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GB/T 19495.4-2018 English PDF (GBT19495.4-2018)

GB/T 19495.4-2018 English PDF (GBT19495.4-2018)

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GB/T 19495.4-2018: Detection of genetically modified organisms and derived products -- Qualitative real-time polymerase chain reaction (PCR) methods

This Part of GB/T 19495 specifies the instruments and equipment, reagents and materials, testing procedures, quality control, pollution prevention, and minimum detection limit of the method related to the real-time fluorescence qualitative polymerase chain reaction (PCR) detection method for the screening and line detection of genetically modified components in plants and their processed products.
GB/T 19495.4-2018
NATIONAL STANDARD OF THE
PEOPLE REPUBLIC OF CHINA
ICS 65.020.01
B 16
Replacing GB/T 19495.4-2004
Detection of Genetically Modified Organisms and Derived
Products ?€? Qualitative Real-Time Polymerase Chain
Reaction (PCR) Methods
ISSUED ON: SEPTEMBER 17, 2018
IMPLEMENTED ON: APRIL 01, 2019
Issued by: State Administration for Market Regulation;
Standardization Administration of the PEOPLE Republic of China.
Table of Contents
Foreword ... 3
1 Scope ... 5
2 Normative References ... 5
3 Terms, Definitions and Abbreviations ... 6
4 Principle of Method ... 8
5 Apparatus and Reagents ... 9
6 Detection Procedures ... 10
7 Judgment of Result ... 12
8 Presentation of Results ... 13
9 Anti-Pollution Measures ... 13
10 Lower Limit of Detection ... 14
Appendix A (Informative) Real-Time PCR Primers and Probes for Screening and Detection of Transgenic Components ... 15
Appendix B (Informative) Primer and Probe for Transgenic Plant Line-Specific Real- Time PCR Detection ... 20
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 equipment, reagents and materials, testing procedures, quality control, pollution prevention, and minimum detection limit of the method related to the real-time fluorescence qualitative polymerase chain reaction (PCR) detection method for the screening and line detection of genetically modified components in plants and their processed products.
This Part applies to the screening and testing of genetically modifies plants, such as soybean, corn, rape, rice, cotton, potato, flax, sugar beet, alfalfa, tomato, papaya, apple, chicory, agrostis matsumurae, tobacco, plum, melon, wheat, eggplant and eucalyptus, etc. It also applies to the line-specific detection of plants, such as soybean, corn, rape, cotton, rice, potato, flax, sugar beet and papaya. etc.
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 document.
GB/T 6682 Water for Analytical Laboratory Use - Specification and Test Methods GB/T 19495.2 Detection of Genetically Modified Organism 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
For the purposes of this Document, the following terms and definitions apply. 3.1.1 Transgene
The technology that makes the DNA sequences that the species have or from other species, through biological engineering technology, transcribe or express it in the species, so that the species can obtain new varieties characteristics.
3.1.2 Real-time polymerase chain reaction
Real-time fluorescent polymerase chain reaction. A method of adding fluorescent groups to the polymerase chain reaction system, using the accumulation of fluorescent signals to monitor the entire PCR process in real time, and quantitatively analyzing the unknown template through the standard curve.
NOTE: The intensity of the fluorescent signal directly reflects the number of templates. 3.1.3 Endogenous gene
A gene that is constant in copy number in the tested species and does not exhibit allelic changes. NOTE: This gene can be used to determine species specificity.
3.1.4 Exogenous gene
Other biological genes that are transferred by bioengineering technology. NOTE: After the exogenous gene is transferred, the biological specie shows new biological characteristics.
3.1.5 Cycle threshold
The number of cycles through which the fluorescent signal in each reaction tube reaches the set threshold.
3.2 Abbreviations
The following abbreviations apply to this Document.
Alfalfa-Acc: Alfalfa Acetyl-CoA carboxylase.
BAR: phosphinothricin acetyltransferase gene.
bp: base pair.
CP4-EPSPS: Agrobacterium tumefaciens strain CP4 Product: herbicide tolerant form of 5- PSSuAra: the small subunit promoter of Arabidopsis.
pTA29: developmentally regulated promoter 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: terminator derived from pea ribulose diphosphate carboxylase gene
Tris: [tris(hydroxymethyl) aminomethane].
tg7: transglutaminase 7 gene.
tNOS: terminator of nopaline synthase gene from Agrobacterium tumefaciens. tOCS: terminator of octopine synthase.
t35S: Cauliflower mosaic virus 35 terminator.
UDG: uracil DNA glycosylase.
UGPase: UDP-glucose pyrophosphorylase gene from Solanum tuberosum.
UNG: uracil-N-glycosylase.
Wx012: waxy wheat genes.
zSS II b: [the endosperm-specific SS (starch synthase) II b].
18S rRNA: 18S ribosomal RNA.
4 Principle of Method
After extracting the sample DNA, the sample DNA was screened and detected by real-time fluorescent PCR technology; and determine whether the sample contained transgenic components according to the real-time fluorescent PCR amplification results. For samples that are positive for exogenous gene detection, or samples known to be positive for transgenes, if further line identification is required, then real-time PCR detection of line-specific fragments is performed; and determine which species(s) of transgenic line components are contained in the sample according to the results.
5 Apparatus and Reagents
5.1 Apparatus
5.1.1 Real-time fluorescent PCR instrument.
5.1.2 Sample crusher or grinder.
5.1.3 Balance: with sensitivity of 0.01g.
5.1.4 Water bath or constant temperature incubator.
5.1.5 Refrigerated centrifuges.
5.1.6 Autoclave.
5.1.7 Vortex shaker.
5.1.8 Biological safety cabinet.
5.1.9 pH meter.
5.1.10 Nucleic acid protein analyzer or UV spectrophotometer.
5.1.11 Micropipettes (2??L, 10??L, 100??L, 200??L, 1000??L)
5.2 Major reagents
Unless otherwise specified, all reagents are of analytically pure or biochemical reagents; and the experimental water shall satisfy the specifications of Grade-1 water in GB/T 6682. 5.2.1 Real-time PCR master mix
A solution prepared by mixing Taq DNA polymerase (5U/??L), PCR reaction buffer, MgCl2 (3 mmol/L~7 mmol/L), dNTPs (containing dATP, dUTP, dCTP, dGTP), UNG enzyme, etc. 5.2.2 ROX
Fluorescence calibration reagent (50??, diluted to 1?? when used).
5.2.3 Primers and probes
5.2.3.1 Screening and detecting primer and probes
The primers and probes for screening and detecting genes were synthesized according to the --- Blank control: Cycle threshold of endogenous gene detection ???40, Cycle threshold of exogenous gene or line-specific detection ???40;
--- Negative control: Cycle threshold of endogenous gene detection ???30, Cycle threshold of transformation event specific detection ???40;
--- Positive control: Cycle threshold of endogenous gene detection ???30, Cycle threshold of transformation event specific detection ???35.
7.2 Judgment of result
If the Cycle threshold of exogenous gene detection in the test sample is ???40, and the Cycle threshold of endogenous gene detection is ???30, it can be determined that the sample does not contain the tested gene or line.
If the Cycle threshold of exogenous gene detection in the test sample is ???35, and the Cycle threshold of endogenous gene detection is ???30, it can be determined that the sample contains the tested gene or line.
If the Cycle threshold of the exogenous gene in the test sample is 35~40, the template concentration shall be adjusted and the real-time PCR shall be redone. If the Cycle threshold of the exogenous gene after re-amplification is still 35~40, it can be determined that the sample contains the tested gene or line. If the Cycle threshold of the exogenous gene after re- amplification is ???40, it can be determined that the sample does not contain the tested gene or line.
8 Presentation of Results
If the result is positive, it shall be expressed as "detection of xxx foreign gene" or "detection of xxx transgenic line".
If the result is negative, it shall be expressed as "no xxx foreign gene detected" or "no xxx transgenic line detected".
For samples whose nucleic acid cannot be effectively extracted, the test result is "no nucleic acid components detected".
9 Anti-Pollution Measures
Measures to prevent cross-contamination during the detection process shall be implemented in accordance with the provisions in GB/T 27403 and GB/T 19495.2.

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