GB/T 19495.3-2004Detection of genetically modified organisms and derived products--Nucleic acid extraction (English PDF)

3.1 General principles

The purpose of nucleic acid extraction is to provide suitable DNA for subsequent analysis. DNA quality includes the average length of extracted DNA molecules, chemical purity, integrity of the DNA sequence and double helix, such as intra- and inter-strand linkages of DNA bases and interstrand linkages. Moreover, these characteristics are sequence-specific and therefore not randomly distributed throughout the genome.

3.2 Extraction

The basic principle of DNA extraction is to release the DNA from the sample, followed by DNA purification to remove PCR inhibitors. The different DNA extraction and purification methods in Appendix A are suitable for different samples.

DNA quantification aids subsequent PCR analysis and can be achieved through physical methods (measuring light absorption at a specific wavelength), chemo-physical methods (binding with fluorescent substances), enzymatic methods (biofluorescence detection), or quantitative PCR method. The latter is most suitable for multi-component samples and DNA with low concentrations or that has been degraded.

5.1 Preparation of test sample

5.1.1 General requirements

Except for agricultural products and minimally processed materials (such as crushed products) at the beginning of the food and/or feed chain; most industrially processed foods undergo physical, chemical, enzymatic treatments. These treatments reduce the content and purity of DNA. Therefore, the applicability and reproducibility of each method will vary depending on the specific sample; the characteristics of a particular DNA extraction method depend on the type of food being studied. The representativeness of laboratory samples shall comply with GB/T 19495.7-2004.

In the methods in Appendixes A ~ E of this Part, a sample size of 250 mg (200 mg ~ 300 mg) is sufficient for DNA-rich raw materials (such as seeds), but insufficient for materials with low DNA content or degradation. In such cases, the sample size may be increased to 1 g ~ 2 g; sample sizes exceeding this upper limit are not within the framework of this Part.

DNA shall be extracted from two separate test samples for each laboratory sample.

The preservation of standards, laboratory samples, test samples shall be carried out in accordance with GB/T 19495.1-2004.

5.1.2 Samples

Laboratory samples shall be thoroughly homogenized before being prepared as test samples.

All test sample preparation operations shall be performed in accordance with GB/T 19495.2-2004, with careful handling to prevent any contamination and alteration of sample composition.

Solid or paste-like samples with high liquid content are difficult to grind into particles of the desired size. They can be treated by removing the liquid during grinding, freezing or freeze-drying before grinding.

For grinding paste-like or viscous samples, one of the following methods can be used.

- Heating (not exceeding 40 °C);

- Homogenizing in a liquid (e.g., in water);

- Freezing (<= -20 °C).

Laboratory samples treated by the above methods may be diluted or concentrated; they shall be mixed thoroughly before preparing two sets of test samples. For liquid samples, shake the container to further homogenize the product. For samples that cannot be homogenized, such as crude oil, ensure that all precipitates have been completely detached from the inner wall of the container.

For solid samples that are not easily suspended, grinding shall be performed to facilitate DNA extraction. In this case, particle size shall be taken into account. The test sample used for DNA extraction shall contain the minimum amount of particles specified in GB/T 19495.7-2004.

Any steps that may generate dust shall be separated from other analytical steps. All removable salts, seasonings, powdered sugars shall be taken into account, according to the methods in the appendix.

For multi-component samples, the target sample (e.g., the bread layer in a fish fillet) can be separated for DNA extraction. The methodological procedure used shall be described in the analytical report.

5.2 DNA extraction and purification

5.2.1 General requirements

To obtain high-quality DNA, the following impurities should be removed and methods shall be employed.

- Treatment with suitable enzymes (pectinase, cellulase, hemicellulose, amylase, etc.) to remove polysaccharides (pectin, cellulose, hemicellulose, starch, thickeners) or extraction with organic reagents (e.g., CTAB/chloroform);

- Treatment with suitable methods, such as RNA enzymes and protease to treat RNA and/or proteins respectively;

- Small amounts of liquid residue can be treated with enzymes or organic reagents (e.g., n-hexane);

- Salts that may interfere with downstream analysis (from extraction buffer, cell division buffer, DNA precipitation steps, etc.).

For solid or dry samples, the volume of cell division buffer or extraction buffer shall be sufficient to ensure DNA dissolution.

DNA purification can be achieved by precipitation with small amounts of solvents such as phenol, chloroform, ethanol, or isopropanol, or adsorption onto solid materials (anion exchange resin, diatomaceous earth, glass glue, membranes, etc.). Several DNA purification methods can be combined. If properly combined, DNA extraction and purification can be performed in the same steps.

To improve DNA recovery, a co-precipitant (such as glycogen, PEG, or t-RNA) can be used in the precipitation step. This co-precipitant shall not contain any nuclease activity, PCR inhibitors, and/or competitors, nor shall it contain any sequences that may be similar to the target DNA sequence in the research and detection. When freeze-drying DNA, cross-contamination shall be avoided.

DNA should be resuspended in water or buffer to prevent DNA degradation.

5.2.2 Extraction blank control

To check for potential contamination during extraction, an extraction control (extraction blank control) shall be set up. The extraction blank control should preferably use buffer or water instead of the sample. Ideally, the extraction blank control can also serve as a reagent control. The tube for the extraction blank control shall be placed last in each extraction series.

Adding heterologous nucleic acids or nucleic acid supports (such as glycogen or PEG) as a co-precipitant may help improve contamination during extraction. The co- […]