GB/T 35918-2018Identification of animal origin in animal products by DNA barcoding—Sanger sequencing (English PDF)

3.1 Terms and Definitions

The following terms and definitions are applicable to this document.

3.1.1 DNA barcode

A fragment of an organism's DNA that is recognized as standard, sufficiently variable, easily amplifiable, and relatively short, serving as a representative marker for that species.

3.1.2 Sanger sequencing

Dideoxy chain termination method

Using single-stranded or double-stranded DNA as a template, and under the catalysis of DNA polymerase, a primer bound to the template is extended until a chain-terminating nucleotide is incorporated. Each reaction mixture contains four types of dNTPs, spiked with isotope- or fluorescence-labeled ddNTPs to induce the selective termination of the elongating oligonucleotides; the base sequence is subsequently determined by separating the resulting fragments using high-resolution denaturing capillary electrophoresis.

3.1.3 DNA barcoding

A technique that employs DNA sequencing technology to identify the DNA barcodes of species, utilizing their intraspecific specificity and interspecific diversity to achieve rapid species identification.

3.1.4 Quality value; QV

During the instrument sequencing process, as the software analyzes the sequencing signals, it assigns a corresponding quality value to each base - based on the predicted base-calling error rate - to assess the sequencing quality of individual bases.

3.2 Abbreviations

The following abbreviations are applicable to this document.

BOLD. The Barcode of Life Data System

bp. Base pair

CTAB. Cetyltrimethylammonium Bromide

DNA. Deoxyribonucleic Acid

dNTP. Deoxyribonucleoside Triphosphate

ddNTP. Dideoxyribonucleoside Triphosphate

EDTA. Ethylene Diaminetetraacetic Acid

NCBI. National Center for Biotechnology Information

QV. Quality Value

Taq DNA Polymerase. Thermostable DNA polymerase

Tris. Tris(hydroxymethyl) Aminomethane

7.1 Sampling

For sampling and subsampling procedures, refer to GB/T 9695.19, GB/T 14699.1, and SN/T 3500-2013.

7.2 Preparation

7.2.1 General

Samples shall be thoroughly mixed and divided into three equal portions to serve as the test sample, retest sample, and reserve sample. Sample preparation procedures must be designed to prevent cross-contamination and alterations to the sample's composition.

7.2.2 Meat products

Collect appropriate quantities of samples from various parts, then combine and mix them. Rinse the samples twice to three times, first with 70% ethanol and subsequently with double-distilled water. Transfer the samples into 50 mL centrifuge tubes or sealed bags, and store them frozen at temperatures below -20°C.

7.2.3 Milk and dairy products

Liquid dairy products shall be mixed thoroughly and then directly aliquoted for frozen storage at temperatures below -20°C. Solid dairy products shall be pulverized, transferred into 50 mL centrifuge tubes or sealed bags, and stored at temperatures between 4°C and 8°C. Powdered milk shall be mixed thoroughly, directly aliquoted into 50 mL centrifuge tubes or sealed bags, and stored at temperatures between 4°C and 8°C.

7.2.4 Hides, horns and bones, internal organs, etc

Rinse the samples twice to three times - first with 70% ethanol and subsequently with double-distilled water - then pulverize them. Transfer the samples into 50 mL centrifuge tubes or sealed bags, and store them frozen at temperatures below -20°C.

7.2.5 Blood

Apply the blood directly onto a blood collection card. After allowing it to air-dry, store it frozen at temperatures below -20°C.

7.2.6 Hair samples

Rinse the hair samples twice to three times - first with 70% ethanol and subsequently with sterile double-distilled water - then transfer them into sealed bags and store them at temperatures between 4°C and 8°C.

7.2.7 Animal feeding stuffs

Animal feeding stuffs shall be mixed thoroughly, directly aliquoted into 50 mL centrifuge tubes or sealed bags, and stored at temperatures between 4°C and 8°C.

8.1 Experimental Setup

The detection setup includes a positive control and a blank control (sterile double-distilled water); DNA extraction is performed once for both the positive and blank controls, while the samples under test are subjected to two replicate extractions. PCR amplification for all DNA samples is performed in duplicate. […]