Protocaol of the polymerase chain reaction for detecting genetically modified components in rapeseeds
Some standard content:
Entry-Exit Inspection and Quarantine Industry Standard of the People's Republic of China SN/T1197—2003
Qualitative PCR for Genetically Modified Components in Rapeseeds
Detection Method
Protocol of the polymerase chain reaction for detecting genetically modified components in rapeseeds2003-03-17 Issued
People's Republic of China
General Administration of Quality Supervision, Inspection and Quarantine
Implementation on September 1, 2003
Appendix A of this standard is an informative appendix.
This standard is proposed and approved by the Certification and Accreditation Administration of the People's Republic of China. The drafting unit of this standard is the Shanghai Entry-Exit Inspection and Quarantine Bureau of the People's Republic of China. The main drafters of this standard are: Ban Liangwen, Shen Zaifei, Chen Jiahua, Hu Yongqiang. This standard is the first published inspection and quarantine industry standard, SN/T1197-2003
「Scope
Qualitative PCR detection method of genetically modified components in rapeseed
SN/T 1197-2003
This standard specifies the qualitative PCR detection method for glyphosate herbicide-resistant, thiophene herbicide-resistant and male-sterile transgenic rapeseed. This standard is applicable to the qualitative PCR detection of glyphosate herbicide-resistant, thiophene herbicide-resistant and male-sterile transgenic oilseed. 2 Normative referencesbzxZ.net
The following clauses are used as clauses of this standard through reference in this standard. For all referenced documents with a date, all subsequent amendments (excluding errata) or revisions are not applicable to this standard. However, the parties to the agreement based on this standard are encouraged to explore whether the latest versions of these documents can be used. The latest versions of the referenced documents with an undated date shall apply to this standard. GB/T6682
Specifications and experimental methods for water used in analytical experiments SN/T1193 Laboratory technology requirements for genetic testing SN/T 1194:
Detection and sampling methods for genetically modified ingredients in plants and their products SN/T1204 Real-time fluorescent CR qualitative test method for genetically modified ingredients in plants and their processed products 3 Terms, definitions and abbreviations
The following terms, definitions and abbreviations apply to this standard. 3.1
Transgene
The functional [DNA sequence] that the species itself does not have but comes from other species is introduced into the species through various means to make it express in the species so that the species acquires new variety characteristics. 32
Polymerase chain reactionpolymerase chain reaction, the PCR template gene sequence is first denatured at high temperature to become a single strand, and under the action of DNA synthase and appropriate reaction conditions, the two primers designed according to the template sequence anneal to the corresponding complementary sequences on the two strands of the template DNA and combine with each other, and then on the DNA Under the action of affinity enzyme, four deoxyribonucleic acid (dNTI) is used as substrate to extend the primer, and then the cycle of denaturation, annealing and extension is repeated continuously, so that the amplified gene fragment is amplified geometrically. 3 3 Abbreviations
3. 3. 1 PCR polymerase chain reaction, referred to as PCR. 3. 3. 2 DNArdeoxyribonucleic acid. 3. 3. 3 dNTP, deaxyrihanucleoside triphosphnte. 3. 3. 4 dATP, deoxyadenosine triphosphate. 3. 3. 5 dcTP, deoxycytidine triphosphate. 3. 3. 6 dGTP, denxyguanosine triphosphate. 3. 3. 7 d'TTP, deoxythymidine triphosphete, deoxycytophosphate 3. 38 dUTPdeoxyuridinetriphoaphate, deoxyuridine = transient acid. 1
SN/T 1197—2003
3, 3. 9 UDG, uracil DNA glycosylae, uracil DNA-glycosylase 3. 3. 10 bp, base pair. 3. 3. 11 EDTA, ethylene diatminetetreacetic acid, ethylenediaminetetraacetic acid. 3. 3. 12 Tagi Ta DNA Polymetege, heat-resistant DNA polymerase 3. 3. 13 TE, Tris-HCl. EDTA buffer 3. 3. 14 SDS, sadium dodecylsulfate, sodium dodecylsulfate. 3.3.15Tri:tris（hydroxymethyl）aminomethane. 3, 3. 16PEP, phoaphoenolpyruvate carboxylase gene. FMV 35S,35S promoter from modified figwort mosaic virus(caulimavirus graup), 3. 17
leaf disease capsule 35S promoter.
gene,
CP4EPSPS,5-enolpyruvylshikimate-3-phosphate synthasegene,5-oxalic acid-3-hydroxylamine synthetase 3. 19 CTP,chloroplast transit peptide gene, 3.3. 20GOX,glyphosate axidoreductase gene. 3 3. 21 E9 3',3' sequence of smnall subunit of rbcS(ribulose-1,5-bisphosphete carboxylage) Eg gene(fram pea), derived from the 3' end sequence of the small subunit of ribulose-1,5-bisphosphete carboxylation E9 gene, 3.3.22 CaMV35S, 35S promoter from cauliflowermogaicvirus cauliflower mosaic virus 35S promoter. 3.3.23PAT, phasphinothricinacetyltransferaseBene fromBacilius amytoliquefaciens, 3.3.24BARNASE, ribonuclease gene from Bacillus amyloliquefaciens, 3.3.25BARSTAR, specific inhibitor of the barnase gene from Batiflu, unylotiyuefaciens, 3.3.26NPTiI, neomycin-3'-phasphotransferaseBene, neomycin-3'-phosphotransferaseBene. 3. 3. 27NOS.promoter of nopaline synthase gene from Agrobacterium tumefaciens, 3. 3. 28NOS 3'+ terminator af nopaline synthase gene, 3.3.29OCS3',3. 3. 30 SsuArat ribulose-1, 5-bisphosphate carboxylese small subunit arslA promoter from Arahi-dapsis thattana. 3. 3. 31 TA29, developmentally regulated promater from anther-specific TA29 gene from Nicotiana tabacum. 3.3.32 TR7 3',3' regulatory region from Agrubacteriur tumefuciens of the T-DNA transcript 7,3' regulatory region from Agrubacteriur tumefuciens of the T-DNA transcript 7,4 Anti-contamination measures
Measures to prevent cross-contamination during the test shall be carried out in accordance with the provisions of SN/T 1193. 5 Sampling and sample preparation
5.1 Sample preparation
Perform the method specified in SN/T 1194. 5.2 Sample preparation
Weigh about 20 g of rapeseed sample, dry heat sterilize (pre-treatment at 150℃ for 2 h) and sterilize at 120℃ for 30 min, then grind the sample powder to 0.5 mm in size in a magnetic or pulverizer. Determination method
6.1 Original
SN/T 1197-2003
After DNA is extracted from the sample, primers are designed for the genes of the foreign genes inserted into the transgenic plants. The DNA fragments of the foreign genes are specifically amplified by PCR technology. The results of ICR amplification are used to determine 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 grade 1 water as specified in GB/T6682. 6. 2.1 1 mnl/I Tris-HCl buffer (pH 8.0), weigh 121.1 B Tris, dissolve in 80 ml water, stir, add 42 ml concentrated dicalcium, cool to room temperature, adjust pH to 4.0 with 2-HCl, sterilize by autoclave after aliquoting for later use. 6220.5 mal/I EDTA (pH 8.0), add 186.1 N2-EDTA·2HU in 800 ml water, stir vigorously on a magnetic stirrer, adjust pH to 8.0 with sodium hydroxide (about 20 ml of sodium hydroxide is needed to make the solution granular), then make up to 1 L, aliquot and sterilize by autoclave for later use.
6 2 3 DNA extraction buffer, measure 100 mL. Inol/I. Tris-Cl, 5n ml, 0.5 mol/l. FDTA, weigh 5.0 g SDS, 16.48 g sodium chloride, make up to 1 L, and sterilize by high pressure after aliquoting.
6 2 4 Tris saturated phenol.
Tris-methane.
6 2 5 Rat.
6. 2. 7 Isopropanol.
6.2.8 70% ethanol,
6.29 TE buffer (pH 8.0), take 10 ml, 1 mol/l Trus-Cl (pH 8.0) and 2 mL 0.5 mol/l EDTA (pH 8.0). Add water to make up to 1000 mL, transfer and sterilize by high pressure for later use. 6.2.10 [0X PCR buffer
contains 500 nunal/l KCl, 100 nimol/l Tris-FiCl (pH 8.3), 15 mmol/L magnesium oxide (MgCl2), 0.1% gelatin 6.211 [0X magnesium fluoride, 25 mmol/L. 6 2 120XdNTP, containing 2.5 mmol/t dATE, dUTP, dL:rF, dGTP6 2 13 Tag 5 Unit/μl..6 2 14 Primers, synthesized according to the sequence in Table 1, such as ultrapure water to 100 μmal/], stored, the primers directly used for PCR test were 5 μol/E
Table 1 Primer sequences required for detection of transgenic rapeseed exogenous genes The detection gene
CnMV35S
Gene to stimulate
Primer sequence
5'-gctagtxtnsaccuguctig 3\
5'-cactllgteicthicerc-
5'-AEERUliRgIRLLRSTRA-3'
G'-eagggcagiccnnaigangh-3'
5'-gcicctacuaatgct Hit 3
5'-gntogtgngallgigtxirur 3'
scatcsrtEcgataaagHtRA
5'-tcaitcrincgtenigir 3'
Expansion length
degrees/bp
Xiaohuo temperature
Screening test
Deselection test
Choose one
Selection tool-
SN/T 1197--2003
Sexuality detection gene
FMY35S
GOX
CP4-EPSPS
(Jinglue)
BARNASE
BARSTAR
Gene source
6. 2. 15 Double distilled water. | |tt | aggatctcgtcgtgacccat-3*
5'-gcicgamgbazcacttce-3\
5'-ggatetcetgtcitet-3'
5'-g.tcatcctgatcga3
5'-uagcctcaacaggtcag-3'
5'-ctgctcgatgttgacaag-3\
5'-angacatccaccgaxgucttu-3'
5'-eggaoagctcttttccacgtt-3\5 -gtcttcgtgttgctgmaccgtt-a\5'-nuactgac is aggcgagage1-3\S'-ctettgttcgtcgtttcatc-a\
5'-gaapcccatccerttgz+gtn-3\5'-gacttgcgtgttcgttcttc-3\||tt ||5'-uncaccgttgagcttgagac-3'
S'-cgcggtttgtaatatcgttaao-3\5'-tcttgcaacctctctagatcatca-3\6'-gtcgacatgtetccgtagug-3\
5'-gcaaccaeceaagggtatc-3'||tt ||5'-accatcgtcaaccLcticuteg-3\5-gcteccaglancecacgtcat-3
5'-acaagoacgatcancitcc-3
5'-acteggccgtccegtegte-3\
5'-ctggg tggcatcananggg*tcc-3\5'-tccggtectguattctgaagcotg-3'5'-tcagangtatcagcgacctccacc-3'5'-angtatgutggtgatgtcgcigce-3\6.2.16RNaeA.2mg/μL.
. 2. 17
Dian ethidium bromide; 10 mg/mL
6. 2. 18 DNA, separation marker 100 bp~2 000 bp6. 2. 19
Agarose.
Amplification growth
Deer/bp
Warm ham
Kang/℃
Cockroach selection detection
Screening detection||tt ||Anti-gralybdenum foot
Anti-gralybdenum jazz
Anti-gralybdenum spleen
Anti-gralybdenum brand
Anti-gralybdenum belly
Anti-gralybdenum Reduce
and barrier
Qingyoulai
anti-grass foot
and the shaking property is not
oil swallow
anti-grass period
and the changing property is not
green oil lettuce
anti-grass Phosphate
and male
oil rapeseed
village to play one
choose to play one
choose one
autumn to play its SN/T 1197—2003 6.2.20 50 × TAE buffer Weigh 484 g Tri9 , Measure 114.2 mL natural acid and 200 mL 0.5 mal/L EDTA (pH 8.0). Dissolve in distilled water, make up to 2 L, divide into portions and sterilize under high pressure for later use. 6.2.2 110× loading buffer: containing 0.25% pyrrol blue, 0.25% dibenzoylmethane FF, 30% glycerol aqueous solution. 6.3 Collection
6 3. 1 Gene amplification instrument.
6.3 .2 Electrophoresis apparatus
6. 3 3 Electrophoresis tank
6. 3. Toxic gel analysis imaging system.
6.3. 5 DNA freezing and drying centrifuge,
6.36 Freezing centrifuge.
6.37 DNA analysis system,
6.3.8 Water-soluble steel.
63.9 Microfurnace.
Micropipette, 0.1 μL~2.5 μL, 0.5 μL~-10 μL, 2 μL--20 μL, 10 μL~100 μL, 20 μL~200 μL, 6.3 . 10
200 μL~1 000 μl.s
6. 3. 11 Dog level sensitivity 0. 001 g
High pressure sterilizer.
6.3. 13PCR reaction tubes, 200μl, 500μl. Two specifications 6. 3. 14Eppendorf centrifuge tubes, 1, 5 mL. 6.4 Detection steps
6.4.1 Extraction of total DNA from wild rapeseed
6.4.1.1 Weigh about 10 μL of oil-stained seed sample and grind it in a mortar. Grind the sample in liquid nitrogen until the powder particles are about 0.5 mm in size. 6.4 1.2 Weigh about 40 mg of the ground sample and transfer it into a 1.5 mL Eppendarf tube. Set up a double extraction experiment. 6.4 1.3 Add Add 100 μL DNA extraction buffer, mix, and inject to prevent the sample from agglomerating. Then add 900 μL DNA extraction buffer, shake for 30 g, and repeatedly balance and mix for 5 minutes. min.6.4.1.4 Centrifuge at 10000°C for 5 min, take 700 μL of supernatant, add 5 μL RNase A (2 mg/mL), and keep warm at 37°C for 30 min.6.4.1.5 Add an equal volume of Tris saturated solution, and mix repeatedly for 10 min. 6. 4. 1. Centrifuge at 610 000 g for 5 min, take 600 μl of the supernatant, add an equal volume of trichloromethane, and filter repeatedly at -1 for 5 min. Centrifuge at 10 000 g for 5 min, take 500 μl of the supernatant , add an equal volume of isopropyl alcohol and mix gently for 3 minutes. 6. 4. 1. 7
6. 4. 1. B10 000 Centrifuge for 10 minutes. There is a white precipitate at the bottom of the tube. 6.4.1.9 Remove the supernatant and add 1 mL of 70% ethanol to wash 1 to 3 times. 6.4.1.10 Pour off the 70% ethanol, centrifuge briefly, and use a pipette to remove as much ethanol as possible. Low-temperature freeze-drying for about 2 minutes, adding 50 μl ITE buffer (pH 8, 0) to dissolve the DNA precipitate. The total DNA of oilseeds can also be extracted using the corresponding commercially available DNA extraction reagent disk. 6 4. 2 PCR detection | |tt||6. 4. 2 1 PCR reaction system
The PCR reaction system used to detect the internal and external test genes in transgenic rapeseed is shown in Table 2. The amount of each reagent in the reaction system can be adjusted according to the specific situation or The total reaction volume was adjusted appropriately. Each reaction system should be set up with 100 replicates and 100 μg/ml of sample. Table 2 PCR reaction system for detecting exogenous genes in transgenic oils. 10X PCR flushing solution. Oxidizing agent (MgCl, )
Preparation temperature
25 mmol/L
25μLReaction system sample volume/uL
50 μl. Reaction system sample volume/ μE5. 0
SN/T 1197—2003
Reagent Name
dNTP contains dUTPY
Taa enzyme
Horizontal plate DNA
Double fuel water
Reserve rescue
2. 5 mol/L
SU/ μL
1u/μl
20 pmal/ μL.
0. 3 μg/ μI,~8 pz/ μL
妻2(继续)||tt| |25 μL reaction system sample injection/ μL2. 5
to 25 μl,
50 μL reaction volume/ μl,5.0
to 50 l| |tt||Genetically modified rapeseed was used as the positive control, non-genetically modified rapeseed was used as the negative control, and double distilled water was used as the blank control. 6. 4. 2.2 PCR reaction parameters
The PCR reaction parameters for detecting exogenous genes in transgenic rapeseed were: 95°C, 5 min; 94°C, 20 5; annealing temperature of primers at 40°C; 72°C, 40 cycles; 72°C, 3 mln; the annealing temperature of the primers is shown in Table 1. The PCR reaction cycle parameters can be adjusted appropriately according to the type of gene amplification instrument.
6.4.2.3 Detection of PCR products by gel electrophoresis: Dilute an appropriate amount of 50×TAE into 1×TAE and prepare a liquefied Z-chain complex of 0.5 P4/tpL. 2% agarose gel, take 15 μL PCR product, add 1.5 μL 10× loading buffer to get the sample and perform electrophoresis, and add DNA marker to determine the fragment size of PCR product. The voltage is determined according to the length of electrophoresis, generally controlled at 3V/cm~5V/cm. The electrophoresis time is determined according to the displacement of phenol blue. The electrophoresis test results are recorded by gel analysis imaging system. 6.5 Result judgment
6.5.1 Whether rapeseed DNA extraction contains PCR amplified fragments Use primers designed for the endogenous reference gene PEP gene of rapeseed (two pairs of primers can be selected) to perform PCR test on rapeseed DNA extraction microplate. Positive control bottle, positive control plate and test sample part amplify 248bp or 121bp PCR product. If no PCR amplification is observed, it means that DNA suitable for PCR test is not extracted during DNA extraction. There is PCR inhibition in the DNA extraction solution. If the reaction material is present, DNA should be collected again until a PCR product of 24B bp or 121 bp is amplified. 6.5.2 Detection of transgenic components in rapeseed For the detection of transgenic components in rapeseed samples, please refer to Appendix A. First, screen and detect CaMV35S, FMV35S, NOS, and NPTII genes. If the detection results of CaMV 35S, NOS, NPTI, and FMV 35S genes are all negative, the test results are directly reported. If the detection of CaMV35S, NOS, NPTI, and FMV35S genes gives two negative results, further detection of one or more genes of the eversion target gene GOX (GOX), CP4-EPSPS (CP4-EPSPS), BAR, PAT, BARNASE, and BARSTAR should be carried out to confirm which trait the transgenic rapeseed is. For samples with positive HCR test results, further confirmation tests should be carried out and the test results should be reported after confirmation. 6.6 Confirmation test The confirmation test method shall be in accordance with SN/T 1204 The medium-term determination of the formula is carried out according to the heat. The number of fruits is increased and the number of fruits is decreased. The representative samples of rapeseed are tested by PCR method, and the ×××× gene is not detected. 7.2 Positive fruit The representative samples of rapeseed are tested by PCR method, and the ×X××X gene is detected. 5
Company Name
Mnabatto
AgtEva
Genetic
Systems
Attached to the gene
(Informative Appendix)
Information on the foreign genes transferred into the transgenic rapeseed A. 1
Improved traits
Anti-gray
Anti-gray
Promoter
1) FMV 35S
2) FMV 35S
1) CnMV 35S
2) NOS
1) TA20
Male sterility and weed resistance!
2) TA29
3) ShuAra
Structural gene
SN/T1197——2003
Bee stopper
CP4 HPSPS((with CTP2)
()X(Modified)(wIh eTPI)
HARNASE
HARSTAR
BAR(With CTP)
CaMV35s
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