Protocol of the polymerase chain reaction for detecting genetically modified components in potatoes
Some standard content:
People's Republic of China Entry-Exit Inspection and Quarantine Industry Standard SN/T 1198—2003
Protocol of the polymerase chain reaction for detecting genetically modified components in potatoes2003-03-17Promulgated
People's Republic of China
General Administration of Quality Supervision, Inspection and Quarantine
2003-09-01Implementation
Appendix A of this standard is an informative appendix.
This standard is proposed and managed by the Certification and Accreditation Administration of the People's Republic of China. The drafting unit of this standard: Shandong Entry-Exit Inspection and Quarantine Bureau of the People's Republic of China. The main drafters of this standard are Gao Hongxi, Liang Chengzhu, Zhang Yibing and Quan Huoxia. This standard is the first issued inspection and quarantine industry standard. SN/T 1198-2003
1 Model
Protocol of the polymerase chain reaction for detecting genetically modified components in potatoesThis standard specifies the qualitative PCR detection method for genetically modified components in potatoes. SN/T1198—2003
This standard applies to the detection of transgenic potato tubers, plants, raw strips, raw potato chips and starch of transgenic potato varieties (NEW LEAF) POTATO LINES BT-06, ATBT04-06, ATBT04-3I, ATBT 04-36 and SPBT 02-05; (NEW LEAF PLUS) POTATO LINES PBMT21-129, PBMT21-350 and PEMT22-82, (NEW LEAFY) POTATO LINES RBMT15-101, SEMT15-02 and SEMT15-15. 2 Normative references
The clauses in the following documents become the clauses of this standard through reference in this standard. For all dated references, all subsequent static amendments (excluding errata) or revisions are not applicable to this standard. However, parties to agreements based on this standard are encouraged to study whether the latest versions of these documents can be used. For all undated references, the latest versions apply to this standard. GB/T6682 Specifications and experimental methods for water for analytical laboratories SN/T 1193 Technical requirements for genetic testing laboratories SN/T1194 Sampling and sample preparation methods for genetically modified components in plants and their products SN/T1204 Qualitative detection methods for genetically modified components in plants and their processed products by real-time fluorescence PCR 3 Terms, definitions and abbreviations
The following terms and definitions (abbreviations) apply to this standard. 3.1
Transgene
The functional DNA sequence from other species that is not present in the species itself is introduced into the species through various means to express it in the species so that the species can obtain new variety characteristics. 3.2
Polymerase chain reaction, PCR template gene sequence is first denatured into single strand by high temperature. Under the action of DNA synthase and appropriate reaction conditions, two primers designed according to the template sequence anneal to the corresponding complementary sequence on the two strands of the template DNA and combine with each other. Then, under the action of DNA synthase, four deoxyribonucleic acids (dNTP) are used as substrates to extend the primers. Then, the cycle of denaturation, annealing and extension is repeated continuously, so that the gene fragment to be amplified is amplified geometrically. 3.3 Abbreviations
3.3.1 Pata; potato species-specific DNA primers, the amplified fragment is the Patatin gene fragment of potato tuber. 3.3.2CaMV 35S: promater from cauliflomer mnosaic virus, cauliflower mosaic virus 35S promoter. 3. 3.3NPT-III; neomycin phasphotransferase-II, 3. 3, 4NOS: nopaline synthase terminator, nopaline synthase 3' transcription terminator. 3.3.5PVYcpipotato Y virus coat protein, 3.3.6 cty 3A: a protein produced by B. thuringiensis subsp, Tenebrionis (B, t, t, ) strain Bl 256-82. This protein has insecticidal activity, especially selectively killing Colorado potato beetle larvae. SN/T 1198--2003
CP4 EPSPS: 5-enolpyrulshikimate-3-phosphate synthase 5-enolpyrul shikimate-3-phosphate3.3.7
synthase, 5-oxalate-3-phosphate synthase from Agrobacterium sp. Strain CP4. 3.3. 8PLRVrep:potato leafroll virus, potato leafroll virus repetitive sequence. E93', the 3' non-translated region of the pea ribulose-l, 5-bisphosphate carboxylase small3.3.9
subunit (rbcS） E9 gene. ,3\ terminal sequence of the small subunit of ribulose-1,5-bisphosphate carboxylase E9 gene from pea.3, 3. 1D ArabssU1A, arabidopsis thaliana ribulose-1*5-bisphosphate carboxylase (Rubisco) smallsubunit ats 1A promoter, arabidopsis thaliana ribulose-1,5-bisphosphate carboxylase (Rubisco) smallsubunit ats 1A promoter. 3, 3. 11 aad: (resides outside T-DNA) Gene encoding streptomycin adenylyltransferase in transposition from Escherichia coli Tn7.
3. 3. 12 oriV: (resides outside T-DNA) A 1.3 kb replication region from the RK2 plasmid of Agrobacterium.
3.3.13ori322: (residesoutsideT-DNA) A 1.8kb fragment derived from the pBR322 plasmid, which contains the replication region and the transfer binding site bom
3. 3, 14 FMV 35S: a promotor derived from figwort masaic virus (FMV), figwort mosaic virus 35S promoter. 4 Anti-contamination measures
The measures to prevent cross-contamination during the detection process shall be implemented in accordance with the provisions of SN/T1193. 5 Sampling and sample preparation
The general sampling method for genetically modified samples shall be implemented in accordance with the provisions of SN/T 1194. 5.1 Sampling quantity of potato products
5.1.1 The sampling quantity of potato tubers, raw French fries, raw potato chips, and plant stalks shall be sampled according to the proportions in Table 1. The unit of the number of samples per package is pieces for tubers, raw French fries, and raw potato chips, and pieces for plants. Table 1 Sampling quantity of tubers, plants and raw strips of marigold Batch/bag
501~ 35 000
≥35001
Sampling quantity of potato starch.
Sampling is carried out according to the proportion in Table 2.
Batch/bag
51~150
151500
501~3200
3 201~35 000
35001~500000
≥500001
Number of packages opened
Sampling quantity of marigold starch
Number of days after opening
Number of samples taken from each bag
Number of samples taken from each bag/g
5.2 Preparation of potato samples
5.2.1 Preparation of potato tubers, raw potato strips, raw slices and plants SN/T 1198—2003
For potato tubers, raw potato strips and raw slices from the same batch, wash each sample and cut a sample of about 1 cm from any part with a knife as a sample for DNA extraction. For potato plants from the same batch, wash a leaf from each sample and cut a leaf of 2 cm with a knife as a sample for DNA extraction.
5. 2. 2 Preparation of potato starch samples
Mix 120g of starch from each package thoroughly and take 100mg from each 120g as a sample. 6 Determination method
6.1 Principle
After DNA is extracted from the sample, primers are designed for the gene sequence of the foreign gene inserted into the transgenic plant. The DNA fragment of the foreign gene is specifically amplified by PCR technology. According to the PCR amplification results, it is determined whether the sample contains transgenic components. 6.2 Reagents and materials
Unless otherwise specified, other reagents are analytically pure or biochemical reagents, and water is first-grade water as specified in GB/T6682. 6.2.1 Primers: Synthesize primers according to the primer sequences provided in Table 3, add deionized water to make 100 pmol/uL. Storage, and make a 20 pmol/μL working solution directly used for PCR reaction. See Appendix A. 3'R 5'-gt gat ttc agg agt tct tcg-3*F 5'-gct cct acs aat gcc etc a-3'R 5'-gat agt ggg att gtg cgt ca-3\F 5'-egt cch aag cct caa cua ggt c-3\R 5'-cat lag tg± gtg ggu tgt cag g-3*F 5'-gaa tec tgl igc cgg tct tg-3R 5*-tta tcc tag t gcg cge te-3*F5'-gge tct cetgtc ate t-3*
R 5'-gat cat cct gat cge c-3'F 5'-trg tcu tta aac tig acg ac-3*R 5'-ct ctt tca cgg agt Icc ag-3\F 5'-gaa tca agg cle tea cgt cc-3*Rs'-cat ccg cac tgc ctc Eta c-3*5'-nga gcc gte gct aac acc #et c-3'5'-tet ggg tgr tgg cet cat cg-3*片
Internal control
Screening, setting detection
NEW LEAF?
Screening and setting test
NEWLEAF*PLUS
NEW LEAF Y
Screening test
NEW LEAF PLUS
NEW LEAFY
NEW LEAF?
Screening test
NEW LEAF Plus
NEW leaF y
NEW LEAF+
Setting test
NEW LFAF PLUS
Identification test
NEW LEAFO Y
Identification test
NFW LFAF PLUS
NEW LEAF Y
NEW LEAF?
SN/T 1198—2003
6. 2. 2 Tag DNA synthase.
6. 2. 3 dNTP: dATP.dTTP,dCTF,dGTP,dUTP.6. 2. 4 Agarose; electrophoresis pure.
6.2.5 Ethidium bromide (EB) or other staining agent. 6. 2. 6 Chloroform,
6. 2. 7 Isopropanol.
6. 2.8 70% ethanol
6. 2. 9 Molecular weight marker; 50 hp~300 bp. 6.2.10 Plant total DNA extraction buffer: Measure 100 mL 1 mol/L Tris-Cl, 50 mL 0.5 mol/L LEDTA, weigh 5.0 g sodium dodecyl sulfate (STS), 16.848 g sodium fluoride, make up to 1 L, and sterilize in aliquots for later use. 6.2.11TE buffer, 10mmol/L Tris-HCl, pH8.0; 1mmol/L EDTA, pH8.0. 6.2.12 10×PCR buffer: 100mmol/L potassium chloride (KCl), 160mmol/L ammonium sulfate (NH4SO4), 20mmol/L magnesium sulfate (MgSO4), 200mmol/L Tris-HCl (pH8.8), 1% Triton x-100, 1mg/mL BSA 6.2.13 5×TBE carboxylic acid wash solution: Tris, 54g, boric acid 27.5g, 0.5mol/L EDTA (pH8.0) 20mL, add distilled water to 1000ml.
6.2.14 10× loading buffer: 0.25% bromophenol blue, 40% sucrose. 6.2.15 RNase (10μg/mL). 6.2.16 Polyvinylpyrrolidone (PVP). 6.3 Only for photography
6.3.1 Solid crusher and grinding machine.
6.3.2 High-speed centrifuge, desktop small centrifuge, Mini personal centrifuge. 6.3.3 Water-rich incubator, constant temperature incubator, constant temperature incubator. 6.3.4 Balance with a sensitivity of 0.001 g.
6.3.5 High-pressure sterilizer
6.3.6 High-temperature dry coal box.
Water purifier, double distilled water purifier.
6.3.8 Cold algae, freezing refrigerator.
6.3.9 Ice maker.
Travel vortex explosion medicine navigation.
Microwave oven.
Gene amplification instrument.
Electrophoresis instrument,
PCR clean bench.
Nucleic acid and protein analyzer.
Micropipette: 0.1 μL~2 μL, 0.5 μL~10 μL, 2 μL~20 μL, 10 μL~100 μL, 20 μL~200 μL, 200 μL~1 000 μL.
Gel imaging system.
Real-time fluorescence PCR instrument.
6.3.19 Eppendorf tube: 1.5 mL~5 mL.6.3.20 PCR reaction tube, 200μL, 500μL. 6.4 Detection method
6.4.1 DNA extraction and chemical analysis of samples
For potato tubers, strips, raw potato slices, and plants, each sample was thoroughly mixed and placed in a mortar, frozen with liquid ammonia, and quickly ground into powder. SN/T 119B-2003
100 mg of the ground powder or starch powder was divided into 1.5 mL eppendorf tubes, 600 μL of preheated extraction buffer was added, and then 200 μL of 20% PVP was added, mixed, and kept warm at 65°C for 20 min, shaking several times during the period. An equal volume of phenol-trifluoromethane was added, and the mixture was gently inverted to mix. Centrifuge at 10,000 1/rmin for 10 min. Take the supernatant, add two-thirds of the volume of ice-cold isopropanol, and mix. Centrifuge at 10,000 r/min for 10 rrin. Remove the isopropanol in the supernatant. There is a white precipitate at the bottom of the tube. Add 10 μL of RNA (ethanol < 10 mg/mL) to the white precipitate.37 Incubate for 30 min. Add 1 mL potassium acetate (5 mnl/L). Centrifuge at 10 000 r/min for 10 min. Discard the supernatant and keep the precipitate. Wash the precipitate three times with 70% ethanol and dry. Dissolve the precipitate with 100 μL TE and use it as a template for PCR reaction. Store at 4℃ for later use. You can also use the corresponding commercial DNA extraction kit to extract DNA. 6. 4. 2 PCR amplification
6. 4. 2. 1 Establishment of experimental controls
Each sample must have two parallel experiments, and three controls must be established for each test: positive control, which is the DNA of the transgenic plant material containing the gene fragment to be detected or the positive plasmid DNA containing the gene fragment to be detected:
negative control, non-transgenic horse genomic DNA: blank control (without DNA template).
6.4.2.2PCR reaction system
In a 0.2 mL PCR reaction tube, add the reactants in the order listed in Table 4, and the total volume is 25 μL. Table 4 Components of PCR reaction system
Components
10×PCR buffer
4×dNTP (2.5 mmol/l)
Primer 1 (20 pmol/μL)
Primer 2 (20 pnol/μL)
TaqDNA 50% ethanol (5 U/μL)
UNG (1 U/μL)
Magnesium oxide (MgCl2) (25 mmol/L)
DNA 50% ethanol
Deionized water
6.4.2.3PCR reaction procedure
25 μL reaction volume
o. 5 μt.
0, 2 μl
D. 5 μL~2. 0 μL(10 ng~50 ng DNA) make the reaction volume reach 25 μl.
To detect different exogenous genes, the PCR amplification conditions are slightly different. The specific amplification procedures for different exogenous genes are shown in Table 5. 5 PCR reaction condition parameters
Amplified exogenous genes
CaMV 35S.NOS,NPTII
FMV 35 5
PI.RVeep,Cry 3Aa
94 c.3 min
94 C.3 min
94 c.3 min
94 C,3 min
Gel electrophoresis detection of PCR amplification products
94 C.40 s+50 C,45 $+72'T.45 s94 C.40 =+54 C.45 =;72 C.45 s94 C,40 8+60 C,45 s;72 C,45 94 .40 s55 C,45 s+72 C,45 s
94 C.40 h±63 ℃,40 $+72 C,40 sNumber of prying rings
Most extended
72 C,7 min
?2 C,7 min
72C,7min
72c,7min
72 ℃,7 min
Dissolve the agarose gel in a microwave oven to prepare a 2.0% agarose gel. Put the prepared agarose gel into the electrophoresis sugar of 0, 5×TBE electrophoresis buffer, take 10 μL of the amplification product and 2 μL of 6× loading buffer with a pipette, mix them, and spot them on 5
SN/T1198—2003
2.0% agarose gel. Use Marker as molecular marker. Conduct electrophoresis at 1V/cm~5V/cm for 40min, use EB to stain, and then observe with a gel imaging system, take pictures and record. 6.5 Result judgment
6.5.1 Detection of endogenous genes
For the internal reference primer PATA of the potato species-specific endogenous gene Patetin fragment, amplify the sample DNA. If the PCR products of the negative control, sample and positive control all show a 214bp band, it means that the extracted sample DNA meets the requirements of the PCR reaction and can be used for exogenous gene detection; otherwise, it cannot be used to detect exogenous genes and the sample DNA should be re-extracted until a 214 bp band is amplified.
6.5.2 Detection of exogenous genes
Perform PCR test on exogenous genes in DNA extract of potato samples. If no amplified bands appear in negative control and blank control, and expected amplified bands appear in positive control and sample to be tested (amplified fragment size see Table 3), it can be preliminarily determined that the sample to be tested contains the suspected exogenous gene. Further confirmation test should be conducted, and the final report should be based on the result of confirmation test. If no PCR amplified product appears in the sample to be tested, it can be concluded that the sample to be tested does not contain the exogenous gene. 6.5.3 Selection of screening test and determination test criteria For the detection of transgenic components in potato samples, the contents of Appendix A can be referred to. First, the CaMV35S, FMV35SNOS, and NPTⅡ genes are screened and tested. If the screening test result is negative, the result is directly reported. If the screening test result is positive, further determination test of PLRVrep, PVYcp.Cry3A genes is required to determine which transgenic potato strain it is.
6.6 Confirmation test
The confirmation test method shall be carried out in accordance with the method specified in SN/T1204. 7 Result expression
7. 1 No XXX gene was detected.
7. 2 XXX gene was detected, (may be further reported) the test sample is a XXX transgenic potato line. 6
Company Name and Relationship
Monsailo
NEW LEAF PLUS
RBMT 21-129
Monsanto
NEW LEAF PLUS
RMT 21-350
Monsanto
NEW LEAF PLUS
RBMT 22-82
Mons&nto
NEW IEAF Y
RBMT 15-101
Monsanto
NEW I.EaFo Y
SEMT15-02
Mansanto
NEW LEAF Y
SEMT15-15
Monganto Company
NEW LEAFD
Appendix A
(Appendix on Material Characteristics)
Information on the foreign genes introduced into common transgenic potatoes Table A.1 Common transgenic potato traits improved by foreign genes
1) Resistance to potato leaf roll
2) Resistance to potato beetle
1) Resistance to potato leaf roll
2) Resistance to potato beetle
1) Resistance to potato leaf roll
2) Resistance to potato beetle
1) Resistance to potato virus Y
2) Resistance to potato beetle
1) Resistance to potato virus Y
2) Resistance to potato beetle
1) Resistance to potato virus Y
2) Resistance to potato beetle
1) Resistance to potato beetle
Promoter
1) FMV 35S
2) rεbSSU1A
3) P-nos
1) FMV 35S
3)Pnox
J) FMV 35S
23 ArabsSUIA
1) FMV 35S
2) ArabsSU1 A
3) P-nog
1) FMV 35S
1 2) ArebSSU1A
[ 3) P-nos
1) FMV35S
2) ArabsSU1A
3>P-nos
1)ArabsSUiA
21 CaMV 35S
structural genes
1) PLRVrep
2) cry 3Aa,
I) PI.RVrep
2) cry 3An.
3)NPTI
2) cry 3Aa.
4) ori-322||tt| |) PVYep | 3A2.
3) NPT1
5) oriv
6)ori322
1) cry 3Aa.
SN/T 1198—2003
terminator
2) nos
1) no8
2) nos
3) nos||tt ||JYE93#
3) E9 3'
11 F9 3*
2) nos
1) E9 3*
2) nos
3) nog
1)E93+
2) nos
3) nos
PV-STMT21
PV STMT21
PV-STMT22
PV-SMT15
PV-STMT15
PV-STMT15
PV-STBT02
SN/T 1198—2003
Company Name and Strain
Monsanto
NEWLEAF?
ATET 04-06
Mpnento
NEWLEAF
ATBT 04-31
Monanto
NEWLEAFO
ATBT04-36
Monsanto
NEW LEAF
SPBT 02-05
SN/T 1198-2003
Improved traits
1) Resistance to potato beetle
1) Resistance to horsetail beetle
1) Resistance to potato mold beetle
1) Resistance to horsetail beetle
Table A, 1 (continued)
Promoter
1) CaMV 35S
2) CaMV 35S
1) ArahsSUIA
2) CeMV 35S
1)AbsSU1A
2) CaMV 35S
1) CaMV 35S
2) CaMV 35S
structural genes
1) cry 3At,
1) cry 3A,
2) NPTI
I) cry 3A
2) NPT1
3) aad
4) ariy
5)ori322www.bzxz.net
1) cry 3Ab,
2) NPT
3)oriy
4)o322
continuation
1) E9 3*
2) nos
2) nos
1) F9 3*
2)nog
PV-STBT04
PV-STBT04
PV-STBT04
PV-STBT02
Copyright exclusive infringement must be investigated
Book number: 155066: 2-15173
Price:
002-86ll L/Ns*
SN/T 1198—2003
terminator
2) nos
1) no8
2) nos
3) nos
JYE93#
3) E9 3'
11 F9 3*
2) nos
1) E9 3*
2 ) nos
3) nog
1)E93+
2) nos
3) nos
PV-STMT21
PV STMT21
PV-STMT22
PV-SMT15
PV-STMT15
PV-STMT15
PV-STBT02
SN/T 1198 —2003
Company Name and Strain
Monsanto
NEWLEAF?
ATET 04-06
Mpnento
NEWLEAF|| tt||ATBT 04-31
Monanto
NEWLEAFO
ATBT04-36
Monsanto
NEW LEAF
SPBT 02- 05
SN/T 1198-2003
Improved traits
1) Resistance to potato beetle
1) Resistance to horsetail beetle
1) Resistance to potato mold beetle
1) Resistance to horsetail beetle
Table A, 1 (continued)
Promoter
1) CaMV 35S
2) CaMV 35S
1) ArahsSUIA
2) CeMV 35S
1)AbsSU1A
2) CaMV 35S
1) CaMV 35S
2) CaMV 35S
Structure Gene
1) cry 3At,
2) NPT11
1) cry 3A,
2) NPTI
I) cry 3A
2) NPT1
3) aad
4 ) ariy
5)ori322
1) cry 3Ab,
2) NPT
3)oriy
4)o322
Cont. stop
1) E9 3*
2) nos
2) nos
1) F9 3*
2)nog
PV-STBT04
PV-STBT04
PV-STBT04
PV-STBT02
Copyright Infringement must be investigated
Book number: 155066: 2-15173
Price:
002-86ll L/Ns*
SN/T 1198—2003
terminator
2) nos
1) no8
2) nos
3) nos
JYE93#
3) E9 3'
11 F9 3*
2) nos
1) E9 3*
2 ) nos
3) nog
1)E93+
2) nos
3) nos
PV-STMT21
PV STMT21
PV-STMT22
PV-SMT15
PV-STMT15
PV-STMT15
PV-STBT02
SN/T 1198 —2003
Company Name and Strain
Monsanto
NEWLEAF?
ATET 04-06
Mpnento
NEWLEAF|| tt||ATBT 04-31
Monanto
NEWLEAFO
ATBT04-36
Monsanto
NEW LEAF
SPBT 02- 05
SN/T 1198-2003
Improved traits
1) Resistance to potato beetle
1) Resistance to horsetail beetle
1) Resistance to potato mold beetle
1) Resistance to horsetail beetle
Table A, 1 (continued)
Promoter
1) CaMV 35S
2) CaMV 35S
1) ArahsSUIA
2) CeMV 35S
1)AbsSU1A
2) CaMV 35S
1) CaMV 35S
2) CaMV 35S
Structure Gene
1) cry 3At,
2) NPT11
1) cry 3A,
2) NPTI
I) cry 3A
2) NPT1
3) aad
4 ) ariy
5)ori322
1) cry 3Ab,
2) NPT
3)oriy
4)o322
Cont. stop
1) E9 3*
2) nos
2) nos
1) F9 3*
2)nog
PV-STBT04
PV-STBT04
PV-STBT04
PV-STBT02
Copyright Infringement must be investigated
Book number: 155066: 2-15173
Price:
002-86ll L/Ns*
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