SN/T 5199-2020 in English
VALIDInspection protocol for livestock-derived and poultry- derived ingredients with DNA barcode method
- Issued on:2020-12-30
- Implemented on:2021-07-01
- File Format:PDF
- Delivery:Via email within 1~3 business days
$175.00
Introduction
Technical background of the standard
This standard replaces SN/T 1162-2002 and introduces DNA barcoding technology to meet the needs of animal-derived ingredient identification in import and export inspection and quarantine. Through sequence comparison of mitochondrial Cytb gene fragments (about 249bp), accurate species identification of livestock (pigs/cattle/sheep, etc.) and poultry (chickens/ducks/geese, etc.) can be achieved.
Core Detection Principle
| Technical Elements | Parameter Requirements | Key Points for Quality Control |
|---|---|---|
| Target Gene | Mitochondrial Cytb Gene | Balance between Conservatism and Variability |
| Primer Design | Universal Primer Pair (F:5'-CCC-CGC-CTG-TTT-ACC-AAA-AAC-AT-3') | Annealing Temperature 58℃ |
| Sequence Alignment | GenBank BLAST | Homology ≥97% |
Application case: In the detection of imported beef products, DNA was extracted by CTAB method and a 249bp band was obtained by PCR amplification. Sequencing comparison showed that the sequence homology with Bos taurus was 99.2%, and it was determined to be a beef component.
Key experimental process
- Sample processing: Solid samples need to be ground to 100mg, and liquid samples need to take 200μL
- DNA extraction: Using the CTAB-phenol chloroform method, A260/A280 needs to be controlled at 1.7-1.9
- PCR system: 25μL reaction system contains 12.5μL of 2×Master Mix and 1μL of each primer
- Amplification program: 35 cycles (94℃ 30s→58℃ 30s→72℃ 30s)
Implementation suggestions
- Quality control requirements: Non-vertebrate negative controls (such as shrimp) and known livestock and poultry positive controls must be set
- Equipment calibration:PCR instruments need to be regularly verified for temperature gradient, and the centrifuge speed error should be <2%
- Database update:It is recommended to synchronize the latest sequence data from GenBank every quarter
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