CN106872395A - A kind of physical detection methods of novel corn transgene component - Google Patents
A kind of physical detection methods of novel corn transgene component Download PDFInfo
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- 235000005822 corn Nutrition 0.000 title claims abstract description 52
- 108700019146 Transgenes Proteins 0.000 title claims abstract description 22
- 238000011202 physical detection method Methods 0.000 title claims abstract description 12
- 238000001514 detection method Methods 0.000 claims abstract description 59
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/35—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light
- G01N21/3581—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light using far infrared light; using Terahertz radiation
- G01N21/3586—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light using far infrared light; using Terahertz radiation by Terahertz time domain spectroscopy [THz-TDS]
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/35—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light
- G01N21/3563—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light for analysing solids; Preparation of samples therefor
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/35—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light
- G01N21/3563—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light for analysing solids; Preparation of samples therefor
- G01N2021/3572—Preparation of samples, e.g. salt matrices
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Abstract
The invention discloses a kind of physical detection methods of novel corn transgene component, comprise the following steps：1）The making of standard specimen and sample；2）The acquisition of the terahertz time-domain spectroscopy data of standard specimen：3）The tera-hertz spectra detection of sample transgene component；4）The qualitative analysis of corn gene composition.The present invention uses terahertz light spectrum detection method, it is whether positive by analyzing the tera-hertz spectra signal detection of corn sample its transgene component, with sample preparation is simple, detection speed fast, high precision, intelligence degree is high, require testing staff the low advantage of level, can effectively solve the problems, such as that the detection speed that conventional biochemical detection method is present is slow, detection process is complicated.
Description
Technical field
The present invention relates to the component detection method of cereal crops corn, specially a kind of thing of novel corn transgene component
Reason detection method.
Background technology
Crops transgenic detection method mainly has 2 classes：1st class method market surpervision detection in it is the most commonly used, be with
The specific dna sequence of foreign gene is the detection technique of object；2nd class is protein detection techniques.
1. the detection method of DNA sequence dna is based on
(1) PCR detection method.It is divided into 4 classes according to its detection object difference：Element specific PCR, gene specific PCR,
Build specific PCR, specificity of transformant PCR.Promoter, mark that transgenosis conversion carrier is carried are detected by round pcr
The particular sequences such as gene, terminator, judge whether it is genetically modified crops.The method specificity is preferable, efficiency is higher, expense
It is low, it is that current China's agricultural transgenosis supervision department carries out the main method that transgenosis supervision is detected, such inspection has been issued at present
Mark standard exceedes 50.The method detected by regular-PCR or real-time fluorescence quantitative PCR to genetically modified crops universal component,
It is a kind of more accurate, efficient method, but defect is that current flux is smaller, the cycle is more long, false positive easily occurs.
(2) gene chips.Gene chips are also called DNA microprobe array methods, and its essence is exactly Highgrade integration
Reverse dot blot hybridization technique, detection chip is made by by the specific segment of foreign gene, is carried out with the DNA of sample to be tested miscellaneous
Hand over, after reaction result scanning, judge whether testing sample is transgenic product by computer software analysis.The method
Flux is high, but detection process is cumbersome, especially costly, high to experimental facilities requirement, can popularization it is relatively low.
2. the detection method of expression product protein is based on.Immunology protein detection side based on antibody, antigen
Method, judges whether crop is transgenic product by the protein of qualitative, quantitative exogenous gene expression generation.Heterogenous expression egg
White detection method has 3 kinds：Biochemical reaction detection method；Immunological detection, mainly has Western hybridization, ELISA method and is immunized
The precipitation method；The detection of heterogenous expression biological activity of albumen.The detection of heterogenous expression albumen is genetically modified crops detection and safety
Property evaluate one of most efficient method, but this kind of method just for some transformation event, it is necessary to take the method for excluding one by one
To reach testing goal, cumbersome, high cost is unsuitable for the blind sample detection of high-volume.In addition it is also contemplated that gene expression after transgenosis
, easily there is missing inspection in the problem of silence.
The detection of corn gene composition, prior art is mainly biochemical detection methods, exist sample pre-treatments it is complicated,
Detection process is cumbersome, it is more to employ reagent, testing cost expensive, detection time is long, false positive easily occur, and testing staff's specialty
The technical problem such as level requirement is high,
The content of the invention
In order to overcome above-mentioned deficiency of the prior art, terahertz light spectrum detection method is used the invention provides one kind,
It is whether positive by analyzing the tera-hertz spectra signal detection of corn sample its transgene component, with sample preparation is simple, inspection
Degree of testing the speed is fast, high precision, intelligence degree is high, require testing staff the low advantage of level, can effectively solve conventional biochemical inspection
The problem that detection speed is slow, detection process is complicated that survey method is present.
The object of the present invention is achieved like this：
A kind of physical detection methods of novel corn transgene component, comprise the following steps：
1) making of standard specimen and sample；
2) acquisition of the terahertz time-domain spectroscopy data of standard specimen：
3) the tera-hertz spectra detection of sample transgene component；
4) qualitative analysis of corn gene composition.
Described step 1) standard specimen and sample making in, standard specimen be used for set up each strain corn (including transgenosis and
It is non-transgenic) Terahertz standard spectrum storehouse；Sample is used for the detection to corn gene composition；Make the reference material of standard specimen
Matter raw material sources in the standard substance of each corn strain, from Institute of Science and Technology, National Food Bureau buy, and has by standard substance
Standard substance certificate；
Standard substance raw material take 120mg after fully crushing, grinding, and add 40mg polyethylene powders, are filled through shaking up device
After dividing mixing, placing tablet press machine carries out compressing tablet.Pressure 10MPa is kept during compressing tablet tri- minutes, take out compressing tablet, use electron helical micrometer
After device measures thickness (being accurate to 0.01mm), place hermetic bag and seal, hermetic bag is labelled dated family name and thickness, extremely
This, standard specimen makes and finishes；Sample making process is with standard specimen manufacturing process；
Standard specimen or sample need to be completed within half an hour, to avoid standard specimen from placing the physic-chemical changes for occurring in atmosphere；
Standard specimen or sample raw material, in 12%-15% or so, if moisture is too high, can influence sample to too using preceding holding moisture
The absorption of Hertzion radiation, it is therefore desirable to carry out sample preparation after being dried using dryer again.The water parameters of 12%-15% determine
Concerned countries standard is stored from grain security；
Described step 2) standard specimen terahertz time-domain spectroscopy data acquisition in：Standard specimen is positioned over terahertz time-domain light
In spectrometer, the terahertz time-domain spectroscopy data of standard specimen are obtained using transmission mode.The environmental condition of detection is set to：Environment temperature
Degree：20 DEG C, ambient humidity：≤ 10%；Every kind of corn strain makes 3 standard specimens, and each standard specimen is measured 3 times, altogether to every kind of jade
Rice strain measures 9 groups of time-domain spectroscopy data, and take 0.2T-1.2T wave bands carries out averagely, obtaining the standard specimen and putting down for 9 times as valid data
Equal time-domain spectroscopy data, recycle spectrometer accompanying software in itself, obtain the absorption spectra data and waveform of the standard specimen, store
To in database；Above-mentioned detection process is repeated to each strain standard specimen, each strain terahertz absorption spectra normal data of corn is set up
Storehouse；
Described step 3) sample transgene component tera-hertz spectra detection in, it is first for the corn sample that need to be detected
First need to carry out decontamination, cleaning, dry, taking fraction carries out determination of moisture, when moisture stabilization is between 12%-15%, then
Crushed, ground, compressing tablet.Compressing tablet is positioned in terahertz light spectrometer by step 2) carry out spectroscopic assay；
Described step 4) corn gene composition qualitative analysis in：(1) with 0.01T as frequency interval, 0.2T- is taken
100 data points of 1.2T terahertz wave band absorption spectras.For sample data, Xi (i=1,2 ..., 100) is designated as；For standard
Data in library of spectra, are designated as Yi (i=1,2 ..., 100)；
(2) coefficient correlation of sample data and corresponding library of spectra standard specimen data is calculated as follows；
In above formula, n=100,It is sample data average value,It is standard specimen statistical average in corresponding library of spectra, r is meter
The coefficient correlation for obtaining；
Related system r1, r2 are calculated to each standard specimen in standard spectrum storehouse ..., rn ...；
From r1, the maximum r values of the middle selection of r2 ..., rn ..., and using corresponding standard specimen as corn gene composition mirror
Determine result.
Positive beneficial effect：The present invention uses terahertz light spectrum detection method, by the terahertz light for analyzing corn sample
Spectrum signal detects whether its transgene component is positive, with sample preparation is simple, detection speed fast, high precision, intelligence degree
It is high, require testing staff the low advantage of level, the detection speed that can effectively solve the presence of conventional biochemical detection method is slow, detection
The complicated problem of process.
Brief description of the drawings
Fig. 1 is four kinds of terahertz time-domain spectroscopies of corn strain of embodiment；
Fig. 2 is the Terahertz absorption spectra of four kinds of corn strains of embodiment.
Specific embodiment
With reference to embodiment, the present invention is described further：
A kind of physical detection methods of novel corn transgene component, comprise the following steps：
1. standard specimen and Sample Prep Protocol：
Standard specimen is used to set up the Terahertz standard spectrum storehouse of each strain corn (including transgenosis and non-transgenic)；Sample
For the detection to corn gene composition.
The standard substance raw material sources of standard specimen are made in the standard substance of each corn strain, standard substance is from national food
The purchase of research institute of office, and with standard substance certificate.
Standard substance raw material take 120mg after fully crushing, grinding, and add 40mg polyethylene powders, are filled through shaking up device
After dividing mixing, placing tablet press machine carries out compressing tablet.Pressure 10MPa is kept during compressing tablet tri- minutes, take out compressing tablet, use electron helical micrometer
After device measures thickness (being accurate to 0.01mm), place hermetic bag and seal, hermetic bag is labelled dated family name and thickness, extremely
This, standard specimen makes and finishes；Sample making process is with standard specimen manufacturing process.
Standard specimen or sample need to be completed within half an hour, to avoid standard specimen from placing the physic-chemical changes for occurring in atmosphere.
Standard specimen or sample raw material, in 12%-15% or so, if moisture is too high, can influence using preceding holding moisture
Absorption of the sample to terahertz emission, it is therefore desirable to carry out sample preparation after being dried using dryer again.The moisture of 12%-15%
Parameter determination stores concerned countries standard from grain security.
2. the acquisition of the terahertz time-domain spectroscopy data of standard specimen
Standard specimen is positioned in terahertz time-domain spectroscopy instrument, the terahertz time-domain spectroscopy number of standard specimen is obtained using transmission mode
According to.The environmental condition of detection is set to：Environment temperature：20 DEG C, ambient humidity：≤ 10%.Every kind of corn strain makes 3 marks
Sample, each standard specimen is measured 3 times, measures 9 groups of time-domain spectroscopy data to every kind of corn strain altogether, takes 0.2T-1.2T wave band conducts
Valid data carry out averagely, obtaining 9 average time-domain spectroscopy data of the standard specimen, in the accompanying software using spectrometer in itself,
The absorption spectra data and waveform of the standard specimen are obtained, in storage to database.Above-mentioned detection process is repeated to each strain standard specimen, is set up
Each strain terahertz absorption spectra standard database of corn
3. the tera-hertz spectra detection of sample transgene component
For the corn sample that need to be detected, need to carry out decontamination, cleaning first, dry, taking fraction carries out determination of moisture,
When moisture stabilization is between 12%-15%, then crushed, ground, compressing tablet.By compressing tablet be positioned in terahertz light spectrometer by
Step 2 carries out spectroscopic assay.
4. the qualitative analysis of corn gene composition
(1) with 0.01T as frequency interval, 100 data points of 0.2T-1.2T terahertz wave band absorption spectras are taken.For sample
Product data, are designated as Xi (i=1,2 ..., 100)；For the standard specimen data in standard spectrum storehouse, be designated as Yi (i=1,2 ...,
100)。
(2) coefficient correlation of sample data and standard specimen data in corresponding library of spectra is calculated as follows.
In above formula, n=100,It is sample data average value,It is standard specimen statistical average in corresponding library of spectra, r is meter
The coefficient correlation for obtaining.
Related system r1, r2 are calculated to each standard specimen in standard spectrum storehouse ..., rn ....
From r1, the maximum r values of the middle selection of r2 ..., rn ..., and using corresponding standard specimen as corn gene composition mirror
Determine result.
Term is explained：
1. THz wave：THz wave refers to frequency in 0.1THz to 10THz (1T=1012Hz) electromagnetic wave of scope, ripple
It is long probably 0.03 to 3mm scopes, between microwave and it is infrared between.
2. terahertz time-domain spectroscopy (THz-TDS)：It is a kind of new, very effective coherent swpectrum Detection Techniques.It is special
Levy：(1) insensitive to black body radiation, signal to noise ratio is far above Ftir Spectroscopy.(2) detectable material is too
The physics and chemical information of hertz wave band.(3) dielectric substance, semi-conducting material, the amplitude of large biological molecule and phase can be measured
Position information.(4) time-resolved measurement can be carried out.
THz wave transmittance is strong, and its transmission capacity can be obtained on sample much stronger than infrared using transmission mode
The tera-hertz spectra of product, Terahertz transmitted spectrum necessarily carries more physical and chemical performances on sample interior, thus
Tera-hertz spectra can more reflect sample interior physics and chemistry information than infrared spectrum, and Qualitive test is carried out to sample using tera-hertz spectra
Also just it is provided with accuracy higher.
Embodiment
By this method to corn gene strain MIR162, Bt-11, MON810 and non-transgenic corn strain
The terahertz time-domain spectroscopy figure of Jinboshi781 is as shown in figure 1, and absorption spectrogram as shown in Figure 2.
The present invention uses terahertz light spectrum detection method, by analyzing the tera-hertz spectra signal detection of corn sample its turn
Whether gene element positive, with sample preparation is simple, detection speed fast, high precision, intelligence degree it is high, will to testing staff
The low advantage of level is sought, can effectively solve the problems, such as that the detection speed of conventional biochemical detection method presence is slow, detection process is complicated.
Above case study on implementation is merely to illustrate the preferred embodiment of the present invention, but the present invention is not limited to above-mentioned embodiment party
Formula, in the ken that the field those of ordinary skill possesses, that is made within the spirit and principles in the present invention is any
Modification, equivalent substitute and improvement etc., are regarded as the protection domain of the application.
Claims (7)
1. a kind of physical detection methods of novel corn transgene component, it is characterised in that comprise the following steps：
1) making of standard specimen and sample；
2) acquisition of the terahertz time-domain spectroscopy data of standard specimen：
3) the tera-hertz spectra detection of sample transgene component；
4) qualitative analysis of corn gene composition.
2. physical detection methods of a kind of novel corn transgene component according to claim 1, it is characterised in that：It is described
The step of 1) standard specimen and sample making in, standard specimen is used to set up each strain corn (including transgenosis and non-transgenic)
Terahertz standard spectrum storehouse；Sample is used for the detection to corn gene composition；Make the standard substance raw material sources of standard specimen
In the standard substance of each corn strain, standard substance is bought from Institute of Science and Technology, National Food Bureau, and with standard substance certificate.
3. physical detection methods of a kind of novel corn transgene component according to claim 2, it is characterised in that：Standard
Material raw material are fully crushing, after grinding, take 120mg, add 40mg polyethylene powders, through shaking up after device is sufficiently mixed, put
Putting tablet press machine carries out compressing tablet；Pressure 10MPa being kept during compressing tablet tri- minutes, taking out compressing tablet, thickness is measured with electronics micrometer caliper
After (being accurate to 0.01mm), place hermetic bag and seal, hermetic bag is labelled dated family name and thickness, so far, standard specimen makes
Finish；Sample making process is with standard specimen manufacturing process.
4. a kind of physical detection methods of the novel corn transgene component according to claim 1-3 Arbitrary Terms, its feature
It is：Standard specimen or sample need to be completed within half an hour, to avoid standard specimen from placing the physic-chemical changes for occurring in atmosphere；Standard specimen
Or sample raw material, in 12%-15% or so, if moisture is too high, can influence sample to Terahertz using preceding holding moisture
The absorption of radiation, it is therefore desirable to carry out sample preparation after being dried using dryer again；The water parameters of 12%-15% determine source
Concerned countries standard is stored in grain security.
5. physical detection methods of a kind of novel corn transgene component according to claim 1, it is characterised in that：It is described
The step of 2) in the acquisition of the terahertz time-domain spectroscopy data of standard specimen：Standard specimen is positioned in terahertz time-domain spectroscopy instrument, is used
Transmission mode obtains the terahertz time-domain spectroscopy data of standard specimen；The environmental condition of detection is set to：Environment temperature：20 DEG C, environment
Humidity：≤ 10%；Every kind of corn strain makes 3 standard specimens, and each standard specimen is measured 3 times, measures 9 groups to every kind of corn strain altogether
Time-domain spectroscopy data, take 0.2T-1.2T wave bands carries out averagely, obtaining 9 average time-domain spectroscopies of the standard specimen as valid data
Data, recycle spectrometer accompanying software in itself, the absorption spectra data and waveform of the standard specimen are obtained, in storage to database；
Above-mentioned detection process is repeated to each strain standard specimen, each strain terahertz absorption spectra standard database of corn is set up.
6. physical detection methods of a kind of novel corn transgene component according to claim 1, it is characterised in that：It is described
The step of 3) in the detection of the tera-hertz spectra of sample transgene component, for the corn sample that need to be detected, need to carry out impurity elimination first
Matter, cleaning, drying, taking fraction carries out determination of moisture, when moisture stabilization is between 12%-15%, then is crushed, ground
Mill, compressing tablet；Compressing tablet is positioned in terahertz light spectrometer by step 2) carry out spectroscopic assay.
7. physical detection methods of a kind of novel corn transgene component according to claim 1, it is characterised in that：It is described
The step of 4) in the qualitative analysis of corn gene composition：(1) with 0.01T as frequency interval, 0.2T-1.2T terahertz wave bands are taken
100 data points of absorption spectra；For sample data, Xi (i=1,2 ..., 100) is designated as；For the standard specimen in standard spectrum storehouse
Data, are designated as Yi (i=1,2 ..., 100)；
(2) coefficient correlation of sample data and corresponding library of spectra standard specimen data is calculated as follows；
In above formula, n=100,It is sample data average value,It is standard specimen statistical average in corresponding library of spectra, r is to calculate
The coefficient correlation for arriving；
Related system r1, r2 are calculated to each standard specimen in standard spectrum storehouse ..., rn ...；
From r1, the maximum r values of the middle selection of r2 ..., rn ..., and using corresponding standard specimen as corn gene composition identification knot
Really.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109001004A (en) * | 2018-07-16 | 2018-12-14 | 湖南农业大学 | Transgene rape standard sample and its method for building up |
| CN113237847A (en) * | 2021-05-07 | 2021-08-10 | 西南科技大学 | Nondestructive testing method and system for hexanitrohexaazaisowurtzitane crystal form |
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| CN109001004A (en) * | 2018-07-16 | 2018-12-14 | 湖南农业大学 | Transgene rape standard sample and its method for building up |
| CN113237847A (en) * | 2021-05-07 | 2021-08-10 | 西南科技大学 | Nondestructive testing method and system for hexanitrohexaazaisowurtzitane crystal form |
| CN113237847B (en) * | 2021-05-07 | 2022-09-02 | 西南科技大学 | Nondestructive testing method and system for hexanitrohexaazaisowurtzitane crystal form |
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