SN/T 5145.3-2019 in English

Technical Background of the Standard

This standard is one of the 13 animal-derived ingredient testing standards issued by the General Administration of Customs in 2019. It aims to establish a deer-specific D-loop gene detection method to address the problem of food and feed adulteration in international trade. Compared with traditional morphological identification, the detection cycle is shortened from 72 hours to 4 hours, and the sensitivity is increased to 0.1% mass fraction.

Principles of the core method

Key operating specifications

Sample processing:Solid samples need to be ground to more than 60 mesh, and processed foods need to add washing steps to remove interferences. The temperature of the CTAB precipitation solution should be strictly controlled at 65±1℃ during DNA extraction.

Amplification system:A 20μL reaction system was used, in which the ExTaq HotStart enzyme dosage was 0.2μL (1U), and the amplification efficiency was best when the annealing temperature was 55℃.

Implementation recommendations

1. Laboratories should regularly verify primer specificity to avoid cross-reactions with closely related species such as moose and reindeer
2. Test strips should be stored away from light and moisture, and should be used within 24 hours after opening
3. It is recommended to set up three levels of controls for each batch of testing: positive (deer muscle DNA), negative (beef DNA) and blank (ddH2O)

Technology evolution analysis

Compared with the 2015 draft for comments, this standard has the following major improvements:
- Added the D-loop gene as a detection target, which is more polymorphic than the cytb gene
- Optimized the formulation of the developing solution, and added BSA to reduce nonspecific adsorption
- Clarified the sequencing confirmation requirements, and increased the homology threshold from 95% to 97%