Codeofchina.com is in charge of this English translation. In case of any doubt about the English translation, the Chinese original shall be considered authoritative.
GB/T 19495 consists of the following parts, under the general title of Detection of genetically modified organisms and derived products:
——GB/T 19495.1 Detection of genetically modified organisms and derived products—General requirements and definitions;
——GB/T 19495.2 Detection of genetically modified organisms and derived products—General requirements for laboratories;
——GB/T 19495.3 Detection of genetically modified organisms and derived products—Nucleic acid extraction;
——GB/T 19495.4 Detection of genetically modified organisms and derived products—Qualitative real-time polymerase chain reaction (PCR) methods;
——GB/T 19495.5 Detection of genetically modified organisms and derived products—Quantitative real-time polymerase chain reaction (PCR) methods;
——GB/T 19495.6 Detection of genetically modified organisms and derived products—Gene-chip detection;
——GB/T 19495.7 Detection of genetically modified organisms and derived products—Methods for sampling and sample preparation;
——GB/T 19495.8 Detection of genetically modified organisms and derived products—Protein based methods;
——GB/T 19495.9 Detection of genetically modified organisms and derived products—Liquid bead array detection for plant products.
This part is Part 4 of GB/T 19495.
This part is developed in accordance with the rules given in GB/T 1.1-2009.
This part replaces GB/T 19495.4-2004 Detection of genetically modified organisms and derived products—Qualitative real-time polymerase chain reaction (PCR) methods. In addition to editorial changes, the following main technical changes have been made with respect to GB/T 19495.4-2004:
——Detection methods for plant endogenous genes are added;
——Detection methods for screening genes of transgenic plants are added;
——Detection methods for structural genes are deleted and detection methods for strain specificity of crops such as soybean, maize, rape seeds, cotton, rice, potato, flax, sugar beet, etc. are added.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. The issuing body of this document shall not be responsible for identifying any or all such patent rights.
This part was proposed by and is under the jurisdiction of the National Technical Committee on Plant Quarantine of Standardization Administration of China (SAC/TC 271).
The previous edition replaced by this part is as follows:
——GB/T 19495.4-2004.
Detection of genetically modified organisms and derived products—
Qualitative real-time polymerase chain reaction (PCR) methods
1 Scope
This part of GB/T 19495 specifies the instruments and apparatus, reagents and materials, detection procedures, quality control, contamination-proof measures and minimum limit of detection of methods related to the screening of transgenic components and strain detection in plants and their processed products by qualitative real-time polymerase chain reaction (PCR) methods.
This part is applicable to transgenic screening and detection of plants such as soybean, maize, rape, rice, cotton, potato, flax, beet, alfalfa, tomato, papaya, apple, endive, bentgrass, nicotiana tabacum, plum, muskmelon, wheat, eggplant and eucalyptus, and is also applicable to the detection for strain specificity of plants such as soybean, maize, rape, cotton, rice, potato, flax, sugar beet and papaya.
2 Normative references
The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies.
GB/T 6682 Water for analytical laboratory use—Specification and test methods
GB/T 19495.2 Detection of genetically modified organisms and derived products—General requirements for laboratories
GB/T 19495.3 Detection of genetically modified organisms and derived products—Nucleic acid extraction
GB/T 19495.7 Detection of genetically modified organisms and derived products—Methods for sampling and sample preparation
GB/T 27403 Criterion on quality control of laboratories—Molecular biological testing of food
3 Terms, definitions and abbreviations
3.1 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1.1
transgene
technique in which a functional DNA sequence possessed by or derived from a species is bioengineered to be transcribed or expressed in the species in order to obtain a new trait of the species
3.1.2
real-time PCR
real-time polymerase chain reaction method in which a polymerase chain reaction system needs to add a fluorescent group whose fluorescence signal monitoring the entire PCR process in real time, and the unknown template is quantitatively analyzed by a standard curve
Note: The strength of the fluorescent signal directly reflects the number of templates.
3.1.3
endogenous gene
gene with constant copies and no allele changes were detected in the tested species
Note: This gene can be used to determine species specificity.
3.1.4
exogenous gene
other biological genes transferred using bioengineering techniques
Note: After transferring to an exogenous gene, the biological variety exhibits new biological traits.
3.1.5
cycle threshold
the number of cycles experienced by the fluorescent signal in each reaction tube reaching a set threshold
3.2 Abbreviations
For the purposes of this document, the following abbreviations apply.
Alfalfa-Acc: Alfalfa Acetyl-CoA carboxylase
BAR: Phosphinothricin acetyl-transference gene
bp: Base pair
CP4-EPSPS: Agrobacterium tumefaciens strain CP4 product: herbicide tolerant form of 5-enolpyruvulshikimate-3-phosphate synthase
Cry IA(c): Bacillus thuringiensis (Bt) subsp. Kurstaki crystal protein IA(c)
Cry3A: Bacillus thuringiensis (Bt) subsp. Tenebrionis crystal protein 3A
CTAB: Cetyhrithylammonium bromide
DNA: Deoxyribonucleic acid
dATP: Deoxyadenosinc triphosphate
dCTP: Deoxyeytidine triphosphate
dGTP: Deoxyguanosine triphosphate
dNTP: Deoxyribonucleosidc triphosphate
dUTP: Deoxyuridine triphosphate
EDTA: Ethylene diaminetetraacetic acid
EPSPS: 5-enolpyruvylshikimate-3-phosphate synthase gene
GAG56: One of 7-gliadin genes
GLuA3: Rice glutelin gene
GOX: Glyphosate oxidoreductase gene
LAT52: Pollen specific protein LAT52 gene
Lectin: Plant lectin gene
NPT II: Neomycin-3'-phosphotransferase gene
PAT: Phosphinothricin acetyltransferase gene
PCR: Polymerase chain reaction
PLD: Phospholipase D family gene
PMI: Phosphomannose-isomerase gene
pCaMV 35S: 35S promoter from cauliflower mosaic virus
pFMV 35S: 35S promoter from a modified figwort mosaic virus
pNOS: Promoter of nopaline synthase gene from Agrobacterium tumefaciens
pSSuAra: The small subunit promoter of Arabidopsis
pTA29: Developmentally regulated pro-rooter from anther-specific TA29 gene from Nicotiana tabacum
pUbi: Promoter of maize Ubiquitin
SAH7: Sinapis arabidopsis homolog 7 gene
SDS: Sodium dodecylsulfate
SPS: Sucrose phosphate synthase gene
Taq: Taq DNA polymerase
TE: Tris-HCl, EDTA buffer
tE9: A terminator derived from the pea ribulose bisphosphate carboxylase gene
Tris: Tris (hydroxymethyl) aminomethane
T97: Transglutaminase 7 gene
tNOS: Terminator of nopaline synthase gene from Agrobacterium tumefaciens
tOCS: Terminator of octopine synthase
t35S: Cauliflower mosaic virus 35 teminator
UDG: Uracil DNA glycosylase
UGPase: UDP-glucose pyroph08phorylase gene from Solanum tuberosum
UNG enzyme: Uracil-N-glycosylase
Wx012: Waxy wheat genes
zSSII b: The endosperm-specific SS (starch synthasc) II b
18S rRNA: 18S ribosomal RNA
4 Principle
After extracting the sample DNA, real-time PCR technology was used to screen and detect the sample DNA, and the results of real-time PCR amplification were used to determine whether the sample contained transgenic ingredients. For samples that are positive for the detection of exogenous gene, or samples that are known to be positive for transgenes, if further strain identification is required, real-time PCR detection of the strain-specific fragments will be performed. Then the results were used to determine which of the transgenic strain(s) was(were) contained in the sample.
5 Instruments and apparatus, and reagents
5.1 Instruments and apparatus
5.1.1 Real-time PCR instrument.
5.1.2 Sample crusher or grinder.
5.1.3 Balance: with a sensibility of 0.01g.
5.1.4 Water bath or thermostat incubator.
5.1.5 Refrigerated centrifuge.
5.1.6 Autoclave.
5.1.7 Vortex oscillator.
5.1.8 Biological safety cabinet.
5.1.9 pH meter.
5.1.10 Nucleic acid protein analyzer or ultraviolet spectrophotometer.
5.1.11 Micropipettes (2 μL, 10 μL, 100 μL, 200 μL, 1 000 μL).
5.2 Main reagents
Unless otherwise specified, all reagents shall be analytical or biochemical reagents, and the experimental water shall meet the specifications of Class I water in GB/T 6682.
Foreword i
1 Scope
2 Normative references
3 Terms, definitions and abbreviations
4 Principle
5 Instruments and apparatus, and reagents
6 Procedure
7 Result judgment
8 Expression of results
9 Contamination-proof measures
10 Minimum limit of detection
Annex A (Informative) Primers and probes for screening and detection of transgenic ingredients by real-time PCR
Annex B (Informative) Primers and probes for detection of transgenic plant strain specificity by real-time PCR
Codeofchina.com is in charge of this English translation. In case of any doubt about the English translation, the Chinese original shall be considered authoritative.
GB/T 19495 consists of the following parts, under the general title of Detection of genetically modified organisms and derived products:
——GB/T 19495.1 Detection of genetically modified organisms and derived products—General requirements and definitions;
——GB/T 19495.2 Detection of genetically modified organisms and derived products—General requirements for laboratories;
——GB/T 19495.3 Detection of genetically modified organisms and derived products—Nucleic acid extraction;
——GB/T 19495.4 Detection of genetically modified organisms and derived products—Qualitative real-time polymerase chain reaction (PCR) methods;
——GB/T 19495.5 Detection of genetically modified organisms and derived products—Quantitative real-time polymerase chain reaction (PCR) methods;
——GB/T 19495.6 Detection of genetically modified organisms and derived products—Gene-chip detection;
——GB/T 19495.7 Detection of genetically modified organisms and derived products—Methods for sampling and sample preparation;
——GB/T 19495.8 Detection of genetically modified organisms and derived products—Protein based methods;
——GB/T 19495.9 Detection of genetically modified organisms and derived products—Liquid bead array detection for plant products.
This part is Part 4 of GB/T 19495.
This part is developed in accordance with the rules given in GB/T 1.1-2009.
This part replaces GB/T 19495.4-2004 Detection of genetically modified organisms and derived products—Qualitative real-time polymerase chain reaction (PCR) methods. In addition to editorial changes, the following main technical changes have been made with respect to GB/T 19495.4-2004:
——Detection methods for plant endogenous genes are added;
——Detection methods for screening genes of transgenic plants are added;
——Detection methods for structural genes are deleted and detection methods for strain specificity of crops such as soybean, maize, rape seeds, cotton, rice, potato, flax, sugar beet, etc. are added.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. The issuing body of this document shall not be responsible for identifying any or all such patent rights.
This part was proposed by and is under the jurisdiction of the National Technical Committee on Plant Quarantine of Standardization Administration of China (SAC/TC 271).
The previous edition replaced by this part is as follows:
——GB/T 19495.4-2004.
Detection of genetically modified organisms and derived products—
Qualitative real-time polymerase chain reaction (PCR) methods
1 Scope
This part of GB/T 19495 specifies the instruments and apparatus, reagents and materials, detection procedures, quality control, contamination-proof measures and minimum limit of detection of methods related to the screening of transgenic components and strain detection in plants and their processed products by qualitative real-time polymerase chain reaction (PCR) methods.
This part is applicable to transgenic screening and detection of plants such as soybean, maize, rape, rice, cotton, potato, flax, beet, alfalfa, tomato, papaya, apple, endive, bentgrass, nicotiana tabacum, plum, muskmelon, wheat, eggplant and eucalyptus, and is also applicable to the detection for strain specificity of plants such as soybean, maize, rape, cotton, rice, potato, flax, sugar beet and papaya.
2 Normative references
The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies.
GB/T 6682 Water for analytical laboratory use—Specification and test methods
GB/T 19495.2 Detection of genetically modified organisms and derived products—General requirements for laboratories
GB/T 19495.3 Detection of genetically modified organisms and derived products—Nucleic acid extraction
GB/T 19495.7 Detection of genetically modified organisms and derived products—Methods for sampling and sample preparation
GB/T 27403 Criterion on quality control of laboratories—Molecular biological testing of food
3 Terms, definitions and abbreviations
3.1 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1.1
transgene
technique in which a functional DNA sequence possessed by or derived from a species is bioengineered to be transcribed or expressed in the species in order to obtain a new trait of the species
3.1.2
real-time PCR
real-time polymerase chain reaction method in which a polymerase chain reaction system needs to add a fluorescent group whose fluorescence signal monitoring the entire PCR process in real time, and the unknown template is quantitatively analyzed by a standard curve
Note: The strength of the fluorescent signal directly reflects the number of templates.
3.1.3
endogenous gene
gene with constant copies and no allele changes were detected in the tested species
Note: This gene can be used to determine species specificity.
3.1.4
exogenous gene
other biological genes transferred using bioengineering techniques
Note: After transferring to an exogenous gene, the biological variety exhibits new biological traits.
3.1.5
cycle threshold
the number of cycles experienced by the fluorescent signal in each reaction tube reaching a set threshold
3.2 Abbreviations
For the purposes of this document, the following abbreviations apply.
Alfalfa-Acc: Alfalfa Acetyl-CoA carboxylase
BAR: Phosphinothricin acetyl-transference gene
bp: Base pair
CP4-EPSPS: Agrobacterium tumefaciens strain CP4 product: herbicide tolerant form of 5-enolpyruvulshikimate-3-phosphate synthase
Cry IA(c): Bacillus thuringiensis (Bt) subsp. Kurstaki crystal protein IA(c)
Cry3A: Bacillus thuringiensis (Bt) subsp. Tenebrionis crystal protein 3A
CTAB: Cetyhrithylammonium bromide
DNA: Deoxyribonucleic acid
dATP: Deoxyadenosinc triphosphate
dCTP: Deoxyeytidine triphosphate
dGTP: Deoxyguanosine triphosphate
dNTP: Deoxyribonucleosidc triphosphate
dUTP: Deoxyuridine triphosphate
EDTA: Ethylene diaminetetraacetic acid
EPSPS: 5-enolpyruvylshikimate-3-phosphate synthase gene
GAG56: One of 7-gliadin genes
GLuA3: Rice glutelin gene
GOX: Glyphosate oxidoreductase gene
LAT52: Pollen specific protein LAT52 gene
Lectin: Plant lectin gene
NPT II: Neomycin-3'-phosphotransferase gene
PAT: Phosphinothricin acetyltransferase gene
PCR: Polymerase chain reaction
PLD: Phospholipase D family gene
PMI: Phosphomannose-isomerase gene
pCaMV 35S: 35S promoter from cauliflower mosaic virus
pFMV 35S: 35S promoter from a modified figwort mosaic virus
pNOS: Promoter of nopaline synthase gene from Agrobacterium tumefaciens
pSSuAra: The small subunit promoter of Arabidopsis
pTA29: Developmentally regulated pro-rooter from anther-specific TA29 gene from Nicotiana tabacum
pUbi: Promoter of maize Ubiquitin
SAH7: Sinapis arabidopsis homolog 7 gene
SDS: Sodium dodecylsulfate
SPS: Sucrose phosphate synthase gene
Taq: Taq DNA polymerase
TE: Tris-HCl, EDTA buffer
tE9: A terminator derived from the pea ribulose bisphosphate carboxylase gene
Tris: Tris (hydroxymethyl) aminomethane
T97: Transglutaminase 7 gene
tNOS: Terminator of nopaline synthase gene from Agrobacterium tumefaciens
tOCS: Terminator of octopine synthase
t35S: Cauliflower mosaic virus 35 teminator
UDG: Uracil DNA glycosylase
UGPase: UDP-glucose pyroph08phorylase gene from Solanum tuberosum
UNG enzyme: Uracil-N-glycosylase
Wx012: Waxy wheat genes
zSSII b: The endosperm-specific SS (starch synthasc) II b
18S rRNA: 18S ribosomal RNA
4 Principle
After extracting the sample DNA, real-time PCR technology was used to screen and detect the sample DNA, and the results of real-time PCR amplification were used to determine whether the sample contained transgenic ingredients. For samples that are positive for the detection of exogenous gene, or samples that are known to be positive for transgenes, if further strain identification is required, real-time PCR detection of the strain-specific fragments will be performed. Then the results were used to determine which of the transgenic strain(s) was(were) contained in the sample.
5 Instruments and apparatus, and reagents
5.1 Instruments and apparatus
5.1.1 Real-time PCR instrument.
5.1.2 Sample crusher or grinder.
5.1.3 Balance: with a sensibility of 0.01g.
5.1.4 Water bath or thermostat incubator.
5.1.5 Refrigerated centrifuge.
5.1.6 Autoclave.
5.1.7 Vortex oscillator.
5.1.8 Biological safety cabinet.
5.1.9 pH meter.
5.1.10 Nucleic acid protein analyzer or ultraviolet spectrophotometer.
5.1.11 Micropipettes (2 μL, 10 μL, 100 μL, 200 μL, 1 000 μL).
5.2 Main reagents
Unless otherwise specified, all reagents shall be analytical or biochemical reagents, and the experimental water shall meet the specifications of Class I water in GB/T 6682.
Contents of GB/T 19495.4-2018
Foreword i
1 Scope
2 Normative references
3 Terms, definitions and abbreviations
4 Principle
5 Instruments and apparatus, and reagents
6 Procedure
7 Result judgment
8 Expression of results
9 Contamination-proof measures
10 Minimum limit of detection
Annex A (Informative) Primers and probes for screening and detection of transgenic ingredients by real-time PCR
Annex B (Informative) Primers and probes for detection of transgenic plant strain specificity by real-time PCR
