Detail of GB/T 19495.4-2018

Introduction of GB/T 19495.4-2018

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