SN/T 5199-2020Technical specification for DNA barcode detection of livestock and poultry components (English PDF)

3.1 Terms and definitions

The following terms and definitions apply to this document.

3.1.1

Livestock

Mammals, such as pigs, cows, sheep, goats, horses, donkeys, mules, and camels, that are domesticated and can be artificially controlled to breed in captivity Camels, rabbits, cats, dogs, etc.

3.1.2

Poultry

The ornithological animals raised in captivity to obtain their meat, eggs and feathers are generally pheasant and ducks, such as chickens, ducks, geese, etc.

There are also other families of birds such as turkeys, pigeons, quail and so on.

3.2 Abbreviations

The following abbreviations apply to this document.

5.1 DNA extraction reagents

5.1.1 Lysis solution, see Appendix A for the preparation method.

5.1.2 Phenol.

5.1.3 Mixture of chloroform and isoamyl alcohol (mixed in a ratio of 24.1).

5.1.4 3 mol/L sodium acetate.

5.1.5 Anhydrous ethanol.

5.1.6 70% ethanol.

5.1.7 TE buffer (Tris-EDTA buffer), see Appendix A for the preparation method.

5.2 PCR amplification reagents

5.2.1 Master Mix (2×).

7.1 Introduction

7.1.1 The process of total DNA extraction, DNA concentration and purity determination, PCR amplification, and gel electrophoresis analysis of the sample shall be in accordance with GB/T 27401 and the requirements of GB/T 27403 shall be carried out.

7.1.2 For dried samples, first wash the surface with water or scrape off the animal or human DNA that may be contaminated on the surface. For fresh samples Samples should be taken from the inside of the sample.

7.1.3 Establish quality control negative control and positive sample control, and use the same method to extract DNA for negative control, positive control and test sample.

The positive control uses any kind of livestock and poultry meat samples (such as cattle, sheep, pigs, chickens, ducks, geese, etc.), and the negative control uses any non-ridged for vertebral meat samples (such as shrimp, crabs, starfish, etc.), the negative samples should be free of other animal and human DNA contamination.

7.1.4 Equivalent DNA extraction reagents can be used for DNA extraction.

7.2 Extraction of sample total DNA

7.2.1 For solid samples, such as muscles, internal organs, fur, teeth, bones, head horns, etc., they should be fully crushed, ground, and mixed.

100 mg is used as the extract; for liquid samples, shake and mix well, and take about.200 µL as the extract.

7.2.2 Transfer the extract to a 1.5 mL centrifuge tube, add 700 µL of lysate (5.1.1), and place it in a 65 °C water bath for 3 h.

Shake and mix from time to time.

7.2.3 After centrifugation at 13 523 g for 5 min, take the supernatant and transfer to a new 1.5 mL centrifuge tube. Add equal volume of phenol (5.1.2), charge After mixing, centrifuge at 13 523 g for 5 min.

7.2.4 Take the supernatant, add an equal volume of the mixed solution of chloroform and isoamyl alcohol (5.1.3), mix well, and centrifuge at 13 523 g for 5 min.

7.2.5 Take the supernatant, add 1/10 volume of sodium acetate solution (5.1.4), mix well, then add 2 times volume of absolute ethanol pre-cooled at -20 °C (5.1.5), let stand at -20 °C for 1 h, centrifuge at 13 523 g for 5 min, discard the supernatant.

7.2.6 Wash once with 70% ethanol (5.1.6), dry at room temperature in an ultraclean bench, add 50 µL TE buffer (5.1.7) to dissolve the precipitate, Make a DNA solution.

7.3 Determination of DNA concentration and purity

Take 5 µL of DNA solution, dilute to 1 mL with double-distilled water (5.2.4), use nucleic acid protein analyzer or UV spectrophotometer to detect 260 nm and the absorbance values A260 and A280 at 280 nm.

7.4 PCR amplification

7.4.1 System

Prepare the following reaction systems in an ice box, each detection reaction system totals 25 µL, including DNA solution (10 ng~100 ng) 2.0 µL. Add the prepared reaction mixture to each PCR reaction tube. Equivalent PCR amplification reagents can be used.

7.4.2 Dosing

Add the prepared sample, negative control and positive control DNA solutions to each PCR reaction tube, and use water as the blank control.

As a template. Tightly cap the tube, mix well, and centrifuge for 5 s to 10 s.

7.4.3 Amplification

Carry out PCR amplification and store at 4 °C after the reaction. The response parameters are.

- Pre-denaturation at 94 °C for 5 min;

- Denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, and extension at 72 °C for 30 s, a total of 35 cycles;

- Extend at 72 °C for 5 min.

7.5 Gel electrophoresis analysis

Take 5 µL of the PCR amplified product and place it in a 2% agarose gel (5.3.1) and TBE (1×) solution (5.3.2).

Perform electrophoresis under pressure for 30 minutes. After the electrophoresis, use the gel imaging analysis system to observe and record the results. Target strips amplified from different animals the tape size varies slightly around 249 bp.

7.6 Sequencing and comparison

Sequencing the amplified samples showing the target bands, the results after sequencing are performed on the sequence using the BLAST function in GenBank Search and match.

8.1 Validity Principle

The following conditions should be met at the same time, otherwise this experiment will not be established.

- Blank control. no specific band;

--Negative control. no specific band;

--Positive control. There is a significant band and the size is correct.

8.2 Judgment of results

The sequencing results are searched and compared according to the method in 7.6, and the sample with the highest homology is judged to be the tested sample with a homology of more than 97% Species.

8.3 Presentation of results

If the PCR amplification of the sample is unsuccessful, the result is expressed as "no livestock or poultry components detected in the sample".

Appendix A

(Normative)

Preparation of DNA extraction lysate, TE buffer and TBE solution (1×) A.1 Lysis Solution A.1.1 Weigh CTAB 10 g, Tris 6.1 g, NaCl 41 g, and Na2EDTA 3.7 g into a 1 L beaker, add about 800 mL of double distilled water, Stir well and mix well.

A.1.2 According to the actual temperature environment, use concentrated HCl and NaOH to adjust the pH to 8.0.

A.1.3 After adding double distilled water to a constant volume to 1 L, store at room temperature.

A.2 TE buffer

A.2.1 Weigh 121.1 g of Tris into a 1 L beaker, add about 800 mL of double distilled water, stir and mix thoroughly, according to the actual temperature environment, Use concentrated HCl to adjust the pH to 8.0, continue to add double distilled water to make the volume to 1 L, and obtain Tris with a concentration of 1 mol/L and a pH of 8.0.

A.2.2 Weigh 186.1 g of Na2EDTA into a 1 L beaker, add about 800 mL of double distilled water, stir and mix thoroughly, according to the actual temperature Environment, use NaOH to adjust the pH value to 8.0, continue to add double distilled water to make the volume to 1 L, and obtain a concentration of 0.5 mol/L and a pH value of 8.0 Na2EDTA.

A.2.3 Mix the above two solutions in proportion to obtain a TE buffer with a final concentration of 10 mmol/L Tris and 1 mol/L Na2EDTA.

Store at room temperature.

A.3 TBE solution

Weigh 54 g of Tris, 27.5 g of boric acid, and 20 mL of 0.5 mol/L Na2EDTA (pH 8.0) described in A.2.2 and place them in a 1 L beaker.

Add 1 000 mL of double-distilled water, stir and mix thoroughly to obtain a TBE solution (1×), and store at room temperature.