CN103924004A - Transgenic tobacco CMV-CP qualitative standard sample and preparation and value defining method thereof - Google Patents

Abstract

The invention discloses a transgenic tobacco CMV-CP qualitative standard sample and a preparation and value defining method thereof and belongs to the technical field of material standard samples for tobacco detection and preparation methods thereof. The preparation and value defining method of the transgenic tobacco CMV-CP qualitative standard sample comprises the steps of raw material selection of a transgenic tobacco CMV-CP strain, preparation of the standard sample, homogeneity and stability inspection, qualitative verification, value defining and the like, wherein the preparation of the sample comprises the steps of smashing and evenly mixing raw materials, performing freeze drying on the mixture through a freeze dryer, split charging the mixture, performing value defining, radiation and identification on the mixture, and storing the mixture at the temperature of 0 DEG C -4 DEG C. Furthermore, the minimum sampling volume of the standard sample and homogeneity examination are performed through a relative G-S index inspection method, and therefore, technical indexes of homogeneity, stability and definite value results of the standard sample can meet requirements of standard sample samples. Furthermore, the transgenic tobacco CMV-CP qualitative standard sample and the preparation and value defining method of the transgenic tobacco CMV-CP qualitative standard sample have important practical significance in actively carrying out the research on the qualitative standard sample for transgenic product testing in China to make up the blank in the measurement field.

Description

The qualitative standard model of transgene tobacco CMV-CP and preparation thereof, valued methods

Technical field

The invention belongs to plnat monitoring material standard sample and preparation method thereof technical field, be specifically related to qualitative standard model of transgene tobacco CMV-CP and preparation method thereof.

Background technology

Nineteen eighty-three, first case transgenic plant are born in the world.1984, English, U.S., Fa Deng state started to carry out taking tobacco as material transgenic research.The people such as Horsh initiate leaf disc transformation method, with agrobacterium tumefaciens infection tobacco leaf explant, obtain transgene tobacco on substratum, and having started with tobacco explant is that material carries out genetically modified new way.1986, the Powell-Abel of the U.S. imported the coat protein gene of tobacco mosaic virus (TMV) (TMV) first tobacco and obtains the transgenic tobacco plant of anti-TMV.From then on, Transgenic Tobacco research has obtained development fast, and has obtained great success.At present, reported in the world the above antiviral tobacco of successful transgenosis of 45 example, nearly hundred routine transgene tobaccos enter field experiment.Transgene tobacco gene used has: coat protein gene, antisense RNA gene, satellite RNA gene, rdrp gene, movement protein gene, toxin-resistant gene, trypsin inhibitor gene, chitinase gene, neuropeptide gene, animal interferon gene, zein spirit-soluble gene etc.Successfully transgene tobacco has: viral diseases tobacco; Antimycotic, Micobial Disease tobacco; Antiweed tobacco; Anti-root nematode disease tobacco; Anti-oriental tobacco budworm evil tobacco; Anti-aphid tobacco, monoclonal antibody tobacco etc.The transgene tobacco of the first approval commercialization plantation is by the antiweed bromoxynil tobacco that turns bxn gene of the SEITA company of European Union's approval in 1994.This transgene tobacco can resist the bromoxynil that exceeds 16 times of typical concentrations, and its economical character, quality, the quality of smokeing panel test again with contrast no significant difference.But the not spread plantation owing to suffering the opposition of some countries.1988, Peking University changed into the oriental tobacco varieties of anti-TMV first with TMV coat protein gene, and in Dandong institute of agricultural sciences field test.In the same year, Institute of Micro-biology of the Chinese Academy of Sciences and the Institute of Plant Protection's cooperation of Henan academy of agricultural sciences have also obtained the dual anti-viral flue-cured tobacco cultivars tobacco of anti-TMV, CMV (cucumber mosaic virus) with the complementary DNA transformation of tobacco of satellite RNA.Many units have carried out the research of shifting to tobacco with the goal gene of different sources subsequently.What Transgenic Tobacco research was carried out in domestic once project verification has 10 more than, and relating to acceptor kind has more than 10, and the research unit relating to has more than 20.

But people exist a lot of arguements to the safety issue of genetically modified food.The ecological risk of genetically modified crops, the environmental problem that may bring, problem as food to Health Impact, product labelling problem, transportation problem, international trade, Intellectual Property Rights etc. have caused the concern of world's popularity.The safety issue of transgenic product, by academic viewpoint difference, develops into even political issue of intellecture property, environmental problem, economic problems.European Union and some countries have put into effect transgenic product laws and regulations on the management in succession, and China comes into effect " agriculture genetically modified organism security control regulations " and three supporting management ways thereof in March, 2002.Calendar year 2001, European Union require genetically modified food is carried out to identity management, and propose to non-transgenic food so long as not intentional pollution, allow to contain 1% transgenic product (threshold value), and need not identify.The limitation threshold value difference of the country variants such as Japan, Korea S, Australia, from 1%-5% not etc.

The detection of genetically modified food is intended to the biological and ecological safety of protection, safeguards consumer rights, gives a impetus to trade and the economic regulation developing in a healthy way and the basic foundation of policy, and its stdn will be for working out, implement and improvement relevant laws and regulations and policy providing strong guarantee.The detection of genetically modified food research is both at home and abroad at the early-stage, and many problems have to be solved, and standard model is still unsolved major issue of this field.The development of carrying out transgene component inspecting standard sample is ensureing comparability and the traceability of test result, is ensuring food safety, settles trade disputes, and the aspect such as promote economic development all has great importance.More severe problem is the reference material that only has at present a few kind such as genetically engineered soybean powder, transgenic corns powder MON810 abroad, and price is very expensive, and formality is various, and the turnover time is long, can not in time meet inspection and quarantine requirements of one's work, Japan is stepping up to develop the molecular dna reference standard material that transgenic product detects, for the detection by quantitative of transgenic product, 2005, the domestic genetically engineered soybean powder national grade ii standard material of having succeeded in developing, fill up the blank of China aspect detection of GMOs standard model, succeed in developing afterwards transgenic rapeseed, transgenic corns, the standard models such as transgene cotton seed, but the development of transgene tobacco standard model or blank, and be a difficult point for the Evaluation for Uniformity of qualitative standard model always, so far there is not the statistical method of science to introduce, the situation of and Evaluation for Uniformity difficulty in short supply in view of domestic transgene tobacco standard model, the preparation of this standard model completes, for the qualitative standard model research of transgene tobacco provides technology of preparing, Evaluation for Uniformity and stability ensure technology, detect the development of qualitative standard model to actively developing China's transgenic product, replenish the blank of this fields of measurement, there is very important realistic meaning.

Summary of the invention

The present invention is in order to complete the preparation work of this standard model, Liaoning Entry-Exit Inspection and Quarantine Bureau has set up development work group, the multinomial work such as the raw material that has carried out transgene tobacco CMV-CP strain detects that primer is determined, raw material chooses, preparation, homogeneity and stability inspection, the definite value of qualitative test, data check, standard model, concrete:

An aspect of of the present present invention relates to the qualitative standard model of transgene tobacco CMV-CP, be by transgene tobacco raw material pulverizing, mix, packing after Freeze Drying Equipment lyophilize, definite value, finally by ⁶⁰c _o4kGy irradiation, sample is sticked on uniqueness mark, under 0-4 DEG C of temperature condition, stores.Its preparation technology's flow process is shown in Fig. 1, and concrete preparation process comprises:

A. after getting 500g transgene tobacco CMV-CP raw material pulverizing, cross 100 mesh standard sieves;

B. the transgene tobacco powder obtaining in step a is added water after (mass ratio 1:1) mixes and be positioned over cryogenic vacuum lyophilize in pilot scale Freeze Drying Equipment, 1 DEG C/min of chilling rate, early stage freezing pt-1 DEG C, 15min, freezing temp-40 DEG C, eutectic point-26 DEG C, 20 DEG C of 120min of Heating temperature are prepared into freeze-dried mixed powder;

C. the freeze-dried mixed powder obtaining in step b is sub-packed in to capping sealing in the clean 7mL Brown Glass Brown glass bottles and jars only of 160 DEG C of dry heat treatment 2h, every bottle of in-built freeze-dried mixed powder sample 1g, then by point sample warp installing ⁶⁰c _oafter 4kGy irradiation 1h, be positioned over 0～4 DEG C of storage in dry place;

D. use the homogeneity of the sample obtaining in relative G-S index testing method checking procedure c.

In technique scheme of the present invention, the homogeneity of the sample obtaining in the relative G-S index testing of described use method checking procedure d refers to the homogeneity of utilizing G-S index method statistical appraisal sample.This is the application's innovation, in existing technical literature, have no report, it compares traditional method significant progress, and usefulness is embodied in the Evaluation for Uniformity of G-S index method being introduced to qualitative standard model, make the Evaluation for Uniformity of qualitative standard model have more science, reliability;

Gini-Simpson index testing method relatively: the two condition attribute situation that only can get "Yes", two values of "No" etc. for quantitative attributes value, adopt quantitative attributes value degree of scatter between Gini-Simpson Index for Calculation article and the degree of scatter of property value in the time being uniformly distributed, the ratio of two indexes can represent relative degree of scatter, i.e. relative uniformity coefficient.Owing to generally representing homogeneity by degree of irregularity, therefore, deduct relative uniformity coefficient with 1 and obtain unevenness.

Sometimes, the ununiformity of standard model under present level may be able to not be detected by existing measuring method, for example, all measuring results are " positive ", at this moment, standard model is diluted or dwindles increment amount until occur that the probability of occurrence of two values of two condition attribute is not all near the scope of 1 (part fields of measurement so-called " Cutoff " value), within the scope of this, adopt relative its ununiformity of Gini-Simpson Index Assessment, the unevenness then getting standard samples according to the relation between unevenness and level.In a lot of situations, the ratio of the unevenness of the standard model being diluted and the unevenness of standard model is about the square root times of thinning ratio.

Account form is:

Each in m sample carried out to n time to measure, use " 1 " and " 0 " to represent respectively the situation of attribute to be measured " existence " and " not existing ", if the ratio that i proficiency testing article are obtained result " 1 " is pi, be calculated as follows Gini-Simpson index (being abbreviated as GS index):

$\mathrm{GS}=1-\underset{i=1}{\overset{m}{\mathrm{\Σ}}}{p}_i^2$

Use average proportions to calculate the maximum GS index being uniformly distributed in situation simultaneously:

${\mathrm{GS}}_m=1-\underset{i=1}{\overset{m}{\mathrm{\Σ}}}{\left(\stackrel{\‾}{p_i}\right)}^2$

In formula: ${\stackrel{\‾}{p}}_i=\underset{i=1}{\overset{m}{\mathrm{\Σ}}}{p}_i/m,$ It is the average proportions of m article

Calculate relative GS index (being abbreviated as RGS)

RGS＝GS/GS _m

RGS has represented the relative uniformity coefficient of property value, and 1-RGS has represented the relative unevenness of property value.

The standard model of preparing according to technique scheme of the present invention is Powdered, net weight 1g, and internal packing is Brown Glass Brown glass bottles and jars only, the hard box of outer packaging plastic cement, built-in specification sheets.Can, for transgenic product qualitative detection, also can be used for quality control.This product is opened, and sampling weighs and can use.Use minimum sample mass is 80mg.Product under drying conditions, shady and cool, sealing, 0-4 DEG C of storage, stable in two years, normal temperature transport.Product uniformity testing and stability test are described: through GS index testing, and minimum sample mass 80mg, this standard sample is even.

Raw material in 1 technique scheme of the present invention is chosen: the flue-cured tobacco positive (CMV-CP strain) of collecting in transgenosis testing.Transgene tobacco CMV-CP strain is that cucumber mosaic virus coat protein gene is imported in tobacco and makes it to have anti-this viral proterties, and the foreign gene containing has: CaMV35S promotor, NOS terminator, NPT II and CMV-CP.

The qualitative test of 2 technique scheme Plays samples of the present invention: using the tobacco positive of collecting as representative sample, employing PCR method is analyzed, amplify CMV-CP strain identified gene fragment, confirm that transgene tobacco sample is transgene tobacco CMV-CP strain.

The preparation of 3 technique scheme Plays samples of the present invention: by raw material pulverizing, mix, store under packing after Freeze Drying Equipment lyophilize, definite value, irradiation, mark, 0-4 DEG C temperature condition.Concrete grammar is: transgene tobacco pulverized and mixed, and packing after Freeze Drying Equipment lyophilize, finally by ⁶⁰c _o4kGy irradiation, sample is sticked on uniqueness mark, is positioned over shady and cool dry place storage.

Minimum sample mass, the homogeneity of the standard model in 4 inspection technique scheme: this research is used relative G-S index testing method inspection, get at random 1 duplicate samples, the different sampling such as 60mg, 80mg, 100mg gradient is set, adopt PCR method to carry out repeated test, the impact of more different sampling amounts on detected result, thereby determine that minimum sample mass is 80mg, and sample is even under this sampling gradient.

The stability of 5 standard models of preparing according to technique scheme of the present invention: from October, 2011, adopt the sample preparation program identical with this project to carry out the test of transgene tobacco CMV-CP strain standard model stability, the standard model of test preparation has experienced the stability test of 2 years, in lucifuge, vacuum-sealing, under 0-4 DEG C of temperature condition, store, stable in 2 years.Therefore it is 2 years that this product validity period is fixed tentatively.

The 6 standard model definite values of preparing according to technique scheme of the present invention: Duo Jia laboratory cooperation definite value, testing method adopts SN/T1200-2003 " transgene component qualitative PCR detection method in tobacco ".This standard model is qualitative detection standard model, thus the process of definite value actual be exactly checking process.

7. every bottle of sample-loading amount of standard model of preparing according to technique scheme of the present invention is 1 gram.

Another aspect of the present invention relates to a kind of transgene tobacco CMV-CP detection kit, and it comprises detection primer: transgene tobacco CMV-CP standard model prepared by SEQ ID NO.1～4 and technique scheme.The base sequence of described CMV-CP primer is SEQ ID NO.1～4.Can be for detection of transgene tobacco CMV-CP strain specificity and tobacco NR native gene.In the preferred case, in this detection kit, can also comprise one or more in other reaction reagents: the dNTPs (dUTP) of 10mmol/ μ L, the SEQ ID NO.1/2 of each 50pmol/ μ L or SEQ ID NO.3/4, the magnesium chloride (MgCl of 25mmol/ μ L ₂), 10 × Buffer, the Taq of 5U/ μ L, the UNG enzyme of 1U/ μ L.

Brief description of the drawings

Fig. 1. the technical process of transgene tobacco powder preparation of standard sample.

Embodiment

Following non-limiting example can make the present invention of those of ordinary skill in the art's comprehend, but does not limit the present invention in any way.

The freeze drier using in the present invention is German Christ MP2-6D freeze drier.

Embodiment 1 preparation of standard sample

1 raw material is chosen: the flue-cured tobacco positive (CMV-CP strain) of collecting in transgenosis testing.Transgene tobacco CMV-CP strain is that cucumber mosaic virus coat protein gene is imported in tobacco and makes it to have anti-this viral proterties, and the foreign gene containing has: CaMV35S promotor, NOS terminator, NPT II and CMV-CP.

2 qualitative tests: using the tobacco positive of collecting as representative sample, adopt PCR method analysis, amplify CMV-CP strain identified gene fragment, confirm that transgene tobacco sample is transgene tobacco CMV-CP strain.

The preparation of 3 standard models, concrete operation step is as follows:

A. after getting 500g transgene tobacco raw material pulverizing, cross 100 mesh standard sieves.

B. transgene tobacco powder is added water after (mass ratio 1:1) mixes and be positioned over (1 DEG C/the min of chilling rate (Cooling Rate) of cryogenic vacuum lyophilize in pilot scale Freeze Drying Equipment, early stage-1 DEG C of 15min of freezing pt (Incipient Freezing Point),-40 DEG C of freezing temps (Cooling temperature),-26 DEG C of eutectic points (Melting Point), 20 DEG C of 120min of Heating temperature (Heating temperature)) be prepared into freeze-dried mixed powder;

C. freeze-dried mixed powder sample is sub-packed in to capping sealing in the clean 7mL Brown Glass Brown glass bottles and jars only of 160 DEG C of dry heat treatment 2h, every bottle of in-built freeze-dried mixed powder sample 1g.The finally all points of sample warps that install ⁶⁰c _o4kGy irradiation 1h;

D. obtain 400 bottles of transgene tobacco powder standard models.Sample after bottled is sticked on uniqueness mark, is positioned over 0 DEG C～4 DEG C storages in dry place.

4. transgene tobacco CMV-CP strain standard model finished product:

It is Powdered that finished product is, net weight 1g, and internal packing is Brown Glass Brown glass bottles and jars only, the hard box of outer packaging plastic cement, built-in specification sheets.Can, for transgenic product qualitative detection, also can be used for quality control.This product is opened, and sampling weighs and can use.Use minimum sample mass is 80mg.Product under drying conditions, shady and cool, sealing, 0-4 DEG C of storage, stable in two years, normal temperature transport.Product uniformity testing and stability test are described: through GS index testing, and minimum sample mass 80mg, this standard sample is even.

Embodiment 2 homogeneities

Standard model minimum sample mass, uniformity testing: the present invention uses relative G-S index testing method inspection.

Gini-Simpson index relatively

Each in m sample carried out to n time to measure, use " 1 " and " 0 " to represent respectively the situation of attribute to be measured " existence " and " not existing ", if the ratio that i proficiency testing article are obtained result " 1 " is pi, be calculated as follows Gini-Simpson index (being abbreviated as GS index):

$\mathrm{GS}=1-\underset{i=1}{\overset{m}{\mathrm{\Σ}}}{p}_i^2$

Use average proportions to calculate the maximum GS index being uniformly distributed in situation simultaneously:

${\mathrm{GS}}_m=1-\underset{i=1}{\overset{m}{\mathrm{\Σ}}}{\left(\stackrel{\‾}{p_i}\right)}^2$

In formula: ${\stackrel{\‾}{p}}_i=\underset{i=1}{\overset{m}{\mathrm{\Σ}}}{p}_i/m,$ It is the average proportions of m article

Calculate relative GS index (being abbreviated as RGS)

RGS＝GS/GS _m

RGS has represented the relative uniformity coefficient of property value, and 1-RGS has represented the relative unevenness of property value.

2. the detection of GMOs result of different sampling amount samples

From a collection of (400 bottles) the freeze-drying sample preparing, extract 1 bottle as test sample, for finding " Cutoff " value in-scope.This bottle of freeze-drying sample arranged to 60mg, 80mg, tri-sampling amount gradients of 100mg, under each sampling gradient, repeat 6 PCR method tests, until occur that under same sampling gradient 6 samples are not exclusively positive, this sampling gradient is the orientation at " Cutoff " value place.If need to find out " Cutoff " value, may need further experiment.All test portions are tested under repeat condition with random order, in same experiment, are tested within a short period of time by the identical testing method of identical librarian use and instrument.

The detected result of different sampling amounts is in table 1, and wherein " 1 " represents positive findings, and " 0 " represents negative findings.

The detection of GMOs result of the different sampling amount samples of table 1 transgene tobacco CMV-CP strain

Result shows: negative findings appears in sampling amount while being 60mg, and therefore 60mg is " Cutoff " value in-scope.

3. sample homogeneity test result

From sample is criticized, choose at random 10 bottles of freeze-drying samples, in test sample under the same gradient at " Cutoff " value place, every bottle of sample divides to be got and repeated test 6 times again, and what obtain the results are shown in table 2, and wherein " 1 " represents positive findings, and " 0 " represents negative findings.

Table 2 transgene tobacco CMV-CP strain sample homogeneity test result

Statistics: equally distributed situation is that the positive contribution rate of 10 samples is 10%, maximum GS index GSm=0.9, GS index=0.897507/0.9=99.7% relatively, the unevenness △ 60=1-99.7%=0.3% under this gradient.

According to the general relationship of unevenness and gradient ratio, by the unevenness under this gradient divided by the square root of gradient ratio the unevenness of raw sample be: △ 80=0.240% when sampling amount 80mg, △ 100=0.215% when sampling amount 100mg, therefore when sampling amount is 60mg in 400 bottles of samples, has an appointment 1.2 bottles and may occur not detecting, when sampling amount is 80mg, have an appointment 0.96 bottle and may not detect, to sum up, this batch sample minimum sample mass is defined as 80mg, the requirement of this batch of freeze-drying sample homogeneity conformance with standard sample preparation under this sampling amount.

Embodiment 3 stability

The selected packaged sample for stability test has been gone through respectively temperature variation and long-time preservation.Choose at random sample and carry out stability test, all samples of being selected are placed at room temperature (18～25 DEG C) 18 days, are then placed in 50 DEG C of insulation cans 4 days, then place at room temperature 18 days, then put into the 0-4 DEG C of medium-term and long-term preservation of refrigerator.Every two months of First Year, Second Year every three months be each to be got at random a sample and carries out detection of GMOs 4 times, and PCR method detects.Identical with the method for uniformity testing, under minimum sample mass, test, if there is negative findings, illustrate that this sample is unsettled with this understanding.In whole stability tests, personnel used, instrument, testing method and laboratory are all identical with uniformity test.Test result is in table 3, and wherein " 1 " represents positive findings, and " 0 " represents negative findings.Result shows, it is unstable that transgene tobacco standard model has no in two years.

The stability test result of table 3 transgene tobacco powder standard model

Embodiment 4 cooperation definite values

Duo Jia laboratory cooperation definite value, testing method adopts SN/T1200-2003 " transgene component qualitative PCR detection method in tobacco ", and concrete operation step is as follows:

(1) instrument and reagent: PE2400 type pcr analysis instrument; Gel imaging system Tocan; Germany Christ MP2-6D freeze drier; DNA extraction test kit: Takara (article No. D9093); PCR Premix:Takara; Primer;

The primer sequence that detects transgene tobacco CMV-CP strain specificity and tobacco NR native gene sees the following form 4.

Table 4 detects the primer sequence of tobacco CMV-CP strain

(2) reaction system

The consumption of each reagent of reaction system is suitably adjusted according to the cumulative volume of reaction system.Each reaction system should arrange two parallel pipes, as table 5

Table 5PCR reaction system 50 μ L

(3) pcr amplification condition

Denaturation: 94 DEG C of 10min

Cycle sets: 94 DEG C of 1min of sex change

Annealing: 55 DEG C of 1min

Extend: 72 DEG C of 2min

Cycle index: 30 circulations

Rear extension: 72 DEG C of 5min

(4) pcr amplification product electrophoresis detection

Claim that agarose is dissolved in 0.5 times of tbe buffer liquid, make 1.5% gel, add EB (0.5 μ g/mL), in the time that being down to 55 DEG C of left and right, temperature pours in the gel mould that is placed with comb, after condensation, extract comb, put into Horizontal electrophoresis tank, get 10 μ L amplified productions and add sample-loading buffer point sample, add DNA standard molecular weight mark electrophoresis, gel imaging system is observed electrophoresis result.

(5) result judgement

At native gene, all in normally amplification situation, if negative and positive and blank are set up, sample to be checked has specific gene band positive, otherwise negative; If negative and positive and blank have one to be false and all will again to test.

Method described in employing above-described embodiment 4, combining 8 laboratories adopts PCR method to carry out cooperation definite value (numbering respectively 1～8 in table 6), the repeated test of 6 times has been carried out respectively in these 8 laboratories to transgene tobacco standard model, test data summary sheet is in table 6.

Table 6 transgene tobacco CMV-CP strain standard model definite value result

In the preparation work of standard model of the present invention, by choosing suitable representative raw material, adopt rationally technical process reliably, fully ensure homogeneity, the stability of standard model, in preparation process, avoid container and environment to pollute finished product.The homogeneity of standard model is tested and carry out statistical study by choosing representational parameter.Choose accurately and reliably measuring method and by multiple laboratories definite value that cooperates, selected laboratory is country or accredited laboratory of department, and value data is carried out to strict statistical treatment, and computation interval.Stability to standard model is tested.Adopt qualified packaged form, be convenient to transport and long-term preservation.Adopt rational banking system to preserve.The requirement of sample thereby the technical indicators such as the homogeneity of guarantee standard model, stability, definite value result are up to state standards.

Claims (2)

1. the qualitative standard model of transgene tobacco CMV-CP, is characterized in that, preparation process comprises:

A. after getting transgene tobacco CMV-CP raw material pulverizing, cross 100 mesh standard sieves;

B. the transgene tobacco powder obtaining in step a is added water, after mixing with mass ratio 1:1, be positioned over cryogenic vacuum lyophilize in pilot scale Freeze Drying Equipment, 1 DEG C/min of chilling rate, early stage freezing pt-1 DEG C, 15min, freezing temp-40 DEG C, eutectic point-26 DEG C, 20 DEG C of Heating temperatures, 120min are prepared into freeze-dried mixed powder;

C. the freeze-dried mixed powder obtaining in step b is sub-packed in to capping sealing in the clean Brown Glass Brown glass bottles and jars only of 160 DEG C of dry heat treatment 2h, then by point sample warp installing ⁶⁰c _oafter 4kGy irradiation 1h, in 0～4 DEG C of storage in dry place;

D. use the homogeneity of the sample obtaining in relative G-S index testing method checking procedure c.

2. a transgene tobacco CMV-CP detection kit, is characterized in that, comprises detection primer: SEQ ID NO.1～4 and CMV-CP standard model claimed in claim 1.