GB/T 38164-2019Identification of animal ingredients from common livestock and poultry - Real-time PCR (English PDF)

1 Scope

This Standard specifies the real-time PCR for the identification of animal ingredient from common livestock and poultry.

This Standard applies to the Taqman probe real-time PCR qualitative determination of species ingredients from cattle, yak, buffalo, sheep, goat, pig, camel, red deer, sika deer, reindeer, rabbit, dog, chicken, duck, goose, quail, pigeon, turkey, cat, fox, mink, raccoon, and rat in meat and processed products, offal, milk, and animal feed. Limit of detection (LOD) 1% (mass fraction).

2 Normative references

The following documents, in whole or in part, are normatively referenced in this document and are indispensable for its application. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies.

GB/T 6682, Water for analytical laboratory use - Specification and test methods

GB/T 27403-2008, Criterion on quality control of laboratories - Molecular biological testing of food

GB/T 34796, Quantification and purity analysis of nucleic acid concentration in solution - Ultraviolet spectrophotometry

GB/T 35918, Identification of animal origin in animal products by DNA barcoding - Sanger sequencing

3 Terms and definitions, abbreviated terms

4 Principle

Perform real-time PCR amplification of animal ingredient from livestock and poultry using species-specific primers and probes; use the fluorescence signal of each cycle product in the PCR amplification reaction for identification, enabling qualitative detection of animal ingredient from common livestock and poultry.

5 Primers and probes for detection

The internal reference primer and probe sequences and common livestock and poultry-specific primer and probe sequences are shown in Table 1.

6 Reagents

6.3 Absolute ethanol.

6.4 Sodium hydroxide solution (10 mol/L). Add 80.0 g of sodium hydroxide (NaOH) to 160 mL of double-distilled water; dissolve; cool to room temperature; then add double-distilled water to fix he volume to 200 mL.

6.5 Na2-EDTA solution (500 mmol/L, pH 8.0). Weigh 18.6 g of Na2-EDTA; add it to 70 mL of double-distilled water; then add an appropriate amount of sodium hydroxide solution (6.4); heat until it is completely dissolved; cool to room temperature; use sodium hydroxide solution (6.4) to adjust the pH to 8.0; add double-distilled water to fix the volume to 100 mL. Sterilize at 103.4 kPa (121 °C) for 20 min.

6.6 Tris-HCl solution (1 mol/L, pH 8.0). Weigh 121.1 g of Tris and dissolve it in 800 mL of double-distilled water; use HCl to adjust the pH to 8.0; add double-distilled water to bring the volume to 1000 mL. Sterilize at 103.4 kPa (121 °C) for 20 min.

6.7 CTAB extraction buffer (pH 8.0). Weigh 4.0 g of CTAB and 16.364 g of NaCl; add 20 mL of 1 mol/L Tris-HCl solution (pH 8.0) (6.6) and 8 mL of 500 mmol/L Na2-EDTA solution (pH 8.0); dissolve in 70 mL of double-distilled water; then bring the volume to 200 mL and sterilize at 103.4 kPa (121 °C) for 20 min.

6.8 CTAB precipitate. Weigh 1.0 g of CTAB and 0.467 g of NaCl; dissolve them in 70 mL of double-distilled water; then bring the volume to 200 mL; sterilize at 103.4 kPa (121°C) for 20 min.

6.9 Proteinase K (670 U/mL). Weigh 0.10 g of proteinase K dry powder with an enzyme activity of 33.5 U/mg (Unit, enzyme unit); add 5 mL of double-distilled water; shake gently until proteinase K is completely dissolved; aliquot into small portions and store at -20 °C.

6.10 PCR Master Mix (2×), or an equivalent real-time PCR premix can be used.

7 Instruments and equipment

7.1 Real-time PCR instrument.

7.2 Nucleic acid protein analyzer or ultraviolet spectrometer.

7.3 Constant temperature water bath.

7.4 Centrifuge: centrifugal force >=12 000 g.

7.5 pH meter: grade 0.01.

7.6 Balance: sensitivity 0.1 g, 0.01 g, 0.001 g.

8 Inspection procedures