SN/T 5334.1-2020 English PDF

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SN/T 5334.1-2020: (Digital PCR detection method for genetically modified plant products - Part 1: General requirements and definitions)
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Basic data

Standard ID: SN/T 5334.1-2020 (SN/T5334.1-2020)
Description (Translated English): (Digital PCR detection method for genetically modified plant products - Part 1: General requirements and definitions)
Sector / Industry: Commodity Inspection Standard (Recommended)
Classification of Chinese Standard: B16
Classification of International Standard: 65.020.01
Word Count Estimation: 8,813
Date of Issue: 2020-12-30
Date of Implementation: 2021-07-01
Regulation (derived from): General Administration of Customs Announcement No. 136 [2020]
Issuing agency(ies): General Administration of Customs

SN/T 5334.1-2020: (Digital PCR detection method for genetically modified plant products - Part 1: General requirements and definitions)

---This is a DRAFT version for illustration, not a final translation. Full copy of true-PDF in English version (including equations, symbols, images, flow-chart, tables, and figures etc.) will be manually/carefully translated upon your order.
The People's Republic of China Entry-Exit Inspection and Quarantine Industry Standards 2020-12-30 release 2021-07-01 implementation Issued by the General Administration of Customs of the People's Republic of China

Foreword

SN/T 5334-2020 "Digital PCR Detection Method for Genetically Modified Plant Products" is divided into 8 parts. --Part 1.General requirements and definitions --Part 2.Genetically modified soybeans; --Part 3.Genetically modified corn; --Part 4.Genetically modified rape; --Part 5.Genetically modified cotton; --Part 6.Genetically Modified Potatoes; --Part 7.Genetically modified alfalfa; --Part 8.Genetically modified sugar beet. This document is part 1 of SN/T 5334-2020. This document was drafted in accordance with the rules given in GB/T 1.1-2020. Please note that some of the contents of this document may involve patents. The issuing agency of this document is not responsible for identifying these patents. This document was proposed and managed by the General Administration of Customs of the People's Republic of China. Drafting organizations of this document. Chinese Academy of Inspection and Quarantine, Nanning Customs Technology Center of the People’s Republic of China, People’s Republic of China Heguo Guangzhou Customs Technology Center. The main drafters of this document. Fu Wei, Zhu Pengyu, Du Zhixin, Wei Shuang, Wang Chenguang, Zheng Minghui, Zhu Shuifang, Huang Wensheng. Digital PCR detection method for genetically modified plant products Part 1.General requirements and definitions

1 Scope

This document specifies the scope of digital PCR detection methods for genetically modified ingredients of genetically modified plant products, normative references, and abbreviations. Languages, terms and definitions, principles, method establishment steps, result determination and sample storage content. This document is applicable to the digital PCR quantitative detection of genetically modified components in genetically modified plant products.

2 Normative references

The contents of the following documents constitute the indispensable clauses of this document through normative references in the text. Among them, dated quotations Only the version corresponding to that date is applicable to this document; for undated references, the latest version (including all amendments) is applicable Used in this document. GB/T 6682 Analytical laboratory water specifications and experimental methods GB/T 19495.1 General requirements and definitions for the testing of genetically modified products GB/T 19495.2 Technical requirements for genetically modified products testing laboratories GB/T 19495.3 Nucleic acid extraction and purification method for detection of genetically modified products GB/T 19495.7 Sampling and sample preparation methods for genetically modified products

3 Terms and definitions

The following terms and definitions apply to this document. 3.1 Transgene Introduce functional gene sequences from other species that do not exist in the parent species into the parent species through various means In the genome, it is expressed in the parent species to obtain the corresponding traits. 3.2 Genetically modified plant events Through transgenic technology, functional gene fragments are introduced into the parent plant genome, so that the parent plant can obtain heritable target traits. The parent plants that have acquired the desired traits become transgenic plant lines. 3.3 Event-specific GMO content detection In transgenic plant lines, the plant endogenous gene sequence next to the foreign gene is called the flanking sequence. For different genetically modified plants Line, the flanking sequence is unique. Therefore, the detection method that amplifies the target fragment located at the junction of the foreign gene and the flanking sequence is called transgenic Specific detection methods for transformation events. 3.4 Digital PCR digital PCR An absolute quantitative PCR technology. Divide the PCR system into a large number of micro-reaction systems for PCR amplification, and then react to the micro-reactions after amplification The system reads the fluorescent signal, and calculates the copy number concentration of the target sequence in the sample by calculating the positive rate and Poisson distribution. 3.5 Conversion factor multiplication factor The coefficient used when converting the ratio of the transformant-specific sequence and the copy number concentration of the internal standard gene into the content of the transgenic component (W/W). 3.6 Limit of quantitation The lowest copy number concentration within the linear dynamic range that meets the precision conditions. 3.7 Limit of detection Within the linear dynamic range, at least 2 microreaction systems can be stably detected as the lowest copy number concentration of the positive signal. 3.8 Repeatability By two different operators, in two different time periods, samples with the same mass fraction (including target concentration samples, detection limit Samples, quantitation limit samples and negative samples).

4 Principle

Digital PCR is a quantitative detection technology of gene copy number developed on the basis of fluorescent PCR, which is used for the absolute copy of nucleic acid template. Determination of the number. By filling the fluorescent PCR reaction system containing template, primer/fluorescent probe, heat-resistant DNA replicase and its buffer Divide and mix, and divide the same amount into a large number (more than 10 000) of micro-reaction systems isolated from each other, so that each template is allocated independently and randomly Into the micro-reaction system; all the micro-reaction systems simultaneously perform PCR amplification reactions under the same specified conditions, according to the set fluorescence threshold Value to judge the amplification results of each micro-reaction system; the digital PCR reaction is calculated according to the positive rate of the micro-reaction system and the Poisson distribution formula Should be the template concentration in the system. 5 Equipment, reagents and consumables 5.1 Instrument and equipment 5.1.1 Analytical balance. Sensitivity 0.1 mg. 5.1.2 Biological safety cabinet (ultra-clean workbench). 5.1.3 Digital PCR system. micro-reaction system generator or other instruments with the same function, micro-reaction system fluorescence detector or its It has the same function instrument. 5.1.4 PCR thermal cycler. 5.1.5 Constant temperature incubator. temperature control accuracy ±1.0 ℃. 5.1.6 Sample crusher. It can grind samples such as cottonseed and cottonseed meal into powder of about 60 mesh. 5.1.7 Micropipette. 100 μL~1 000 μL, 20 μL~200 μL, 10 μL~100 μL, 0.5 μL~10 μL, 0.1 μL~2.5 μL. 5.1.8 Double distilled water generator or pure water meter. 5.1.9 Vortex oscillator. 5.1.10 Nucleic acid detector. nucleic acid/protein content determination spectrophotometer, micro-spectrophotometer or other nucleic acid detector. 5.1.11 Centrifuge. centrifugal force ≥ 12 000 g. 5.2 Reagents and consumables Unless otherwise specified, the quality of reagents and materials should meet the requirements of GB/T 19495.1.Double distilled water or meet the requirements of GB/T 6682 The first-level water requirements. 5.2.1 CTAB method genome extraction reagent or equivalent DNA extraction kit. 5.2.2 The digital PCR micro-reaction system generates row tubes and membranes or chips. 5.2.3 96-well fluorescent quantitative PCR reaction plate and sealing membrane. 5.2.4 0.2 mL and 1.5 mL centrifuge tubes. 5.2.5 Digital PCR reaction master mix. contains magnesium ions, dNTPs, hot-start Taq enzyme and buffer with 5'-3' exonuclease activity Premix reagents for digital PCR.

6 General operation steps

6.1 Sampling In accordance with the provisions of GB/T 19495.1 and GB/T 19495.7. 6.2 Sample preparation In accordance with the provisions of GB/T 19495.1 and GB/T 19495.7. 6.3 Sample pretreatment In accordance with the provisions of GB/T 19495.1 and GB/T 19495.3. 6.4 DNA template preparation Follow the method of GB/T 19495.1 and GB/T 19495.3 or use a plant genomic DNA extraction kit with the same effect DNA extraction. Measure the absorbance at 260 nm and 280 nm of the extracted DNA with an ultraviolet spectrophotometer, and calculate the purity of the nucleic acid respectively And concentration. DNA purity is expressed by OD260/OD280, the value should be 1.7~1.9; DNA concentration=50×OD260 mg/mL, the concentration is not lower 20 ng/L.

7 Method establishment steps

7.1 General requirements The extraction and content determination of DNA in the test sample shall be carried out in accordance with the provisions of GB/T 19495.3.When performing DNA extraction on a sample It is necessary to ensure the stability of the DNA quality and concentration to ensure the stability and repeatability of the digital PCR reaction. Detect sample DNA Parallel experiments are required for digital PCR detection. The digital PCR reaction system includes digital PCR amplification master mix, primers and probes for exogenous genes and internal standard genes, DNA Template and water. The final concentration of the digital PCR amplification premix is 1×, and the final concentration of the primers for the exogenous gene and the internal standard gene is 0.3 μmol/L ~ 1 μmol/L, the final concentration of the probe is 0.15 μM ~0.5 μM, and the amount of DNA template is 10 ng ~100 ng. The reaction program is based on different primer probes And the amplified fragment is different, the annealing temperature is 55 ℃ ~ 65 ℃, and the extension depends on the length of the fragment. 7.2 Digital PCR reaction design 7.2.1 Primer probe requirements 7.2.1.1 Principles of primer design Primer design should follow the following principles. a) The primer design is designed with the specific sequence of the transformation event of each strain as a template. Similar to conventional quantitative PCR, requires an amplified piece The segment needs to be less than.200 bp, and the shortest should not be less than 50 bp; b) The length of the primer sequence used for digital PCR quantification should be 15 bp ~ 30 bp, and the length of the probe used should be 20 bp ~ 35 bp; c) The GC content in primers and probes should be 40%~60%; d) The Tm value of the digital PCR primers should be in... ......

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