GB/T 38505-2020General detection methods for genetically modified products (English PDF)

1 Scope

China's national general detection methods for genetically modified products. It specifies the terms and definitions, the reagents and materials, the instruments and equipment, and the procedures. This is the umbrella document beneath which the specific event detection methods sit, and it covers what they have in common: how a sample is taken and homogenised so that it represents the lot, how DNA is extracted from materials as different as seed, flour, oil and processed food, how its quantity and quality are checked, and how the screening reactions are set up and controlled. Screening comes first in practice because most transgenic events share a small number of regulatory elements - the promoters and terminators used to drive the inserted gene - so a handful of reactions can establish whether a sample contains any GM material at all, before the more specific and expensive event identification is undertaken.

This standard specifies the qualitative testing methods for genetically modified products. This standard is applicable to the general testing of genetically modified components in rice, corn, soybean, rapeseed, potato, sugar beet, alfalfa, and their processed products by real-time fluorescent PCR. The minimum detection limit of the method of this standard is 0.1% (mass fraction).

2 Normative references

The following documents are essential to the application of this document. For the dated documents, only the versions with the dates indicated are applicable to this document; for the undated documents, only the latest version (including all the amendments) is applicable to this standard.

GB/T 6682 Water for analytical laboratory use - Specification and test methods

GB/T 19495.1 Detection of genetically modified organisms and derived products - General requirements and definitions

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

3 Terms and Definitions

The following terms and definitions apply to this document. 3.1 18S rRNA gene Transcribed from 18S rDNA, one of the components of the ribosome, which exists as a plant endogenous gene. 3.2 35S promoter from Cauliflower mosaic virus The 35S promoter that is from the cauliflower mosaic virus (CaMV). 3.3 35S terminator from Cauliflower mosaic virus The 35S terminator that is from the cauliflower mosaic virus (CaMV).

3.4 terminator of nopaline synthase gene The terminator that is from the nopaline synthase gene.

3.5 terminator of ribulose-1,5-biphosphate carboxylase small subunit It is from the 3' terminator sequence of the ribulose-1,5-biphosphate carboxylase small subunit gene of peas.

3.6 phosphinothricin acetyltransderase gene It is from the Streptomces viridochromogenes and encoded as phosphinothricin acetyltransderase (PAT).

Note. The pat gene is tolerant to the herbicide "glyphosate".

3.7 terminator of proteinase inhibitor II The terminator that is from the proteinase inhibitor II (Pin II) of potatoes.

3.8 promoter of ribulose-1,5-biphosphate carboxylase small subunit 1A The promoter that is from the encoding gene of the ribulose-1,5-biphosphate carboxylase small subunit 1A (RbcS4) of the Arabidopsis thaliana. 3.9 5'-end event-specific sequence of DAS40278 The sequence of the junction region between the 5' end of the exogenous insert of DAS40278 transformant and the maize genome.

Note. This sequence includes part of the vector sequence of the exogenous insert and part of the sequence of the maize genome. 3.10 3'-end event-specific sequence of DP305423 The sequence of the junction region between the 3' end of the exogenous insert of DP305423 transformant and the soybean genome.

Note. This sequence includes part of the vector sequence of the exogenous insert and part of the sequence of the soybean genome. 3.11 5'-end event-specific sequence of CV127 The sequence of the junction region between the 5' end of the exogenous insert of CV127 transformant and the soybean genome.

Note. This sequence includes part of the vector sequence of the exogenous insert and part of the sequence of the soybean genome.

3.12 cycle threshold The number of cycles through which the fluorescent signal in the reaction tube reaches the set threshold.

4 Reagents and materials

Unless otherwise specified, all reagents shall be analytical reagents or biochemical reagents. The water used in the test shall meet the specifications of Grade 1 water in GB/T 6682.

4.1 Real-time fluorescent PCR premix solution Use a real-time fluorescent PCR premix solution that has been verified to meet the requirements of real-time fluorescent PCR. 4.2 500 mmol/L ethylenediaminetetraacetic acid disodium solution (pH 8.0) Carry out the operation in accordance with the provisions of GB/T 19495.1 and GB/T 19495.3.

6.4 DNA template preparation Carry out the operation in accordance with the provisions of GB/T 19495.1 and GB/T 19495.3.Alternatively, DNA template preparation can be performed by using a plant genomic DNA extraction kit with the same effect.

6.5 DNA concentration determination Determine the DNA concentration with UV spectrophotometry; dilute the DNA solution appropriately, measure its absorbance at 260 nm, and calculate the DNA concentration according to the measured OD (at 260 nm, 1 OD=50 µg/mL double- stranded DNA); the OD shall be in the range of 0.2~0.8.Measure its absorbance at 280 nm, and calculate the OD260 nm/OD280 nm ratio of the DNA solution according to the measured OD; the ratio shall be in the range of 1.8~2.0.

6 Operation steps...