CN102690885A - Standard sample of transgenic rice flour and establishing method and application of standard sample - Google Patents
Standard sample of transgenic rice flour and establishing method and application of standard sample Download PDFInfo
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Abstract
The invention discloses a preparation method and an establishing method for a standard sample of transgenic rice flour BT63 as well as application of the standard sample in detection of the transgenic rice flour BT63. The preparation method comprises the following steps: respectively grinding and sieving the transgenic rice flour BT63 as a raw material; adding water and stirring to obtain lyophilized powder; and carrying out split charging and storing the lyophilized powder at the temperature of 0-4DEG C. The standard sample of the transgenic rice flour BT63, which is obtained by the method, can be also applied to a Real-Time PCR (Polymerase Chain Reaction) detection kit of the transgenic rice flour BT63 and has important real significance for comprehensively and deeply searching to solve a preparation technology and a stable ensuring technology of the standard sample for detecting transgenic components, actively developing the research on the standard sample for detecting transgenic products and filling up the blank of the measurement field.
Description
Technical field
The invention belongs to genetically modified foodGMF detection technique field, be specifically related to standard model preparation, its establishment method and the application in detecting transgenic ground rice BT63 thereof of transgenic ground rice BT63.
Background technology
Paddy rice is the staple food grain of people's lives, and China is again the main producing region of paddy rice, and annual China exports to products such as the brown rice, rice of countries such as Japan, Korea S and accounts for nearly 1/3 of China's rice yield.For improving rice yield, China has carried out research extensively and profoundly aspect transgenic paddy rice research, and has obtained gratifying achievement, but the commercialization plantation is not carried out in approval.Yet, last year on home markets such as Hubei, occurred sale, and published by the company of detection abroad without the commentaries on classics BT gene rice of commercialization approval plantation.This has brought very bad influence for the trade of agricultural products in China.For this reason, state such as Korea S, Japan batch implements genetically modified detection to product requirements such as the brown rice of China outlet, rice.
Transgenic Bt Rice as yet not in the whole world any area get permission the plantation, for the Bt transgenic paddy rice also not through any environmental impact assessment and human food prods's safety assessment.But, from the result of study of other Bt genetically modified crops (like corn and cotton), can infer that the Bt paddy rice can cause great influence to environment, and cause human food prods's safety-problems.
Since 2006, repeatedly detected the transgenic paddy rice composition by European Union and Japan in the rice made products of China's outlet, wherein mostly is the transgenic paddy rice Shanyou 63 number (BT63) of China's research and development.Because China provides the Biosafety certificate to transgenic paddy rice as yet, detect the transgenic paddy rice composition in the outlet rice made products rice made products export trade of China has been caused certain influence.Similar with the outlet rice made products, China's inspection and quarantine department has also detected the antiweed transgenic paddy rice composition that enters the territory without China's approval from the rice of U.S.'s import.
The development of carrying out transgene component inspecting standard sample ensures food safety at the comparability and the traceability that guarantee test result, settles trade disputes, and aspect such as promote economic development all has great importance.2008, we developed transgenic corns Mon810 strain, NK603 strain, T25 strain, BT11 strain, transgene rape RT73 strain standard model and corresponding molecular dna standard model first, have filled up domestic blank.These standard models have been brought into play huge effect in the transgenic product testing.Yet; After China's rice made products outlet is detected transgenic BT63 composition; Each food safety management mechanism and feeler mechanism begin to carry out in large quantities the detection of transgene component BT63 in the rice made products, but the domestic and international market do not have the standard model of transgenic BT63 all the time both at home and abroad.Domestic transgenic BT63 strain standard model is in short supply.
Summary of the invention
In view of domestic transgenic paddy rice BT63 strain standard model situation in short supply; We have developed transgenic ground rice BT63 strain standard model; Among the present invention, described transgenic ground rice BT63 is that transgenic paddy rice BT63 grinds powdering product afterwards, and the preparation of this standard model is accomplished; To the deep comprehensively technology of preparing of researching and solving transgene component examination criteria sample and stability assurance technology; Actively develop the development of China's transgenic product examination criteria sample, replenish the blank of this field of measurement, have very important practical sense.
The brief introduction of transgenic paddy rice BT63 strain:
The foreign gene that transgenic paddy rice BT63 changes over to when carrying out gene transformation is anti insect gene cry1Ab/cry1Ac, is regulated and control by the NOS terminator.The present invention realizes through following technological line:
One side of the present invention is: disclose a kind of transgenic ground rice BT63 standard model, it is prepared by following method, and step comprises:
A. between aseptic technique, use mortar grinding paddy rice sample, cross 100 mesh standard sieves, processed ground rice, in powder, add the water of 4 times of quality, stir;
B. biased sample is positioned over that the cryogenic vacuum lyophilize is prepared into freeze-dried mixed powder in the pilot scale Freeze Drying Equipment;
C. freeze-dried mixed powder sample is sub-packed in capping sealing in the Brown Glass Brown glass bottles and jars only of the cleaning of 160 ℃ of dry heat treatment 2h, adorns freeze-dried mixed powder sample 1g in every bottle.
D. obtain 500 bottles of transgenic ground rice standard models.Sample after bottled is sticked on the uniqueness sign, is positioned over shady and cool dry 0 ℃ ~ 4 ℃ storages in place.
Each described freeze-drying in the above-mentioned steps (a) ~ (d), its concrete steps are following:
Cryogenic vacuum lyophilize program:
1. sample pre-freeze: earlier sample at-80 ℃ of freezing 2h;
2. refrigerating process: closed the kiln door of frost drying machine, beginning kiln refrigeration; When the kiln temperature is reduced to-35 ℃, begin to cool down the trap refrigeration; When the cooling pit refrigeration temperature is reduced to-40 ℃, the sample that 1. step is handled is put into kiln rapidly, closed the kiln door; Starting vacuum pump begins to vacuumize; When vacuum tightness is reduced to 0.5Torr, finish refrigerating process;
3. sample drying: the temperature of kiln is set to 15 ℃, and when vacuum tightness was reduced to 0.1Torr, sample was dry good;
4. close vacuum pump, kiln communicated with ambient atmosphere, treat inside and outside air pressure balance after, open the kiln door, take out sample, the bottle cap of screwing rapidly.
Another aspect of the present invention is: discloses the Real-Time PCR detection kit of a kind of transgenic paddy rice BT63, comprises that transgenic ground rice BT63 detects gene and positive criteria reference substance, wherein,
Positive criteria reference substance: be the transgenic ground rice BT63 standard model that utilizes the method preparation of above-mentioned a ~ d;
Transgenic ground rice BT63 detects gene, comprising:
The detection primer and the probe sequence of SPS gene:
Forward primer 5 '-ttgcgcctgaacggatat-3 ' SEQ ID NO:1
Reverse primer 5 '-cggttgatcttttcgggatg-3 ' SEQ ID NO:2
Probe 5 '-FAM-tccgagccgtccgtgcgtc-TAMRA-3 ' SEQ ID NO:3
The detection primer and the probe sequence of cry1Ac/NOS gene:
Forward primer 5 '-gactgctggagtgattatcgacaga-3 ' SEQ ID NO:4
Reverse primer 5 '-agctcggtacctcgacttattcag-3 ' SEQ ID NO:5
Probe 5 '-FAM-tcgagttcattccagttactgcaacactcgag-TAMRA-3 ' SEQ ID NO:6
Wherein: FAM is 6-carboxyfluorescein, and TAMRA is 6-carboxytetramethylrhodamine.
Among the present invention; In the above-mentioned detection kit, adopted open description form, its implication is the compositions such as other damping fluids that all do not limit in the detection kit; Because of it can be confirmed according to prior art; And through configuration or commercial sources purchase acquisition, the method for use of detection kit and testing conditions, the technician can do reference frame adjustment according to listed condition among the embodiment.These believe that about the selection of preparation way and method those skilled in the art can be enlightened fully from prior art, the present invention repeats no more.
The method of use of test kit: be that genome with testing sample is a template; Utilize transgenic ground rice BT63 to detect gene; Through Real-Time PCR reaction; Finish the amplification curve that Real-Time PCR is confirmed in the back, first production standard curve can utilize formula to calculate the content of transgene component in the testing sample at last.Perhaps through specific primer of PCR and probe sequence, amplification testing sample and positive criteria reference substance, and the result that will increase compares, and whether contains transgenic paddy rice BT63 kind in the confirmatory sample, specifically making and detection method are with reference to embodiment.The described method of use of preceding text is the mode that those skilled in the art use always, and as example, those skilled in the art can carry out the more expansion through the common practise that combines prior art more.
Character of innovation of the present invention is:
The foundation of preparation completion of this standard model and preparation method thereof; Technology of preparing to solving transgenic BT63 strain detection molecules standard model guarantees technology with stability; Carry out the development of China's transgenic product examination criteria sample; Replenish the blank of this field of measurement, have very important practical sense and using value.
Description of drawings
Fig. 1: the preparation of transgenic ground rice BT63 standard model, characterization processes flow process.
Embodiment
Following non-limiting example can make those of ordinary skill in the art more fully understand the present invention, but does not limit the present invention in any way.
The foreign gene that transgenic paddy rice BT63 changes over to when carrying out gene transformation is anti insect gene cry1Ab/cry1Ac, is regulated and control by the NOS terminator.China does not provide transgenic paddy rice BT63 Biosafety certificate as yet.Transgenic paddy rice BT63 strain raw material is received the Entry-Exit Inspection and Quarantine Bureau in Liaoning;
Mortar, 100 mesh standard sieves, DNA extraction reagent and PCR reaction reagent are all purchased in precious biotechnology (Dalian) ltd;
DNA extraction test kit: purchase in precious biotechnology (Dalian) ltd (article No. D9093);
TaqMan Universal Master Mix: purchase company in ABI;
Primer and probe: precious biotechnology (Dalian) ltd is synthetic;
Real-time fluorescence quantitative PCR appearance: ABI 7500 types;
Nucleic acid-protein analyser: HIT.
Embodiment 1
The preparation process of transgenic ground rice shape standard model
A. between aseptic technique, use mortar grinding paddy rice sample, cross 100 mesh standard sieves, in powder, add the water of 4 times of quality, stir;
B. biased sample is positioned over that the cryogenic vacuum lyophilize is prepared into freeze-dried mixed powder in the pilot scale Freeze Drying Equipment;
C. freeze-dried mixed powder sample is sub-packed in capping sealing in the Brown Glass Brown glass bottles and jars only of the cleaning of 160 ℃ of dry heat treatment 2h, adorns freeze-dried mixed powder sample 1g in every bottle.
D. obtain 500 bottles of transgenic ground rice standard models.Sample after bottled is sticked on the uniqueness sign, is positioned over shady and cool dry 0 ℃ ~ 4 ℃ storages in place.
Each described freeze-drying in the above-mentioned steps (a) ~ (d), its concrete steps are following:
Cryogenic vacuum lyophilize program:
1. sample pre-freeze: earlier sample at-80 ℃ of freezing 2h;
2. refrigerating process: closed the kiln door of frost drying machine, beginning kiln refrigeration; When the kiln temperature is reduced to-35 ℃, begin to cool down the trap refrigeration; When the cooling pit refrigeration temperature is reduced to-40 ℃, the sample that 1. step is handled is put into kiln rapidly, closed the kiln door; Starting vacuum pump begins to vacuumize; When vacuum tightness is reduced to 0.5Torr, finish refrigerating process;
3. sample drying: the temperature of kiln is set to 15 ℃, and when vacuum tightness was reduced to 0.1Torr, sample was dry good;
4. close vacuum pump, kiln communicated with ambient atmosphere, treat inside and outside air pressure balance after, open the kiln door, take out sample, the bottle cap of screwing rapidly is prepared into freeze-dried mixed powder.
E. freeze-dried mixed powder sample is sub-packed in capping sealing in the Brown Glass Brown glass bottles and jars only of the cleaning of 160 ℃ of dry heat treatment 2h, adorns freeze-dried mixed powder sample 1g in every bottle, obtain 500 bottles of transgenic ground rice standard models.Sample after bottled is sticked on the uniqueness sign, is positioned over shady and cool dry 0 ℃ ~ 4 ℃ storages in place.
Embodiment 2 valued methods
Adopt " transgene component real-time fluorescence PCR detection method in paddy rice and products thereof " SN/T 2584-2010 and " transgenic product detects nucleic acid quantification PCR detection method " GB/T 19495.5-2004 to carry out definite value.
(a) DNA extraction test kit: precious biotechnology (Dalian) ltd (article No. D9093)
(b) TaqMan Universal Master Mix:ABI company
(c) real-time fluorescence quantitative PCR appearance: ABI 7500 types
(d) nucleic acid-protein analyser: HIT
The primer sequence and the probe sequence that detect transgenic paddy rice BT63 strain specificity and endogenous SPS gene see the following form 1.
Table 1 detects the primer sequence and the probe sequence of transgenic paddy rice BT63 strain specificity gene
Wherein, FAM is 6-carboxyfluorescein, and TAMRA is 6-carboxytetramethylrhodamine.
The real-time fluorescence PCR reaction system is seen table 2.
Table 2 real-time fluorescence PCR reaction system
The real-time fluorescence PCR reaction parameter is seen table 3.
Table 3 real-time fluorescence PCR reaction parameter
D. the result calculates
Reaction finishes the amplification curve that Real-Time PCR is confirmed in the back, production standard curve etc.Calculate the content of transgene component in the testing sample according to formula.
(a) calculation formula:
Remarks: different machines has different interior mark ratios, and concrete method of calculation are: get 100% genetically modified sample and survey the OD value, press four different gradients of OD value dilution then, example: 500ng/ul, 100ng/ul, 50ng/ul, 10ng/ul.Calculate the ratio of copy number of copy number and native gene of the foreign gene of four different gradient samples respectively, try to achieve the mean number of four numbers at last, this mean number be exactly in the mark ratio.Calculate by following formula.
Interior mark than calculation formula is:
(b) method of calculation:
1. the copy number of standard model being got denary logarithm as the X coordinate axis, is the Y coordinate axis with the CT value, obtains an endogenous typical curve and an external source typical curve respectively.Simultaneously also can obtain two linear equation with one unknown, one is endogenous equation, and one is the external source equation;
2. the Y in the equation calculates the X value with the CT value substitution (the endogenous equation of endogenous substitution, external source substitution external source equation) of sample;
3. the inverse logarithm of obtaining the X value again is the copy number of sample;
4. the copy number of using external source then is divided by endogenous copy number, and again divided by interior reference, the numerical value that obtains multiply by 100% again and is genetically modified percentage composition in this sample.
Embodiment 3 uniformity testings
From the transgenic ground rice BT63 sample of final packaging 1g/ bottle, randomly draw 15 bottles of samples, every bottle of sample is divided into 2 one's share of expenses for a joint undertaking appearance to be tested with real-time fluorescent PCR quantitative detection method, sampling amount 100mg, the content of analysis transgenic paddy rice BT63 strain.All test portions are tested under repeated condition with random order, promptly in same laboratory, use identical testing method and instrument test within a short period of time by identical personnel.Detected result uses variance analysis method to carry out Evaluation for Uniformity.
The uniformity result of transgenic ground rice BT63 standard model sees table 4, and its Evaluation for Uniformity result sees table 5.
Can know from table 5, not have significant difference between two sample sets, can think that sample is uniform.
Visible from table 5, through the F check, calculating the F ratio is 1.21, and less than the resulting F (14,15)=2.42 that tables look-up, interpret sample is uniform.Sample (no) homogeneity standard uncertainty is 0.008%.
Table 4 transgenic ground rice BT63 standard model uniformity result
| Sample number | Increment 1 test result (%) | Increment 2 test results (%) |
| 1 | 1.21 | 1.13 |
| 2 | 1.20 | 1.25 |
| 3 | 1.19 | 1.18 |
| 4 | 1.23 | 1.19 |
| 5 | 1.15 | 1.19 |
| 6 | 1.23 | 1.20 |
| 7 | 1.23 | 1.21 |
| 8 | 1.17 | 1.21 |
| 9 | 1.19 | 1.18 |
| 10 | 1.21 | 1.18 |
| 11 | 1.23 | 1.20 |
| 12 | 1.18 | 1.19 |
| 13 | 1.18 | 1.21 |
| 14 | 1.23 | 1.22 |
| 15 | 1.16 | 1.19 |
Table 5 transgenic ground rice BT63 standard model Evaluation for Uniformity result
Embodiment 4 stable preliminary experiments
For the inspection of the stability of standard model, the preliminary experiment that we at first passed through 4 years continues to measure stable, and on this basis, the standard model of batch preparations has passed through stability test and the stability experiment in 1 year under the transport condition.Standard model is stored 0-4 ℃ of sealing, and is stable in 4 years.The tentative validity period of these article is 4 years.The picked at random sample carries out stability test, and in every three months, second carried out content measuring in 1 year per two months,, adopts the real time fluorescent PCR method definite value.In whole stability tests, used personnel, instrument, testing method and laboratory are all identical with uniformity test.Test result is seen table 6.
The stable preliminary experiment result of table 6 transgenic ground rice BT63 standard model
Owing to there is not a kind of physical/chemical model can describe the mechanism of degradation of this candidate's standard model truly, adopt straight line as empirical model.In fact, for the characteristic value in this matrix, ideal value is: intercept (in uncertainty) equals to measure the value that obtains, and slope levels off to zero.
Slope can be used computes:
In the formula:
Intercept is by computes:
The standard deviation of the point on the straight line can be by computes:
Get its square root s=0.01399%, the uncertainty relevant with slope used computes:
Degree of freedom is that the student of n-2 and p=0.95 (95% confidence level) the t-factor that distributes equals 2.11.
Because | b
1|＜t
0.95, n-2S (b
1)
So slope is inapparent.Thereby do not observe unstable in the stable preliminary experiment.
Stability test under embodiment 5 transport conditions
4-7 was between the month in 2011; Adopt synchronous design method in-80 ℃ ~ 55 ℃ scopes, (to select-80 ℃ ,-20 ℃, 4 ℃, 25 ℃, 55 ℃) and carried out standard stability of sample research under the transport condition, by the resulting data initial analysis of stable preliminary experiment, the standard model in 0-4 ℃ of scope does not observe unstable; Therefore; The standard model that use stores in this TR is as " basic point ", and the standard model of other temperature condition held is a research object, and each temperature condition is 3 bottles of standard models of test down; Every bottle of repeated test twice, the condition of going through and test result such as table 7.
Stability study condition and test result thereof under the table 7 synchronous design transport condition
Do not consider the interaction of ununiformity and temperature effective between bottle, adopt two-way analysis of variance (seeing table 8), obtain that the unstable standard deviation is 0.0023 under the transport condition.
The stable analysis of variance table of table 8
The check of embodiment 6 permanent stability
The selected packaged sample that is used for stability test has been gone through long-time preservation under 0 ~ 4 ℃ of coldcondition.The picked at random sample carries out stability test.In every three months, second carried out content measuring in 1 year per two months,, adopted the real time fluorescent PCR method definite value.In whole stability tests, used personnel, instrument, testing method and laboratory are all identical with uniformity test.Test result is seen table 9, and stable analysis of variance table is seen table 10, and unstable standard deviation is 0.0068.
The stability test result of table 9 transgenic ground rice BT63 standard model
The stable analysis of variance table of table 10
Embodiment 7 standard values and uncertainty thereof detect
Adopt the real-time fluorescence quantitative PCR method to carry out definite value.Respectively transgenic ground rice BT63 standard model has been carried out 6 times repeated test, the test data summary sheet is seen table 11.
Through Grubbs check and Cochran check, all data all can be used as the definite value foundation; Through test for normality, these data fit normal distributions.
Requirement according to GB/T 15000-94 is added up data, calculates the standard value and the uncertainty of transgenic ground rice BT63 standard model, and the result sees table 12.
Table 11 transgenic ground rice BT63 standard model definite value is summary sheet as a result
Table 12 transgenic ground rice BT63 standard model definite value is statistical analysis table as a result
In the test for normality, coefficient of skewness A is less than 95% threshold value, and kurtosis B falls within 95% fiducial interval.Test for normality is the result show, the value data accord with normal distribution.
Test of outlier is the result show, do not have outlier in the value data.
The standard model of considering this development only uses as qualitative detection, therefore the uncertainty of uniformity testing, stability test is introduced in the synthetic uncertainty of definite value, selects k=2 for use, can satisfy use.
The uncertainty that homogeneity and stability are caused is synthetic with the mensuration uncertainty; Obtain result's combined standard uncertainty; Get that to comprise the factor be 2, obtain result's expanded uncertainty, the definite value result of transgenic ground rice BT63 standard model is: 1.20% ± 0.02%.The result who obtains proves that homogeneity is fine with stability.
Embodiment 8
In the Real-Time PCR detection kit of transgenic paddy rice BT63, comprise that transgenic ground rice BT63 detects gene and positive criteria reference substance,
Transgenic ground rice BT63 wherein detects gene, comprising:
The detection primer and the probe sequence of SPS gene:
Forward primer 5 '-ttgcgcctgaacggatat-3 ' SEQ ID NO:1
Reverse primer 5 '-cggttgatcttttcgggatg-3 ' SEQ ID NO:2
Probe 5 '-FAM-tccgagccgtccgtgcgtc-TAMRA-3 ' SEQ ID NO:3
The detection primer and the probe sequence of cry1Ac/NOS gene:
Forward primer 5 '-gactgctggagtgattatcgacaga-3 ' SEQ ID NO:4
Reverse primer 5 '-agctcggtacctcgacttattcag-3 ' SEQ ID NO:5
Probe 5 '-FAM-tcgagttcattccagttactgcaacactcgag-TAMRA-3 ' SEQ ID NO:6
Wherein: FAM is 6-carboxyfluorescein, and TAMRA is 6-carboxytetramethylrhodamine.
Comprise that also the positive criteria reference substance is a transgenic ground rice BT63 standard model, its concrete preparation method such as above-mentioned embodiment 1.
Get transgenic ground rice BT63 and non-transgenic paddy rice and do blind appearance; Respectively through the real-time fluorescence quantitative PCR assay determination; The result is: detect the anti insect gene cry1Ab/cry1Ac and the NOS terminator gene that contain BT63 in the sample of transgenic ground rice BT63, can draw as drawing a conclusion: raw material contains transgenic paddy rice BT63 article set member.
Among the present invention, relate to the method such as experimental procedure, experiment reagent preparation of genetically engineered operating aspect,, be routine techniques like no specified otherwise.PCR, Real-Time PCR react reaction reagent, reaction system and reaction parameter settings such as used damping fluid, all like above-mentioned embodiment.
The method of use of test kit: be that genome with testing sample is a template; Utilize transgenic paddy rice BT63 to detect gene; Through Real-Time PCR reaction; Finish the amplification curve that Real-Time PCR is confirmed in the back, first production standard curve can utilize formula to calculate the content of transgene component in the testing sample at last.Concrete making and detection method are with reference to embodiment.
The preparation of the Real-Time PCR detection kit of transgenic paddy rice BT63 is accomplished; Testing sample through the Real-TimePCR reaction, is finished the amplification curve that Real-Time PCR is confirmed in the back, the production standard curve; Can utilize formula to calculate the content of transgene component in the testing sample at last; This invention comprises the preparation of standard model and the preparation of detection kit, to the deep comprehensively technology of preparing of researching and solving transgene component, examination criteria sample and stability assurance technology, to the development of China's transgenic product examination criteria sample; Replenish the blank of this field of measurement, have very important practical sense.
Claims (2)
1. transgenic ground rice BT63 standard model is characterized in that, by following method preparation, step comprises:
With the raw material grind into powder, cross 100 mesh sieves, with the powder collection after sieving, in powder, add the water of 4 times of quality, after stirring, be positioned over that the cryogenic vacuum lyophilize is prepared into lyophilized powder in the pilot scale Freeze Drying Equipment; After the packing in 0 ℃ ~ 4 ℃ storages;
The cryodesiccated step of above-mentioned cryogenic vacuum comprises:
1. sample pre-freeze: earlier sample at-80 ℃ of freezing 2h;
2. refrigerating process: closed the kiln door of frost drying machine, beginning kiln refrigeration; When the kiln temperature is reduced to-35 ℃, begin to cool down the trap refrigeration; When the cooling pit refrigeration temperature is reduced to-40 ℃, the sample that 1. step is handled is put into kiln rapidly, closed the kiln door; Starting vacuum pump begins to vacuumize; When vacuum tightness is reduced to 0.5Torr, finish refrigerating process;
3. sample drying: the temperature of kiln is set to 15 ℃, and when vacuum tightness was reduced to 0.1Torr, sample was dry good;
4. close vacuum pump, kiln communicated with ambient atmosphere, treat inside and outside air pressure balance after, open the kiln door, take out sample, the bottle cap of screwing rapidly.
2. the Real-Time PCR detection kit of a transgenic paddy rice BT63 is characterized in that, comprises that transgenic ground rice BT63 detects gene and positive criteria reference substance:
Positive criteria reference substance: be the described transgenic ground rice of claim 2 BT63 standard model;
Transgenic ground rice BT63 detects gene, comprising:
The detection primer and the probe sequence of SPS gene
Forward primer SEQ ID NO:1
Reverse primer SEQ ID NO:2
Probe SEQ ID NO:3
The detection primer and the probe sequence of cry1Ac/NOS gene
Forward primer SEQ ID NO:4
Reverse primer SEQ ID NO:5
Probe SEQ ID NO:6.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103060459A (en) * | 2013-01-17 | 2013-04-24 | 中国检验检疫科学研究院 | Primer, probe, kit and method for detecting #8 Kefeng transgenic rice strain |
| CN103882126A (en) * | 2014-03-12 | 2014-06-25 | 中国检验检疫科学研究院 | Primer, method and kit used for detecting trans-mCrylAc-gene insect-resistant corn strain |
| CN103882126B (en) * | 2014-03-12 | 2016-04-06 | 中国检验检疫科学研究院 | For detecting the primer, method and the test kit that turn mCry1Ac gene pest-resistant corn strain |
| CN103924004A (en) * | 2014-05-12 | 2014-07-16 | 蒋丹 | Transgenic tobacco CMV-CP qualitative standard sample and preparation and value defining method thereof |
| CN103924004B (en) * | 2014-05-12 | 2015-10-28 | 蒋丹 | Transgene tobacco CMV-CP qualitative criteria's sample and preparation thereof, valued methods |
| CN109001004A (en) * | 2018-07-16 | 2018-12-14 | 湖南农业大学 | Transgene rape standard sample and its method for building up |
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