GB/T 38505-2020 PDF English
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General detection methods of genetically modified products
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GB/T 38505-2020: General detection methods of genetically modified products---This is an excerpt. Full copy of true-PDF in English version (including equations, symbols, images, flow-chart, tables, and figures etc.), auto-downloaded/delivered in 9 seconds, can be purchased online: https://www.ChineseStandard.net/PDF.aspx/GBT38505-2020
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
ICS 07.080
A 40
General detection methods of genetically modified products
Issued on: MARCH 6, 2020
Implemented on: MARCH 6, 2020
Issued by. State Administration for Market Regulation;
Standardization Administration of PRC.
Table of Contents
Foreword... 3
1 Scope... 4
2 Normative references... 4
3 Terms and Definitions... 4
4 Reagents and materials... 6
5 Instruments and equipment... 9
6 Operation steps... 9
7 Results analysis... 11
8 Judgment and presentation of results... 12
9 Storage period of samples for inspection... 13
10 Transformant information... 13
Appendix A (Informative) Information on transformants covered by this standard... 14
General testing method for genetically modified products
1 Scope
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.
6.6 Real-time fluorescent PCR detection
6.6.1 Setting of negative control, positive control, and blank control
Take the non-transgenic sample as the negative control; take the corresponding
transgenic plant sample strain, or the genomic DNA of the transgenic plant sample
containing the corresponding exogenous gene, or the plasmid standard molecular DNA
containing the above fragments as the positive control; take the water or TE buffer as
the blank control.
6.6.2 Real-time fluorescent PCR reaction system
See Table 3 for the PCR reaction system, or prepare it according to the recommended
system of a kit that has been verified to meet the requirements. Make 2 parallel tubes
for each DNA sample. When the sample is added, the sample DNA solution shall be
completely added to the reaction solution, and it shall not stick to the tube wall. After
the sample is added, the tube cap shall be tightly closed as soon as possible.
Blank control. No typical amplification curves appear in the detection of the internal
reference gene, and no typical amplification curves appear in the detection of all
exogenous genes, or the cycle threshold is greater than or equal to 40.
Negative control. A typical amplification curve appears in the detection of the internal
reference gene, and the cycle threshold is less than or equal to 30; no typical
amplification curves appear in the detection of all exogenous genes, or the cycle
threshold is greater than or equal to 40.
Positive control. A typical amplification curve appears in the detection of the internal
reference gene, and the cycle threshold is less than or equal to 30; a typical amplification
curve appears in the detection of all exogenous genes, and the cycle threshold is less
than or equal to 34.
8 Judgment and presentation of results
8.1 Result judgment
All the test samples are tested in parallel, if no typical amplification curves appear in
the exogenous gene detection, or the cycle threshold is greater than or equal to 40; and
if a typical amplification curve appears in the endogenous gene detection and the cycle
threshold is less than or equal to 30, then it can be determined that the sample does not
contain the tested exogenous genes.
All the test samples are tested in parallel, if a typical amplification curve appears in the
exogenous gene detection and the cycle threshold is less than or equal to 36, and if a
typical amplification curve appears in the endogenous gene detection and the cycle
threshold is less than or equal to 30, then it can be determined that the sample contains
the corresponding exogenous gene.
All the test samples are tested in parallel, if a typical amplification curve appears in the
exogenous gene detection, but the cycle threshold is in the range of 36~40; and if a
typical amplification curve appears in the endogenous gene detection and the cycle
threshold is less than or equal to 30, then the sample shall be reprocessed and tested
again after exclusion of contamination. After re-amplification, if a typical amplification
curve appears in the endogenous gene detection and the cycle threshold is less than or
equal to 30, and if a typical amplification curve appears in the exogenous gene detection
and the cycle threshold is still less than 40, then it can be determined that the sample
contains the tested exogenous genes. After re-amplification, if a typical amplification
curve appears in the endogenous gene detection and the cycle threshold is less than or
equal to 30, and if no typical amplification curves appear in the exogenous gene
detection, or the cycle threshold is greater than or equal to 40, then it can be determined
that the sample does not contain the tested exogenous genes.
......
Source: Above contents are excerpted from the full-copy PDF -- translated/reviewed by: www.ChineseStandard.net / Wayne Zheng et al.
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