing; Ambient temperatare puts 60min; The centrifugal 5min of 12000g, supernatant discarded; Add 350 μ L sodium-chlor (1.2M) dissolution precipitations; Vibrate after adding 350 μ L chloroforms 30s; The centrifugal 10min of 12000g, is transferred in a new centrifuge tube by upper phase; Add the Virahol of 0.6 times of volume, after putting upside down mixing, room temperature places 20min; The centrifugal 10min of 12000g, supernatant discarded; Add 70% ethanol 500 μ L, mixing; The centrifugal 10min of 12000g, supernatant discarded; After drying precipitated, with the heavy molten DNA of 100 μ L sterilizing TE.To the nucleic acid nucleic acid-protein analyser (BeckmanCoulter extracted, DU800) mensuration of concentration and purity is carried out, guarantee that the A260/A280 extracting nucleic acid is between 1.8-2.0, concentration answers > 100ng/ μ L, saves backup in-20 DEG C.
2. the ddPCR reaction system of 20 μ L is prepared according to table 2
3. droplet generates
The ddPCR reaction system of 20 μ L and 70 μ L droplets are generated oil content not join in 8 hole droplets generation cards, be placed in droplet instrument for generating (QX100, Bio-Rad, Pleasanton, CA) and generate droplet.
4. amplified reaction
The water in oil droplet (40 μ L) generated slowly is transferred in EppendorfTwinTecSemi-Skirted96 orifice plate, sealer is placed on PCR instrument (GeneAmp9700, AppliedBioSystems, FosterCity, CA) carry out amplified reaction on, amplification condition is as shown in table 3.
5. result judges
96 orifice plates after amplification are placed in droplet reader (QX100, Bio-Rad, Pleasanton, CA), utilize QuantaSoft software to carry out result reading and analysis.In droplet reader, two signalling channels read the FAM of native gene zein and the HEX fluorescent signal of foreign gene pat-T35S respectively, positive droplet containing amplified production and the difference that can not present fluorescence signal intensity containing the negative droplet of amplified production can the vertex of negative droplet bunch fluorescence amplitude be that boundary is to set threshold line.Calculate the copy number obtaining corn inside and outside source gene according to Poisson's distribution principle, and then obtain the percentage composition of transgenic corns T25 by following formulae discovery.
The present invention is also optimized ddPCR testing conditions, such as annealing temperature.Sample thief S2, S3, as template, carry out amplified reaction respectively under the annealing temperature of 50 DEG C, 53 DEG C and 55 DEG C, compare the detected result of ddPCR under different condition.As seen from Figure 1, under 50 DEG C of conditions, the amplification efficiency of native gene zein is on the low side, and positive droplet and negative droplet are demarcated not obvious; Under 53 DEG C and 55 DEG C of conditions, the amplification efficiency of zein and pat-T35S is higher, can occur obvious positive droplet bunch, and the fluorescent signal increased under 53 DEG C of conditions is a little more than 55 DEG C, therefore, using 53 DEG C as the most fiery temperature of the best.
The present invention also compares list/double reaction effect.Prepare zein, pat-T35S, zein/pat-T35S tri-kinds of reaction systems respectively, often kind of system establishes 8 to detect multiple hole, wherein a hole is used as negative control, all the other 7 the DNA profiling 75ng that Kong Zejun contains sample U6 again (estimate that zein gene 27523 copies, pat-T35S gene 430 copies, T25 content 1.56%), carry out ddPCR detection.Found that zein copy number (deviation=1.1%), pat-T35S copy number (deviation=4.3%) and T25 percentage composition (deviation=2.8%) that substance and dual ddPCR detect all do not have significant difference (table 4), and the measured value variation coefficient of double reaction is compared with substance reaction (table 4) less than normal, illustrates to have better repeatability.
The definite value effectiveness comparison of table 4 substance and dual ddPCR
Embodiment 4 detection method Evaluation on specificity
Standard substance/reference sample
T25 maize leaf genomic dna (AOCS0306-H6, 120ng/ μ L) etc. certified reference material (certifiedreferencematerials, CRM) all purchased from American Oil Chemists association (AOCS) or Joint Research Centre of executive committee of European Union standard and measurement Research institute (IRMM), such as Bioscein tomato is provided by Guangdong Entry-Exit Inspection and Quarantine Bureau (GDCIQ) other transgenic positive samples, transgenic paddy rice BT63, rich No. 6 co-verifications test (CAIQ-CT) organized from China Inst. of Quarantine Inspection Sciences with Kemingdao of section, non-transgenic cotton seed and wheat-flour then preserve sample for Beijing Administration for Entry-Exit Inspection and Quarantine (BJCIQ) in addition, details are in table 5.
Table 5 experiment sample list
In employing table 5, listed sample comprises the transgenic corns of other strains, non-transgenic corn, genetically engineered soybean, transgenic paddy rice, transgenic Fructus Lycopersici esculenti, transgenic Rhizoma Solani tuber osi, non-transgenic cotton and wheat-flour etc., get its DNA, regulate concentration to 70ng/ μ L, be respectively used to ddPCR detect, 2 parallel repetitions established by each sample, replace template as negative control using sterilized water simultaneously, using AOCS0306-H6 (60ng/ μ L) as positive control, judge the specificity of the method.Found that positive amplification signal all appears in AOCS0306-H6 and the transgenic corns of other strains, the zein gene of non-transgenic corn, but in these corn samples, only have the pat-T35S gene test of AOCS0306-H6 to be positive; In addition, zein and the pat-T35S gene test of other non-corn samples is all negative, no positive droplet (result does not show).Prove that the method has good specificity.
Embodiment 5 detects dynamicrange, lowest detectable limit, minimum quantitative limit
Maize leaf genomic dna (AOCS0306-H6) is 120ng/ μ L, 1 copy Maize genome is 2.725pg (Arumuganathan & Earle, 1991), therefore the content of corn native gene zein and foreign gene pat-T35S is 44037 copies/μ L in AOCS0306-H6.AOCS0306-H6 TE damping fluid is diluted by a certain percentage, obtain the standard substance S1-S6 (table 6) of serial different concns, each dilute sample carries out dual ddPCR detection respectively, 5 repetitions established by each sample, arrange 1 blank (replacing template DNA with deionized water) simultaneously.
Dilution and the preparation list of table 6 standard model
Found that zein and pat-T35S gene content all presents extraordinary linear relationship respectively within the scope of 8.8 ~ 29904 and 8.6 ~ 26376 copy numbers, its coefficient R in the dual ddPCR reaction system of 20 μ L
2reach 0.9999 and 1 (Fig. 2) respectively.It detects dynamicrange more than 4 orders of magnitude, covers transgenosis Routine Test Lab and detects target gene concentration scope required for (usual foreign gene with native gene copy number ratio within the scope of 0.1%-100%).
Lowest detectable limit (LOQ) refers in acceptable precision and levels of accuracy, and template to be measured can by the minimum copy number level of reliable definite value.Specifically refer in detection dynamicrange, record the lowest detection level of variation coefficient CV≤25% of copy number.According to this standard, the dual ddPCRLOQ of corn native gene zein and foreign gene pat-T35S is respectively 23 and 25 copies (table 7 and table 8).
Lowest detectable limit (LOD) refers in a sample and can reliably be detected, and the minimum copy number of not necessarily definite value.When usually the positive droplet number that 5 are repeated all being more than or equal to 2, corresponding minimum concentration is decided to be LOD.Therefore, in this research, the LOD of zein and pat-T35S is respectively 8.8 and 8.6 copies (table 7 and table 8).
Table 7ddPCR detects dynamicrange, LOD and LOQ analytical results I.
Note: add No. * for LOD value, adds No. # for LOQ value, all carries out 5 repeated tests (comprising NTC) to each diluent.
Table 8ddPCR detects dynamicrange, LOD and LOQ analytical results II.
Note: add No. * for LOD value, adds No. # for LOQ value, all carries out 5 repeated tests (comprising NTC) to each diluent.
Embodiment 6 accuracy
Accuracy (truness) refers to the degree of consistency between the mean value of one group of test result and acceptable reference value.According to the standard of European Union and Codex, the criterion of accuracy is in whole detection dynamicrange, and measured value should in reference value ± 25% scope.
AOCS0306-H6 (GMO content > 99.99%) and non-transgenic corn leaf DNA standard substance AOCS0306-C2 (GMO content < 0.01%) is utilized to mix by a certain percentage, prepare a series of nucleic acid concentration and be 75ng/ μ L, the transgenic sample U1-U8 (table 6) of different T25 percentage composition, each sample carries out dual ddPCR detection respectively, 7 repetitions established by each sample, arrange 1 blank (replacing template DNA with deionized water) simultaneously.
Found that in the T25 content range of 0.11%-50%, the ddPCR measured value of corn native gene zein copy number, foreign gene pat-T35S copy number and T25 percentage composition all with predicated value closely, its standard deviation is < 10% (table 9 and table 10) substantially all, much smaller than the criterion of 25%, illustrate that the method has extraordinary accuracy, and can 0.11% be reached to the minimum quantitative concentrations of this corn strain, meet in the world to the detection by quantitative demand of GMO.
In addition, show equally in the result of the AOCS0306-H6 of gradient dilution being carried out to ddPCR detection, in whole detection dynamicrange, the percentage composition of the T25 measured by different concns template all standard reference point (being similar to 100%) ± 25% scope in (Fig. 3), meet in the world to the accuracy criterion of quantitative detecting method.
The ddPCR detected result I of table 9 different %T25 content transgenic corns
The ddPCR detected result II of table 10 different %T25 content transgenic corns
Embodiment 7 repeatability
The experimental data of above-mentioned axially viewedplasma, LOQ and LOD and the detected result of different %T25 content sample U1-U8 all can be used to the repeatability assessing whole detection.From table 7 to table 10, in whole detection by quantitative dynamicrange, mensuration variation coefficient CV for corn native gene zein copy number, foreign gene pat-T35S copy number and T25 percentage composition is all less than 25%, meet in the world to the checking requirement of transgenosis measuring method, when proving that the method is used for T25 transgenic corns detection by quantitative, there is good repeatability.
Obviously, the above embodiment of the present invention is only for example of the present invention is clearly described, and is not the restriction to embodiments of the present invention.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here cannot give exhaustive to all embodiments.Every belong to technical scheme of the present invention the apparent change of extending out or variation be still in the row of protection scope of the present invention.
Claims (6)
1. one group for detecting primer and the probe of Transgenic corn lines T25, it is characterized in that: be by for the primer of the foreign gene pat-T35S that increases and probe, and for the primer of the native gene zein that increases and probe composition, wherein, nucleotides sequence shown in sequence table SEQ IDNo.1 to sequence table SEQ IDNo.2 is classified as the primer for the foreign gene pat-T35S that increases, and the nucleotides sequence shown in sequence table SEQ IDNo.3 is classified as the probe for the foreign gene pat-T35S that increases; Nucleotides sequence shown in sequence table SEQ IDNo.4 to sequence table SEQ IDNo.5 is classified as the primer for the native gene zein that increases, and the nucleotides sequence shown in sequence table SEQ IDNo.6 is classified as the probe for the native gene zein that increases.
2. according to claim 1 one group for detecting primer and the probe of Transgenic corn lines T25, it is characterized in that: the probe 5 ' for the foreign gene pat-T35S that increases shown in sequence table SEQ IDNo.3 end mark HEX, 3 ' end mark BHQ; The end of the probe 5 ' for the native gene zein that increases flag F AM shown in sequence table SEQ IDNo.6,3 ' end mark BHQ.
3. accurate quantification detects a test kit of Transgenic corn lines T25, it is characterized in that, described test kit comprises described in claim 1 or 2 one group for detecting primer and the probe of Transgenic corn lines T25.
4. test kit according to claim 3, is characterized in that, described test kit also comprises positive control, ddPCRmastermix, magnesium acetate, droplet generates oil, droplet generates card, 96 orifice plates and aluminium foil thermal sealer.
5. accurate quantification detects a detection method of Transgenic corn lines T25, and it is characterized in that, the method comprises:
(1) testing sample DNA is extracted;
(2) dual ddPCR reaction system is prepared, wherein, described reaction system comprises the primer for the foreign gene pat-T35S that increases shown in SEQ ID NO:1 to SEQIDNO:3 and probe, and the primer for the native gene zein that increases shown in SEQ ID NO:4 to SEQIDNO:6 and probe;
(3) dual ddPCR reaction system step (2) prepared and droplet generate oil and add in droplet generation card, are placed in droplet instrument for generating and generate droplet;
(4) droplet that step (3) generates is transferred in 96 orifice plates, carries out amplified reaction;
(5) result judges.
6. detection method according to claim 5, is characterized in that, in step (5), calculates the copy number obtaining corn inside and outside source gene according to Poisson's distribution principle, and then obtains the percentage composition of transgenic corns T25 by following formulae discovery:
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| CN106222300A (en) * | 2016-08-04 | 2016-12-14 | 北京出入境检验检疫局检验检疫技术中心 | The test kit of a kind of accurate quantification detection A rotavirus and detection method |
| 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 |
| CN106596489A (en) * | 2016-12-19 | 2017-04-26 | 中国科学院苏州生物医学工程技术研究所 | Processing method of fluorescence intensity data in fluorescence droplet detection |
| CN108830046A (en) * | 2018-05-08 | 2018-11-16 | 中国农业科学院生物技术研究所 | A kind of biological technology products DNA content estimation method |
| 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 |
| 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 |
| 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 |
| 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 |
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