YC/T 149-2002 Determination of genetically modified organisms in tobacco and tobacco products
Some standard content:
ICS65.160
Registration number: 10586-2002
Tobacco Industry Standard of the People's Republic of China
YC/T 149-2002
Tobacco and its product-
Determination of genetically modified organism2002-09~12 Issued
State Tobacco Monopoly Administration
2002-12-01 Implementation
YC/T149-2002
This standard is proposed by the Agricultural Sub-Technical Committee of the National Tobacco Standardization Technical Committee. This standard is under the jurisdiction of the National Tobacco Standardization Technical Committee. The drafting unit of this standard: China Tobacco Northeast Agricultural Experiment Station. The main drafters of this standard: Jiao Qingming, Guo Zhaokui, Yan Xinfu, Wan Xiuqing, Yu Yanhua, Wei Jicheng. 648
1 Scope
Determination of genetically modified
Tobacco and tobacco products
This standard specifies the determination method of genetically modified tobacco and tobacco products. This standard applies to tobacco and tobacco products. 2 Terms and definitions
The following terms and definitions apply to this standard. 2.1
genetic engineering
Genetic engineering
YC/T 149—2002
Recombinant DNA technology using vector systems, as well as the technology of introducing recombinant DNA into organisms using physical, chemical and biological methods.
Genetically modified tobaccoGenetically modified tobaccoTobacco obtained by genetic manipulation using genetic engineering, excluding tobacco obtained by natural occurrence, artificial selection and cross breeding technologyDoes not include tobacco obtained by chemical or physical mutagenesis; does not include tobacco obtained by organ, tissue or cell culture and protoplast fusion, chromosome ploidy manipulation
3 Laboratory requirements
3.1 Laboratory space design
Sample pretreatment, DNA extraction, PCR reaction, reagent preparation and gel observation should be carried out in different isolated laboratories, and the laboratory should be regularly exposed to ultraviolet light overnight. 3.2 Operation requirements
The laboratory table should be wiped and disinfected with sodium hypochlorite before and after use, and the sample grinding should be carried out in a strong fume hood; the PCR reaction solution should be added in the clean bench; plastic gloves should be replaced once for each sample operation during sample preparation; the sample should not be weighed in the balance room for weighing reagents, and a special balance should be set up to avoid contaminating the reagents. 3.3 Sample duplication
Each sample to be tested is randomly divided into two groups, and each group is subjected to DNA extraction and PCR analysis respectively. 3.4 Control sample
3.4.1 Positive control
The positive standard push sample is a transgenic tobacco sample prepared with a known content, and its content is determined to be 0.1%, 0.5%, 1%, 2%, and 5% respectively.
3.4.2 Negative control
Negative control is a tobacco sample that has been tested as non-GMO in advance. 3.4.3 Reagent control
This control does not contain tobacco sample, and uses the same procedure as the sample to handle various reagents to control the impact of the reaction reagents on the test results.
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3.4.4 Extraction blank
From the time the sample is ground to the end of the extraction, two centrifuge tubes are placed in the work area and PCR amplified together with the sample DNA to determine the possibility of cross contamination.
3.5 Observation of PCR amplification results
In each PCR reaction, the electrophoresis results can only be recognized when all controls appear normal, at which time the positive sample has a band at a specific position, while other positions and negative controls and blank controls have no amplified bands. If any control is abnormal, re-test to avoid false results.
3.6 Prevention of contamination
All consumables and tools used for DNA extraction and PCR amplification must be sterilized before use. Pipette tips and various centrifuge tubes can only be used once. The work surface and the operation room must be exposed to ultraviolet light overnight after use. Sample pretreatment and grinding must be carried out in a fume hood and disposable gloves must be worn.
3.7 Safety protection
Work clothes and gloves must be worn during the entire experimental operation, especially when contacting EB, chemically resistant gloves and masks should be worn.
4 Reagents
4.1 Chemical reagents
The following reagents are of analytical grade or above. 4. 1. 1
Hexadecyl trimethyl ammonium bromide CTAB.
Methyl chloride.
Ethylenediaminetetraacetic acid disodium NazEDTA.
Ethanol.
Sodium fluoride.
Tris Base.
Isoamyl alcohol.
Agarose.
Boric acid.
Bromophenol blue.
Glacial acetic acid.
Ethidium bromide.
Sodium acetate.
Hydrochloric acid.
Sodium citrate.
Sodium dodecyl sulfate SDS.
4.2 Biochemical reagents
4.2.1 PCR Markel.
Taq DNA enzyme 2 u/μL.
4.2.3 Restriction endonuclease Asp700 (Xmn1), Nsil. 4.3 Buffer
4. 3. 1 CTAB extraction buffer (pH 8) contains CTAB (g = 20 g/L), NaCl (c = 1. 4 mol/L), Tris (c = 0. 1 mol/L) and Na,EDTA (c = 20 mmol/L).
4.3.2 CTAB precipitation buffer contains CTAB (g = 5 g/L) and NaCl (c = 0.04 mol/L). 4.3.3 High salt TE contains NaCl (c = 1.2 mol/L). 650
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4.3.4 DNTPs contains dATP, dCTP, dTTP and dGTP, each at 2.5 mmol/L. 4.3.5 AE buffer (pH7.85) contains Tris (c = 40 mmol/L), sodium acetate (c = 20 mmol/L) and NazEDTA (c = 2 mmol/L).
4.3.6 TBE buffer (pH8.0) contains Tris/boric acid (c = 45 mmol/L) and NazEDTA (c = 1 mmol/L). 4.3.7 Labeled DNA probe.
4.3.8 2XSSC buffer (pH7.0) contains NaCl (c = 3 mol/L) and sodium acetate (c = 0.5 mol/L). 4.3.9 Prehybridization solution (5 times solution, pH7.0) contains N-sarcosin (g 1 g/L) and SDS (g = 0.2 g/L). 4.3.10 Washing solution 1: SSC buffer (2 times solution, pH 7.0) contains NaCl (c = 3 mol/L), sodium acetate (c = 0.3 mol/L) and SDS (q = 1 g/l.).
4.3.11 Washing solution 2 SSC buffer (0.1 times solution, pH 7.0) contains NaCl (c = 3 mol/L), sodium acetate (c = 0.3 mol/L) and SDS (q = 1 g/L).
5 Instruments and consumables
5.1 High-speed refrigerated centrifuge (maximum speed 30,000 r/min) 5.2 Vortex oscillator
5.3 UV spectrophotometer
5.4 Constant temperature water bath
5.5 Electronic analysis instrument (sensitivity 0.0001 g) 5.6 Microcentrifuge
5.7 PCR instrument
5.8 Electrophoresis system
5.9 UV analyzer
5.10 Constant temperature water Bath
5.11 Nylon membrane
Hybridization box
2mL centrifuge tube
5.14 Pipette tip
0.20mL thin-walled PCR tube
6 Sample sampling method
6.1 Fresh tobacco leaves
Random sampling is carried out based on the variety and seed origin. For each sample, one bud or leaf is taken from 10 different plots. After natural dehydration for 2 days, the bud or leaf is numbered and the variety name, seed origin, and sampling location are indicated. 6.2 First flue-cured tobacco (hand-cured tobacco)
6. 2.1 Quantity
Take the inspection batch (origin) as the unit. The origin and grade of each inspection batch should be the same. Take 2 pieces for less than 50 pieces, 4 pieces for 51-100 pieces, 5 pieces for 101-150 pieces, 6 pieces for 151-200 pieces, and 1 piece for every 50 pieces added for more than 200 pieces (less than 50 pieces will be counted as 50 pieces). Take a little leaf from each and mix them as a sample. 6.2.2 Method
Open one end of a tobacco leaf and randomly take 3 points from the left, middle and right parts using a long simple sampler (with a pointed tube with an inner diameter of 1cm). Use the quartering method to reduce the sample taken from each piece of the batch to 2 equal samples (each 250g) as laboratory samples for inspection and re-inspection. Laboratory samples must be sealed and labeled with the origin, grade inspection number, sample delivery unit and sampler. 651
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6.3 Machine-punched tobacco
For packed machine-punched tobacco, take the box (containing 200kg tobacco leaves) as the sampling unit, use a long-shaped sampler to randomly take about 2g of tobacco leaves from each box, and take nearly 200g of tobacco leaves from 99 boxes of tobacco leaves in each container as a test sample; sampling can also be combined with moisture determination in the redrying processing workshop, and the origin, grade and box number of the tobacco leaves must be noted. 6.4 Cigarettes
Sampling should be carried out in accordance with the method specified in IS08243 "Cigarettes - Sampling". 7 DNA extraction method for tobacco leaf samples
7.1 Sample pretreatment
7.1.1 Fresh tobacco leaves
Randomly take about 1g of fresh leaf samples, add liquid nitrogen in a mortar and grind into powder, transfer to a weighed 1.5mL centrifuge tube, and weigh again to determine the mass of leaf tissue.
7.1.2 Cured tobacco leaves
Randomly take about 20g of tobacco leaf samples, wrap them with sulfuric acid paper, put them in a paper bag, put them in a square plate and cover them, dry them in a 40℃ constant temperature box overnight, grind them into powder in a sterilized mortar, store them in a 20ml centrifuge tube, take about 100mg from them and put them in two 1.5mL centrifuge tubes respectively. 7.2 DNA extraction
7.2.1 Fresh tobacco leaves
7.2.1.1 Take 100 mg of powder ground with liquid nitrogen, add 100 uL 2×CTAB extraction buffer, mix thoroughly and place in a water bath at 65°C for 10 min.
7.2.1.2 Add an equal volume of chloroform/isoamyl alcohol (volume fraction is 24:1), mix and centrifuge at 12000 r/min for 4 min. 7.2.1.3 Take the supernatant, add 1/10 volume of 10×CTAB extraction buffer, add an equal volume of chloroform/isoamyl alcohol (volume fraction is 24:1), mix and centrifuge at 12000 r/min for 4 min. Take the supernatant, add an equal volume of CTAB precipitation buffer, mix and centrifuge at 15000 r/min for 8 min. 7.2.1.4
Discard the supernatant, add 100μL high salt TE to the precipitate, and place in a water bath at 65°C for 10 min. 7.2.1.5
Add twice the volume of cold anhydrous ethanol (stored in a refrigerator at -20°C), and place in a water bath at -40°C for 30 min. 7.2.1.7
Centrifuge at 15000r/min for 8 min, and dissolve the precipitate in 70uL ultrapure water. 7.2.2 Flushed tobacco leaves
7.2.2.1Add 700μL 0.75× CTAB extraction buffer to the 1.5mL centrifuge tube containing the sample to be tested, shake and mix, and place in a constant temperature water bath at 65°C for 10 min.
7.2.2.2 Add 700μul. chloroform/isoamyl alcohol (volume fraction is 24:1), mix well and centrifuge at 12000r/min for 5min. 7.2.2.3 Take the supernatant, add 1/10 volume of 10% CTAB extraction buffer, add an equal volume of chloroform/isoamyl alcohol (volume fraction is 24:1), mix well and centrifuge at 12000r/min for 5min. 7.2.2.4 Take the supernatant, add an equal volume of CTAB precipitation buffer, mix well and centrifuge at 15000r/min for 8min. 7.2.2.5 Remove the supernatant, add 100uL high salt TE buffer, and water bath at 65C for 10min. 7.2.2.6 Add twice the volume of cold anhydrous ethanol and cool at -40℃ for 30min. 7.2.2.7 Centrifuge at 15000r/min for 8min, and dissolve the precipitate in 50μL ultrapure water. 7.3 Determination of DNA content and purity
7.3.1 Scanning wavelength in the ultraviolet region
The yield and purity of tobacco leaf DNA extract are determined by scanning with an ultraviolet spectrophotometer at a wavelength between 220nm and 320nm. 7.3.1.1 Zero the ultraviolet spectrophotometer with ultrapure water and calibrate the baseline. 7.3.1.2 Add 10μL of DNA extract to the colorimetric solution, add 1990μL of ultrapure water, mix with a pipette, and scan at a wavelength between 220nm and 320nm.
7.3.1.3 The yield of extracted DNA is calculated based on the absorbance at 260nm. An OD value of 1 is equivalent to 50μg of double-stranded DNA per milliliter of solution.
YC/T149—2002
7.3.1.4 The purity of DNA extract is estimated by the value of OD260/280. The ratio is between 1.7 and 1.9, which indicates good purity. When the ratio is greater than 1.9, the extracted DNA contains RNA. When the ratio is less than 1.7, the extract contains some impurities such as proteins, and the DNA needs to be re-extracted. 7.3.2 PCR amplification of nitrate reductase gene sequence The quality of the extracted DNA can be determined by amplifying the nitrate reductase gene sequence. The primer sequence is: NR-1 GTG TCA TGA ACA GAT GGC TCNR-2 GAT TAC TCG TCG AGT GAA GA The length of the nitrate reductase gene sequence amplified by this primer is 104bp. This is used to determine whether the extracted DNA can be used as a template for detection.
8 Detection method of transgenic tobacco leaves
8.1 PCR with common sequence primers Detection
8.1.1 Primer design
The common markers used for transgenic tobacco detection are mainly: cauliflower mosaic virus 35S promoter, Agrobacterium tumefaciens nos terminator sequence and kanamycin resistance gene (NPTII). PCR primers are designed based on these nucleotide sequences for tobacco transgenic detection. 8.1.1.1 35S promoter sequence amplification
35S-15'GCTCCTACAAATGCCATCA-335S-25'GATAGTGGGATTGTGCGTCA-3 The amplified fragment length of this primer is 195bp, which is used as a marker for transgenic tobacco detection. 8.1.1.2 nos terminator sequence amplification
n0s-15'GATTGAATCCTGTTGCCGGT-3'nos-25'GTAACATAGATGACACCGCG-3' The amplified fragment length of this primer is 213bp, which is used as a marker for transgenic tobacco detection. 8. 1. 1. 3 NPTII gene sequence amplification NPT-1 5'GCCCTGAATGAACTGCAGGACGAGGC-3' NPT-2 5'GCAGGCATCGCCATGGGTCACGACGA-3' The amplified fragment length of this primer is 411bp, which can also be used as a marker for transgenic tobacco detection. 8.1.2 PCR amplification system
For the 35S promoter sequence primers 35S-1, 35S-2 and nos terminator primers nos-1, nos-2 and NPT-1, NPT-2, the following amplification system can be used for amplification reaction. According to the molar number or concentration of the following system, According to the number of tubes to be amplified, calculate the total volume of each component to be added, add the sample in a sterile room, and then divide it into each 0.20mL amplification tube, then add the template according to the nanogram number of the template designed in the system, add mineral oil and centrifuge, and then put it into the PCR instrument for amplification. Table 1 PCR amplification system
Addition order
Reaction components
10xbuffer
Primer 1
Primer 2
Taq enzyme
Ultrapure water
DNA template
Total volume
Concentration or volume (per reaction tube)
220 μmol
25 pmol
25 pmol
37~38μl
YC/T 149—2002
8.1.3PCR amplification conditionsWww.bzxZ.net
For 35S promoter sequence primers 35S-1, 35S-2 and nos terminator primers nos-1, nos-2 and NPT-1, NPT-2, the following cycle program can be used for amplification:
Pre-denaturation: 95℃ for 5 min;
Cycle setting: denaturation at 94℃, 30 s;
Annealing at 64℃, 45 s;
Extension at 72℃, 45 s;
Number of cycles: 35 cycles;
Post-extension: 72℃ for 5 min.
8.1.4 Electrophoresis and gel observation
Weigh agarose and dissolve it in 0.5×TBE buffer. Dissolve it to make 1.5% gel. Add EB (0.5μg/mL). When the temperature drops to 45°C, pour it into the gel mold with a comb. After condensation, pull out the comb and put it into the horizontal electrophoresis tank. Take 10uL of the amplified product and add the sample buffer (containing bromophenol blue indicator) to spot the sample. Use PCR Marker as a molecular weight marker for electrophoresis. Observe the electrophoresis results with a UV analyzer. 8.2 PCR detection of specific gene sequences 8.2.1 Tobacco common mosaic virus coat protein gene TMV-CPTMV1 5'GTGTTCTTGTCATCAGCGTGGGC-3°TMV2 5'CACCGTTGCGTCGTCTACTCTACG-3' The PCR amplification product fragment length is 202bp8.2.2 Cucumber mosaic virus coat protein gene (CMV-CP) CMV1 5'-ACCCAACCTTTGTAGGGAGTGAGCG-3' CMV2 5'-ACATAGCAGAGATGGCGGCAACG-3' The length of its PCR amplification product is 263bp. 8.2.3 Potato mosaic virus coat protein gene (PVY-CP) PVY1 5'-GATATTTCAAATACTCGGGCA-3' PVY2 5'-GCATAACGCGCTAAACCCAC-3' The length of its PCR amplification product is 362bp. 8.2.4 Tobacco mosaic virus replicase gene (TMV54KD) T54D1 5'-GAGTTGTCTGGCATCATTGA-3 T54D2 5'-ACAATGGTCAAAGCCGGGTA-3' The length of its PCR amplification product is 295bp. 8.2.5 Bacillus thuringiensis insecticidal protein gene (BT) BT-1 5'CCCACTAGTTAACAATTTGATTGGA-3BT-25'CCGGAAGCTTTAGGACTGTAGGT-31 The length of the PCR amplification product is 299bp. 8.3 Result verification test
If the PCR amplification of the 35S promoter and (or) nos terminator sequence primers of the sample shows a positive band, the result verification test should be carried out while re-extracting the DNA and repeating the above PCR test. 8.3.1 Restriction endonuclease digestion reaction verification 8.3.1.1 Purification of PCR product
Transfer the PCR product to another sterilized 1.5mL centrifuge tube, add 100μL pre-cooled anhydrous ethanol, precipitate the PCR product DNA fragment, freeze at -80℃ for 1h, centrifuge at 15000r/min for 8min, discard the supernatant, add ultrapure water after the ethanol evaporates. 654
8.3.1.2 Enzyme digestion reaction
8.3.1.2. 135S promoter
YC/T149—2002
For samples with positive 35S PCR reaction, restriction endonuclease digestion analysis should be performed using Asp700 (Xmn1). The method is to add 5u of Asp700 to 10mL of purified PCR product, incubate in a 37℃ constant temperature water bath for 3h, and then perform 1.2% agarose electrophoresis and UV analysis. If the 195bp fragment can be digested by enzymes, 115bp and 80bp can be identified as 35S specific amplification products. 8.3.1.2.2nos terminator
For samples with positive nos primer PCR reaction, the 180bp fragment can be cut into two fragments of 96bp and 84bp using restriction endonuclease Nsil, the method is the same as the 35S digestion reaction. 8.3.2 Nested or semi-nested PCR reaction verification For samples with very weak or no PCR amplification fragment brightness, the nested or semi-nested PCR method should be used to design primers for the amplification product for a second PCR reaction to improve the sensitivity of the detection. In the nested or semi-nested PCR method, the second amplification is based on the product of the first amplification as a template, and the primers are designed based on the target gene sequence of the first amplification product, so that the second amplification range is within the first amplification fragment range. 8.3.2.1 35S nested PCR
The length of the amplification product of 35S nested PCR is 110bp. 35S-nt-15'CGTTCACCCTAACTACACTAT-3 35S-nt-25'GTGACTGCCGTCTCCGTAGA-3' 8.3.2.2 35S semi-nested PCR
In the 35S semi-nested PCR reaction, one of the primers in the second PCR is the same as the first PCR, and the other primer is located inside the first amplification fragment. The length of the amplification product fragment is 134bp. 35S-sn-15'ACAGTGGTCCCAAAGATGGA-3'35S-sn-25'GATAGTTGGGATTGTGCGTCA-3'8. 3. 2. 3 nos terminator nested PCR used the first amplification product as template and the following pair of primers for the second amplification. The length of the amplified fragment was 104bp. nos-NT-15'GAATCCTGTTGCCGGTCTTG-3nos-NT-25'CCCATCTCATAAATAACGTC-3'8.3.3 Probe hybridization verification
Probe hybridization is the formation of base pairing between a single-stranded DNA (probe) labeled with a specific sequence and another fixed DNA with a complementary sequence. The presence of a specific DNA sequence is detected by radioactive autoradiography or chemiluminescence. The experimental operation is as follows; 8.3.3.1 Probe labeling: Use the nick translation chemical fluorescein label and detection kit to prepare an alkaline phosphatase labeled probe. The sequence of the fluorescent labeled DNA probe (H-35S-a1) is 5'-F1-GGGTCTTGCGAAGGATAGTG-3. 8.3.3.2 DNA fragment transfer: Use the capillary method or vacuum transfer method to transfer DNA from an agarose gel to a nitrocellulose membrane or a nylon membrane.
8.3.3.3 Probe hybridization: Wet the membrane with fixed DNA with 2×SSC, add 20mL/100cm pre-hybridization solution, hybridize at 50°C for 1h, replace the pre-hybridization solution with 2.5mL/100cm membrane hybridization solution at 50°C for 4h, remove the hybridization solution, wash twice with 0.2×SSC/0.1%SDS, 5min each time, and then wash with 0.2×SSC/0.1%SDS at 50°C for 20min. 8.3.3.4 Detection of DNA probe: Wrap the wet membrane with Saran cling film, leave it at room temperature overnight, remove bubbles as much as possible, and expose it with MPX film in a Vinyl-covered film holder at room temperature for 4min to 6min. 8.3.4 Sequencing verification of amplified products
Use the primer set 35S1, nos2 whose amplified fragment can contain the target gene for amplification, recover the band with a fragment length greater than 1KB, purify and sequence it, and determine the type of genetically modified organism. 655
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8.4 GMO detection standards and quantitative PCR
8.4.1 Qualitative standards for GMO detection
By blindly testing 30 labeled samples (20 of which are positive samples with a content of 0.1% and 0.5%, and the rest are negative), the positive and negative detection rates are both 100%, proving that this method can accurately detect positive samples with a content of more than 0.1%; and false positive and negative reactions will not occur. 0.1% is determined as the qualitative standard for positive GMO detection. Any sample with a PCR-specific band brightness equal to or higher than the 0.1% reference control brightness is determined to be a positive band. If it is weaker than this brightness, it is necessary to re-extract IDNA and repeat the test. 8.4.2 Quantitative detection of GMOs
8.4.2.1 External standard quantitative detection method
8.4.2.1.1 Preparation of standard samples
Use 100% positive and negative materials to prepare positive samples with known contents (5%, 2%, 1%, 0.5% and 0.1%). 8.4.2.1.2 PCR detection
Extract DNA from the sample to be tested and the positive standard sample. According to the DNA template amount of 300ng, the sample to be tested and the standard sample are amplified in the same system and the same amplification conditions at the same time. Compare the brightness of the gradient electrophoresis bands of the sample to be tested and the standard after amplification to determine the content of the positive components in the sample to be tested.
8.4.2.2 Internal standard quantitative detection method
8.4.2.2.1 Preparation of standard template
Use transgenic tobacco as the material, and prepare a competitive template with a 20bp fragment of 35S promoter and nos terminator inserted by overlapping extension amplification.
8. 4.2.2.2 PCR amplification
In the PCR system, according to the template amount of 500ng, determine the number of copies of the standard template with the brightness of the competitive PCR band containing 10%, 5%, 1%, 0.5% and 0.1% of the positive template through preliminary experiments. According to the amount of this standard template, add the standard DNA template to the reaction solution at the same time as the DNA to be tested, compare the brightness of the target fragment and each standard fragment, and determine the positive content in the sample to be tested. 8.4.2.3 Real-time PCR quantitative method
Use Taqman quantitative PCR analyzer (such as ABI PRISM7700 or Lightcycler) for real-time PCR quantitative determination. 656
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