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

1 Scope

China's national method for identifying animal ingredients from common livestock and poultry species by real-time PCR. It specifies the terms and definitions, the abbreviations, the principle, the primers and probes, and the procedure. The method answers a question that food inspection asks constantly: what species is this meat. DNA survives processing that destroys every other identifying characteristic - mincing, cooking, emulsifying into a sausage - and species-specific PCR detects it in a few hours. The applications are commercial fraud, where a cheaper species is substituted for a dearer one; religious and dietary compliance, where the presence of pork or its absence is the whole question; and allergen and feed control. The horsemeat episode in Europe made the technique routine everywhere, and the reason it is written as a standard rather than left to laboratories is that the primer sequences and the reaction conditions determine whether a result can be relied on.

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

3.1 Terms and definitions For the purposes of this document, the following terms and definitions apply.

3.1.1 real-time PCR Add a fluorescent group to the PCR reaction system, and monitor the entire PCR amplification process in real time by the accumulation of fluorescence signals.

3.1.2 cycle threshold The number of cycles required for the fluorescence signal in each reaction tube to reach the set threshold.

3.2 Abbreviated terms For the purposes of this document, the following abbreviated terms apply. ATP8.ATP synthase subunit 8 COI. Cytochrome Coxidase I CTAB. Cetyltrithylammonium bromide cytb. Cytochrome b DNA. Deoxyribonucleic acid D-loop. Displacement loop region EDTA. Ethylene diaminetetraacetic acid Na2-EDTA. Ethylenediaminetetraacetic acid disodium salt ND

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.

6.3 Absolute ethanol.

6 Reagents...

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.