GB/T 38132-2019Quantitative determination of genetically modified plants by digital PCR (English PDF)

1 Scope

China's national method for quantifying genetically modified plant material by digital PCR. Labelling rules for GM content are expressed as percentages, so enforcing them requires measuring how much of a sample is transgenic rather than merely whether any is. Conventional real-time PCR does that by comparing amplification against a calibration curve built from reference materials, which works but depends on those materials and on the assumption that the sample amplifies like them. Digital PCR removes both dependencies: the reaction is divided into thousands of tiny partitions, each either containing a target molecule or not, and counting the positive partitions gives an absolute number of copies without any calibration curve. That makes it the natural method for a regulatory measurement, where the result must be defensible and independent of a reference material that may not exist for a newly approved event.

This Standard specifies the quantitative determination of genetically modified plants by digital PCR method. This Standard applies to the digital PCR method for quantitative determination of genetically modified maize MON810, MON89034, MIR162, genetically modified soybean GTS-40-3-2, genetically modified rice KMD event, genetically modified cotton GHB119, and genetically modified oilseed rape RT73 in seeds and physically-processed seeds. The quantitative detection limit of this method is 0.1% (mass fraction).

2 Normative references

The following documents are indispensable for the application of this document. For dated references, only the dated version applies to this document. For undated references, the latest edition (including all amendments) applies to this document.

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 SN/T 4853, Quantitative detection of genetically modified rice - Digital PCR

3 Terms and definitions, abbreviations

3.1 Terms and definitions The following terms and definitions are applicable to this document.

3.1.1 Event specific sequence The neighbor-joining sequence that is recombined and produced after the exogenous DNA fragment is inserted into the recipient crop genome.

3.2 Abbreviations The following abbreviations are applicable to this document. Adh-1: Alcohol dehydrogenase

1 Adhc: Alcohol dehydrogenase C gene PCR: Polymerase chain reaction PEP: Phosphoenolpyruvate carboxylase gene PLD: Phospholipase D gene

4 Principle

The technical principle of digital PCR is to segment the original PCR reaction system, and then to amplify and detect all small reaction systems. Through the limited segmentation of the reaction system, the entire reaction system can be more tolerant to the nucleic acid inhibitor, and can more stably, accurately, and rapidly perform a precise identification of the genetically modified organism of the trace. At present, digital PCR includes chip-type digital PCR and droplet-type digital PCR. The chip-type digital PCR uses a microfluidic chip to realize the segmentation of the original reaction system; this segmentation method has the advantages of good stability and uniformity; but the test cost is relatively high. The droplet-type digital PCR generates tiny water-in-oil system to realize the segmentation of the reaction system; this segmentation method has fast response speed and lower segmentation cost. In order to achieve quantitative determination of genetically modified plants by digital PCR, this Standard directly obtains the copy number content of exogenous genes (event specific sequence) and endogenous genes of genetically modified plants through digital PCR amplification reactions. The ratio (percentage) of the copy number of the exogenous gene to the endogenous gene in the sample DNA is the relative percentage content of the corresponding genetically modified plants in the sample.

7.5.2 Digital PCR reaction procedures The digital PCR reaction procedures are shown in Tables 4 and 5.

7.5.3 Setting of the control digital PCR reaction A positive control, a negative control and a blank control shall be set in the test. Use the genomic DNA of genetically modified plants that contains event specific sequence as the positive control; use the genomic DNA of non-genetically modified plants that contains the same endogenous genes as a negative control; use water as a blank control. In each control PCR reaction system, except for the template, the remaining components and PCR reaction conditions are the same as

7.5.1 and 7.5.2.